Anti his hrp

Plates were precoated with 5 µg/mL of hACE2 receptor overnight and washed with phosphate-buffered saline with 0.05% Tween (PBS-T) buffer and blocked with TBS Startblock blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA). Histidine-tagged S-RBD-Rhizavidin was threefold serially diluted and added to coated wells for 2 h at RT. Following washing, 100 µL of anti His-HRP (Bio-Rad, Hercules, CA, USA) was added to the plate. Plates were incubated for 1 h at RT, while SureBlue TMB Microwell Peroxidase Substrate (VWR, Radnor, PA, USA) equilibrated to RT. After a final wash, 100 µL TMB substrate was added to wells and development was stopped with 100 µL of 1N hydrochloric acid after 10 min at RT. The ELISA plates were read at an absorbance of 450 nm on a SpectraMax i3x Plate Reader using Softmax Pro 7.0.

A sandwich ELISA method was utilized to determine the specific antigenic content of viral samples [35 (link)]. Briefly, ELISA plates were coated overnight with polyclonal rabbit anti-EVA71 immune sera at a 1 : 2000 dilution. Samples were added to wells and incubated at 37 °C for 1.5 h. The 16-2-2D scFv fragment or mAb 979 were used to detect NAg and HAg particles, respectively, and were incubated at 37 °C for 1 h. Anti-His HRP was used to detect scFv at a 1 : 1000 dilution (Bio-Rad). Anti-mouse HRP was used at 1 : 1000 dilution (Merck) to detect mAb 979. Samples were detected using OPD and the OD492 nm measured using the Biotek PowerWave XS2 plate reader. Data was graphed using GraphPad Prism software.

A sandwich ELISA method was utilized to determine the NAg content of viral samples. Briefly, ELISA plates were coated overnight with polyclonal rabbit anti-EVA71 immune sera at a 1:2,000 dilution. Twofold dilutions of clarified virus culture supernatant (5 × 10⁶ TCID₅₀/mL to 6.25 × 10⁵ TCID₅₀/mL) or PBS alone were added to wells and incubated at 37°C for 1.5 h. Twofold dilutions of scFv (40 μg/mL to 2.5 μg/mL) or PBS alone were added to wells and incubated at 37°C for 1 h, and anti-His-HRP was used to detect scFv at a 1:1,000 dilution (Bio-Rad). Samples were detected using o-phenylenediamine dihydrochloride (OPD), and the OD₄₉₂ nm was measured using the Biotek PowerWave XS2 plate reader. Raw data were graphed, and a suitable dynamic range for the scFv was determined to be between 5 μg/mL and 20 μg/mL. All further assays were carried out following the same protocol using an scFv concentration of 10 μg/mL.
To detect expanded antigenic conformations, MAb 979 was used in place of the scFv in the aforementioned method at 1:1,000 dilution (Merck). The presence of MAb 979 was detected using anti-mouse IgG-HRP conjugate as described above.