Diethanolamine buffer

96-well NUNC immunoplates (Thermo Fisher Scientific) were coated with 50 μL of either native state or denatured (boiled at 95 °C for 5 min and reduced using 5 mM dithiothreitol) CyRPA at 2 μg/ml overnight at 4 °C. The plates were then washed with PBS/Tween-20 (PBS-T) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.05 % Tween-20) three times. Next, plates were blocked with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄) with 1% casein for 60 min at room temperature. Plates were then washed with PBS-T before the addition of 50 μL of mAb at 10 μg/ml, diluted in PBS-casein. mAb solution was incubated for 60 min at room temperature before washing with PBS-T. Next, 50 μL of goat anti-human whole IgG alkaline phosphate conjugate (Sigma-Aldrich), diluted 1/1000 in PBS with 1% casein, was added to each well and then incubated for 60 min at room temperature, then washed. Next, a 20 mg tablet of 4-Nitrophenyl phosphate (pNPP, Sigma-Aldrich) was dissolved in 4 ml of diethanolamine buffer (Thermo Fisher Scientific) with 16 ml deionized water. 100 μL pNPP was then added to each well and plates were allowed to develop until the positive control (mAb c12) reached an OD₄₀₅ of approximately 2.0. The anti-RH5 mAb, R5.011^{5 (link)}, was used as a negative control as it should not bind to CyRPA.

Recombinant RVFV Gn ectodomain (UniProt accession number P21401, residues 154–560) and Gc ectodomain (UniProt accession number P21401, residues 691–1120) were expressed in HEK293 cells as previously described [26 (link)]. This protein was used to perform ELISAs as previously described [26 (link)] with the exception of the following modifications. A positive reference serum made from a pool of high responding mice was included on each plate as a standard curve (titrated 1:2 and added in duplicate). The secondary antibody used (goat antimouse whole IgG-alkaline phosphatase, Sigma, St. Louis, MO, USA) was diluted 1:1000 in casein. Plates were developed by adding 100 µL/well of 4-nitrophenyl phosphate disodium salt Hexahydrate (Sigma) in diethanolamine buffer (ThermoScientific). Optical density (OD) was read at 405 nm using a BioTek ELx808 plate reader until the internal control (reference serum diluted 1:800) had reached an OD of 1. OD values of reference serum titrations were then fitted to a 4-parameter standard curve using Gen5 software (v3.09 BioTek). Test sera antibody units were calculated from their OD values using the estimated parameters from the standard curve.

Three Nunc-immuno maxisorp plates were coated with 2 μg/mL Oxford, NIH, or WRAIR AMA1 protein in DPBS. The same independent standard curve (10 points) was run on each plate in duplicate, using a previously described high-titer anti-AMA1 human serum reference sample [14 (link)]. Otherwise, the same ELISA method for this experiment was performed on all three plates in parallel: plates were left at RT overnight, then washed 6 times with PBS containing 0.05% Tween 20 (PBS/T) and blocked for 1 h with Casein block solution (Pierce). After another wash step, samples were added to each plate for 2 h. Plates were washed again and alkaline phosphatase-conjugated goat anti-human IgG (γ-chain) (Sigma) diluted 1:1000 in Casein block solution was added for 1 h, before development with p-nitrophenylphosphate substrate (Sigma) diluted in diethanolamine buffer (Thermo Fisher Scientific). Optical density at 405 nm (OD₄₀₅) was read using a microplate reader (Biotek) and Gen5 v1 software.