Development of a Double-Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies.
Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time-consuming and require BSL3 facilities. VP2 protein is considered the major serotype-defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV-8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti-VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti-VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti-VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.
UMR 1161 VIROLOGIE ANSES-INRA-ENVA, Université Paris Est, ANSES, Maisons-Alfort, France.
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Breard
UMR 1161 VIROLOGIE ANSES-INRA-ENVA, Université Paris Est, ANSES, Maisons-Alfort, France.
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Garnier
UMR PIMIT (I2T), Université de La Réunion, INSERM U1187, CNRS 9192, IRD 249, technology platform CYROI, Saint-Clotilde, La Reunion, France.
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Despres
UMR 1161 VIROLOGIE ANSES-INRA-ENVA, Université Paris Est, ANSES, Maisons-Alfort, France.
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Blaise Boisseau
ID VET, Montpellier, France.
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Comtet
UMR 1161 VIROLOGIE ANSES-INRA-ENVA, Université Paris Est, ANSES, Maisons-Alfort, France.
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Viarouge
UMR 1161 VIROLOGIE ANSES-INRA-ENVA, Université Paris Est, ANSES, Maisons-Alfort, France.
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Bakkali-Kassimi
ID VET, Montpellier, France.
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Pourquier
MERIAL S.A.S., Lyon, France.
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P
Hudelet
UMR 1161 VIROLOGIE ANSES-INRA-ENVA, Université Paris Est, ANSES, Maisons-Alfort, France.
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Vitour
Unité Sanitaire de la Faune, Office National de la Chasse et de la Faune Sauvage, Gap, France.
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Rossi
Unité de Pathologie du Bétail, Ecole Nationale Vétérinaire d'Alfort, Université Paris-Est, Maisons-Alfort, France.
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Belbis
UMR 1161 VIROLOGIE ANSES-INRA-ENVA, Université Paris Est, ANSES, Maisons-Alfort, France.
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Sailleau
UMR 1161 VIROLOGIE ANSES-INRA-ENVA, Université Paris Est, ANSES, Maisons-Alfort, France.
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Zientara
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000276:immunology*; D019921:Biotinylation; D001819:Bluetongue / Q000175:diagnosis* / Q000821:virology; D001820:Bluetongue virus / Q000276:immunology* / Q000302:isolation & purification; D036022:Capsid Proteins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005260:Female; D007118:Immunoassay / Q000662:veterinary; D008297:Male; D008863:Microspheres; D011994:Recombinant Proteins; D012418:Ruminants; D012680:Sensitivity and Specificity; D065288:Serogroup; D012756:Sheep; D014758:Viral Core Proteins / Q000276:immunology*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; C442520:VP2 protein, Bluetongue virus; D014758:Viral Core Proteins; C066862:VP7 protein, orbivirus

ELISA
; 
SNT
; bluetongue virus; diagnostic; recombinant antigen