Development of a multiplex assay for antibody detection in serum against pathogens affecting ruminants

Abstract

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Multiplex Assay for Simultaneous Detection of Antibodies against Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein and Glycoproteins in Ruminants

“…The production of CCHFV N protein was described recently ( 32 ). Briefly, cDNA designed to express the full-length N protein of CCHFV strain Baghdad 12 (GenBank accession CAD61342.1 , but with conservative substitutions T111I, R195H, and H445D) was generated synthetically (Genewiz).…”

“…The purification of the CCHFV N protein and its purity were assessed by SDS-PAGE followed by Coomassie staining. This purification was also followed by Western blotting with an anti-CCHFV N protein mAb (2G10) developed at Eurofins-INGENASA S.A. ( 32 ). Finally, to confirm the detection of the recombinant CCHFV N protein, a WB with CCHFV-positive field sera was performed.…”

“…Serial dilutions of mAbs specific to each protein, or to its GST tag, were used to perform a confirmation assay in order to assess the coupling efficiency. The mAbs were produced at Eurofins-INGENASA S.A.: 2G10 anti-CCHFV N protein for the CCHFV N protein and 3H11 anti-GST for CCHFV GP38 and G N e ( 32 ). After the confirmation assay, these mAbs were used to optimize the coupling concentration of each antigen to its corresponding bead region.…”

“…The assay conditions have been described recently ( 32 ) with some modifications to reduce the background of the assay. Plates (96 wells; Stripwell microplate medium binding polystyrene, Costar) previously blocked for 30 min with StabilZyme SELECT stabilizer (Surmodics) were used for the assay.…”

A multiplex assay for the detection of antibodies to relevant swine pathogens in serum

“…This technology combines fluorescent-dyed microspheres, a detection instrument based on lasers read-out, digital signal processing and an analysis software (Christopher-Hennings et al, 2013;Graham et al, 2019). While BBMAs are widely applied in human health, with the development of methods for drug discovery (Komnatnyy et al, 2018), diseases diagnosis (Grignard et al, 2019;Lu et al, 2005) and immune response characterisation (Jones et al, 2002) among others, work in the veterinary field has been more limited, although interest has been rising recently (Chen et al, 2016;Fabian et al, 2020;Hoste et al, 2020;Laamiri et al, 2016;Ragan et al, 2018). To date, there are a few commercial assays available (References TRD-500 and TRD-502, Biovet Inc. Saint-Hyacinthe, Canada).…”