Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

cg.contactk.makkouk@cgiar.orgen_US
cg.contributor.centerInternational Center for Agricultural Research in the Dry Areas - ICARDAen_US
cg.contributor.centerJulius Kühn-Institut - JKI (Germany)en_US
cg.contributor.funderInternational Center for Agricultural Research in the Dry Areas - ICARDAen_US
cg.contributor.projectCommunication and Documentation Information Services (CODIS)en_US
cg.contributor.project-lead-instituteInternational Center for Agricultural Research in the Dry Areas - ICARDAen_US
cg.creator.idKumari, Safaa: 0000-0002-4492-6257en_US
cg.date.embargo-end-dateTimelessen_US
cg.identifier.doihttps://dx.doi.org/10.1046/j.1439-0434.2001.00674.xen_US
cg.isijournalISI Journalen_US
cg.issn0931-1785en_US
cg.issn1439-0434en_US
cg.issue9en_US
cg.journalJournal of Phytopathologyen_US
cg.subject.agrovocantibodiesen_US
cg.subject.agrovocdetectionen_US
cg.subject.agrovocserologyen_US
cg.volume149en_US
dc.contributorMakkouk, Khaleden_US
dc.contributorKatul, L.en_US
dc.contributorVetten, H. J.en_US
dc.creatorKumari, Safaaen_US
dc.date.accessioned2021-07-27T23:51:52Z
dc.date.available2021-07-27T23:51:52Z
dc.description.abstractSpecific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM. About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.en_US
dc.identifierhttps://mel.cgiar.org/dspace/limiteden_US
dc.identifier.citationSafaa Kumari, Khaled Makkouk, L. Katul, H. J. Vetten. (20/12/2001). Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus. Journal of Phytopathology, 149 (9), pp. 543-550.en_US
dc.identifier.statusTimeless limited accessen_US
dc.identifier.urihttps://hdl.handle.net/20.500.11766/13528
dc.languageenen_US
dc.publisherWiley (12 months)en_US
dc.sourceJournal of Phytopathology;149,(2001) Pagination 543-550en_US
dc.subjecthis-tag fusion proteinen_US
dc.subjectnanovirusen_US
dc.subjectrecombinant proteinen_US
dc.titlePolyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virusen_US
dc.typeJournal Articleen_US
dcterms.available2001-12-20en_US
dcterms.extent543-550en_US
mel.impact-factor1.789en_US

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