Goat anti mouse igg1 hrp

Peanut-specific IgE and IgG₁ were measured by ELISA. High binding 96-well plates (Corning) were coated with CPE (20 μg/mL) in coating buffer at 4 °C overnight. Coated plates were blocked with FBS (10%) in PBS for 2 h at room temperature. Plates were washed and incubated with serum samples overnight at 4 °C. After washing, detection antibody (rat anti-mouse IgE-HRP (Southern Biotech) or goat anti-mouse IgG₁-HRP (Thermo Fisher Scientific)) was added and incubated for 2 h at room temperature. O-phenylenediamine (OPD) was used to develop the assay and H₂SO₄ (3 N) was added to stop the reaction for absorbance reading at 492 nm.

Western blots were carried out as previously described^{26 (link)}. Primary antibodies used were anti-HA (Sigma, H3663-200UL; 1:1000 dilution), anti-FLAG (Sigma, F1804-200UG, 1:2000 dilution) and secondary antibody Goat anti-Mouse IgG1, HRP from Thermo Fisher Scientific, PA1 74421, (1:10000 dilution). Full scan uncropped Western blots are provided (Source Data 1). Immunolocalization of RAP2.3 was carried as described^{70 (link)}. Four-day-old seedlings containing the 35S:RAP2.3^3XHA transgene were treated for 3 h with 50 μM bortezomib or DMSO (control). Anti-HA primary antibody (Roche, Lewes, UK) and Alexa-Fluor-488-coupled anti rat secondary antibody (1:200 dilution) (Molecular Probes, Carlsbad, CA) were used for detection. Seedlings were counter stained using Propidium Iodide and visualised using confocal microscopy.

For determination of total IgG antibodies titers against AP205-RBM in sera of immunized mice, ELISA plates were coated with 0.1 µg/mL and 1.0 μg/mL spike RBD or full spike proteins (Sinobiological, Beijing, China) overnight, respectively. Plates were washed with PBS-0.01% Tween and blocked using 100 µL PBS-Casein 0.15% for 2 h in RT. Sera from immunized mice were diluted 1/20 initially and a 1/3 dilution chain was performed. Plates were incubated for 1 h at RT. After washing with PBS-0.01%Tween (PBST), goat anti-mouse IgG conjugated to Horseradish Peroxidase (HRP) (Jackson ImmunoResearch, West Grove, PA, USA) was added 1/2000 and incubated for 1 h at RT. Plates were developed and OD 450 reading was performed. Antibody titers were determined as the concentrations of antibody at 50% maximum optical density (OD50). IgG subclasses were measured from day 35 sera using the same ELISA protocol with the following secondary Abs: goat anti-mouse IgG1-HRP and goat anti-mouse IgG2a-HRP (1:1000) (Thermo Fischer Scientific, Waltham, MA, USA), goat anti-mouse IgG2b-HRP (SouthernBiotech, Birmingham, AL, USA) 1:4000, rat anti-mouse IgG3-HRP (Becton, Dickinson, Franklin Lakes, NJ, USA) 1:2000 incubated at RT for 1 h.