A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses - PubMed (original) (raw)

doi: 10.1155/2015/678084. Epub 2015 Sep 17.

Philippe Desprès 2, Sylvie Paulous 3, Jessica Vanhomwegen 3, Steeve Lowenski 1, Norbert Nowotny 4, Benoit Durand 1, Annabelle Garnier 1, Sandra Blaise-Boisseau 1, Edouard Guitton 5, Takashi Yamanaka 6, Stéphan Zientara 1, Sylvie Lecollinet 1

Affiliations

A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses

Cécile Beck et al. Biomed Res Int. 2015.

Abstract

West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using the Drosophila S2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.

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Figures

Figure 1

Figure 1

12% polyacrylamide gel electrophoresis showing the recombinant WNV.sE-SNAP, SNAP-WNV.EDIII, SNAP-JEV.EDIII, and SNAP-TBEV.EDIII proteins after purification. _∗_MW: molecular weight marker.

Figure 2

Figure 2

Presentation of the flavivirus microsphere immunoassay (MIA) with four beads coupled to four antigens (WNV.sE, WNV.EDIII, JEV.EDIII, and TBEV.EDIII).

Figure 3

Figure 3

MIA fluorescence results, expressed as the ratio of the sample MFI over the positive control MFI X 100 (S/P ratio) on 20 ELISA positive (P), three doubtful (D), and five negative (N) horse samples from Austria, as determined with the ID Screen West Nile competition ELISA kit (ID Vet). The cut-off for the TBEV.EDIII antigen was determined to be 61.

Figure 4

Figure 4

Reference equine sera sampled from ponies experimentally infected by different flaviviruses: TBEV (a), JEV (b), WNV lineage 1 (c), and WNV lineage 2 (d) collected on different days after infection and tested by flavivirus MIA with four antigen coupled beads (JEV.EDIII, WNV.EDIII, WNV.sE, and TBEV.EDIII). The mean and standard error of the S/P ratio of Ag “_x_” divided by the ROC cut-off value for Ag “_x_” is displayed. A sample was considered positive for the bead if its ratio was greater than one. The assay was carried out twice in separate experiments.

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