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 : 001241 ( Main/Exploration ); précédent : 001240; suivant : 001242Construction 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 [Inde] ; J. Charumathi [Inde] ; R. Ravishankaran [Inde] ; P. Kaliraj [Inde] ; S. Meenakshisundaram [Inde]Source :
- Journal of helminthology [ 1475-2697 ] ; 2016.
Descripteurs français
- KwdFr :
- Animaux, Anticorps à chaîne unique (analyse), Anticorps à chaîne unique (génétique), Anticorps à chaîne unique (immunologie), Antigènes d'helminthe (génétique), Antigènes d'helminthe (immunologie), Antigènes d'helminthe (sang), Escherichia coli (génétique), Escherichia coli (métabolisme), Expression des gènes, Filariose lymphatique (diagnostic), Filariose lymphatique (parasitologie), Filariose lymphatique (sang), Humains, Protéines recombinantes (génétique), Protéines recombinantes (immunologie), Souris, Test ELISA, Wuchereria bancrofti (génétique), Wuchereria bancrofti (immunologie), Wuchereria bancrofti (isolement et purification).
- MESH :
- analyse : Anticorps à chaîne unique.
- diagnostic : Filariose lymphatique.
- génétique : Anticorps à chaîne unique, Antigènes d'helminthe, Escherichia coli, Protéines recombinantes, Wuchereria bancrofti.
- immunologie : Anticorps à chaîne unique, Antigènes d'helminthe, Protéines recombinantes, Wuchereria bancrofti.
- isolement et purification : Wuchereria bancrofti.
- métabolisme : Escherichia coli.
- parasitologie : Filariose lymphatique.
- sang : Antigènes d'helminthe, Filariose lymphatique.
- Animaux, Expression des gènes, Humains, Souris, Test ELISA.
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
Affiliations:
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Kamatchi</name><affiliation wicri:level="1"><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>Centre for Biotechnology, Anna University,Chennai600 025</wicri:regionArea><wicri:noRegion>Chennai600 025</wicri:noRegion></affiliation></author><author><name sortKey="Charumathi, J" sort="Charumathi, J" uniqKey="Charumathi J" first="J" last="Charumathi">J. Charumathi</name><affiliation wicri:level="1"><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>Centre for Biotechnology, Anna University,Chennai600 025</wicri:regionArea><wicri:noRegion>Chennai600 025</wicri:noRegion></affiliation></author><author><name sortKey="Ravishankaran, R" sort="Ravishankaran, R" uniqKey="Ravishankaran R" first="R" last="Ravishankaran">R. 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Meenakshisundaram</name><affiliation wicri:level="1"><nlm:affiliation>Centre for Biotechnology, Anna University,Chennai600 025,India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>Centre for Biotechnology, Anna University,Chennai600 025</wicri:regionArea><wicri:noRegion>Chennai600 025</wicri:noRegion></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="KwdFr" xml:lang="fr"><term>Animaux</term><term>Anticorps à chaîne unique (analyse)</term><term>Anticorps à chaîne unique (génétique)</term><term>Anticorps à chaîne unique (immunologie)</term><term>Antigènes d'helminthe (génétique)</term><term>Antigènes d'helminthe (immunologie)</term><term>Antigènes d'helminthe (sang)</term><term>Escherichia coli (génétique)</term><term>Escherichia coli (métabolisme)</term><term>Expression des gènes</term><term>Filariose lymphatique (diagnostic)</term><term>Filariose lymphatique (parasitologie)</term><term>Filariose lymphatique (sang)</term><term>Humains</term><term>Protéines recombinantes (génétique)</term><term>Protéines recombinantes (immunologie)</term><term>Souris</term><term>Test ELISA</term><term>Wuchereria bancrofti (génétique)</term><term>Wuchereria bancrofti (immunologie)</term><term>Wuchereria bancrofti (isolement et 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 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d'helminthe</term><term>Protéines recombinantes</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="isolement et purification" xml:lang="fr"><term>Wuchereria bancrofti</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="parasitologie" xml:lang="fr"><term>Filariose lymphatique</term></keywords><keywords scheme="MESH" qualifier="parasitology" xml:lang="en"><term>Elephantiasis, Filarial</term></keywords><keywords scheme="MESH" qualifier="sang" xml:lang="fr"><term>Antigènes d'helminthe</term><term>Filariose lymphatique</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><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Expression des gènes</term><term>Humains</term><term>Souris</term><term>Test ELISA</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><affiliations><list><country><li>Inde</li></country></list><tree><country name="Inde"><noRegion><name sortKey="Kamatchi, R" sort="Kamatchi, R" uniqKey="Kamatchi R" first="R" last="Kamatchi">R. Kamatchi</name></noRegion><name sortKey="Charumathi, J" sort="Charumathi, J" uniqKey="Charumathi J" first="J" last="Charumathi">J. Charumathi</name><name sortKey="Kaliraj, P" sort="Kaliraj, P" uniqKey="Kaliraj P" first="P" last="Kaliraj">P. Kaliraj</name><name sortKey="Meenakshisundaram, S" sort="Meenakshisundaram, S" uniqKey="Meenakshisundaram S" first="S" last="Meenakshisundaram">S. Meenakshisundaram</name><name sortKey="Ravishankaran, R" sort="Ravishankaran, R" uniqKey="Ravishankaran R" first="R" last="Ravishankaran">R. Ravishankaran</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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