Hrp conjugated anti human igg antibody

The RVFV purified truncated Gns (Gn1a–Gn10a) and RVFV Gn were coated at 0.2 μg per well on 96-well plates at 4 °C overnight. The plates were washed three times with PBST (0.2% Tween 20 in PBS) and blocked with 2% BSA at 37 °C for 1 h. The plates were washed three times with PBST, and the threefold serial dilutions of serum samples in PBST containing 0.2% BSA (starting at 1:100 dilution) were added for 1 h at 37 °C. Plates were washed three times with PBST, and an HRP-conjugated anti-human IgG antibody (Abcam, Cambridge, UK) was added for 1 h at 37 °C. Then, 3,3′,5,5′-tetramethylbenzidine substrate (Solarbio, Beijing, China) was added after being washed three times and 2 M H₂SO₄ was added to stop the reaction. The optical density (OD) was measured at 450–630 nm (OD₄₅₀–OD₆₃₀).

The polypeptides ⁴⁰⁹GSKKCTGDAAFCSAY⁴²³, contained in segment Gn9a, were subjected to alanine-scanning mutagenesis (alanine residues were mutated to serine) to generate 15 segments, named Gn9b–Gn9p. Each segment was also individually cloned into the EcoRI and XhoI restriction sites of the pET-32a vector, expressed via a prokaryotic expression system, and purified using a HisTrap HP column (GE Healthcare, Chicago, IL, USA). Then, these purified segments were coated on 96-well plates (2 μg/mL, 100 μL/well) and incubated with 1332F11 (0.5 μg/mL) and 1331E4 (0.01 μg/mL). NAb concentrations were determined using median effective concentration (EC50) against Gn9a to ensure that signals were within the linear range of detection. NAbs were detected using an HRP-conjugated anti-human IgG antibody (Abcam, Cambridge, UK). Antibody reactivity against each mutant segment was calculated relative to wild-type Gn9a reactivity by normalizing to the OD₄₅₀–OD₆₃₀ from wild-type Gn9a. Mutations were considered critical to the NAb epitope if they did not support reactivity of the NAb.