ELISA for Viral Glycoprotein Detection

Microtitre plates (Nunc Immuno MaxiSorp, ThermoFisher, Waltham, MA, USA) were coated with recombinant NiV G (0.86 mg/mL) [18 (link)], NiV F (0.8 mg/mL, DAG-WT633, Creative Diagnostics, Shirley, NY, USA), NiV N (0.85 mg/mL, NativeAntigen Company, Kidlington, UK, Cat# REC31746), or EBOV GP (1.015 mg/mL, IBT Bioservices, Rockville, MD 20850, USA, Cat# 0511-015) in a carbonate/bicarbonate buffer (pH 9.6) overnight at 4 °C. Following overnight incubation, plates were washed five times with 0.01 M phosphate-buffered saline with 0.1% Tween 20 (PBS-T). The plates were blocked with casein-blocking buffer (Sigma-Aldrich, St. Louis, MO, USA and Burlington, MA, USA) and incubated for 60 min at 37 °C with gentle shaking. Detection mAbs (1:50) were then added, followed by HRP-conjugated anti-mouse IgG (1:2000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Then 3,3′, 5,5′ tetramethylbenzidine dihydrochloride (TMB, Pierce Biotechnology, Inc., Rockford, IL, USA) was added. Each incubation and wash step was as described above. The reaction was stopped with 2 M sulfuric acid and an optical density at a wavelength of 450 nm (OD₄₅₀) was read in an Emax microplate reader (Molecular Devices, San Jose, CA, USA).