F96 maxisorp nunc immuno plate

F96 Maxisorp Nunc-Immuno Plates (Thermo Fisher Scientific) were coated with recombinant RRV 26–95 gH-FcStrep/gL (described previously [15 (link)]) at 1μg/ml in PBS overnight. After three washes with PBS-T, the wells were blocked with 10% FBS in PBS for 2h. Incubation with Plxdc1 ectodomain or Plxdc2 ectodomain was performed for 2h at room temperature in 10% FBS in PBS. The plates were washed three times with PBS-T. Bound protein was detected via the C-terminal 6XHis Tag using 6XHis Antibody MA1-135 (Invitrogen) followed by three washes in TBS-T and incubation with donkey anti-mouse horseradish peroxidase (HRP)-coupled secondary antibody (Dianova). After three washes, 3,3′,5,5′-Tetramethylbenzidin (TMB) substrate (Thermo Fisher Scientific) was added and the reaction was stopped by adding 1M HCl. The plates were imaged on a Biotek Synergy 2 plate reader.

A direct ELISA using walnut-specific multimerized JrBSF-scFv (Juglans regia Biotinylated Soluble Fragment-single chain antibody, multimerized with ExtrAvidin-HRP) was also used to detect walnut protein [15 (link)].
The wells of microtiter plates (F96 MaxiSorp Nunc immunoplates, Thermo Fisher Scientific, Waltham, MA, USA) were coated for 16 h at 4 °C with 100 µL of the protein extracts diluted 1:100 in PBS. After washing three times with TBS, they were blocked with 200 μL of 3% bovine serum albumin (BSA) in TBS for 1 h at 37 °C and washed again. One hundred microliters of multimerized scFv (2 mg mL⁻¹) diluted 1:500 (v/v) in TBST containing 1% BSA was added to each well, and the plates were shaken for 2 h at room temperature and washed again. Then, 100 μL TMB substrate solution was added to each well, and the plates were incubated with shaking for 10 min before addition of 50 μL 1 M sulfuric acid and measurement of Absorbance at 450 nm. All experiments were performed in duplicate on three different days. Three different concentrations of walnut in corn flour (10⁵, 10³, and 0 mg kg⁻¹) were included in each plate as internal controls. A commercial product was considered positive for walnut when its absorbance value was higher than that of the 10³ mg kg⁻¹ standard.

F96 Maxisorp Nunc-Immuno plates (Thermo Fisher Scientific) were coated with 2 mg/mL of recombinant RBD or N proteins overnight at 4°C. After washing with PBS, the plates were blocked with 1% BSA in PBS for 1.5 h at room temperature. Heat-inactivated plasma and reference monoclonal antibodies (mAbs) against RBD (COVA1-18) (Brouwer et al., 2020 (link)) or against N (SKOT-9) (Ohnishi et al., 2005 (link)) were serially-diluted in PBS containing 1% BSA and 0.05% Tween 20 (eight 4-fold serial dilutions starting at 1:20 dilution for plasma, or eight 4-fold serial dilutions starting at a 1 mg/mL for mAbs), and then incubated overnight at 4°C. The following day, the plates were washed with PBS containing 0.05% Tween 20. HPR-conjugated goat anti-human IgG (Southern Biotech) was diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (TOYOBO) and incubated for 1.5 h at room temperature. HRP-activity was visualized by the addition of OPD substrate (Sigma-Aldrich), and OD490 was measured using iMark microplate reader (Bio-Rad).