Hrp conjugated goat anti mouse igm or igg antibodies

For the detection of TNP-specific antibodies by ELISA, plates were coated with TNP₃₃-BSA. ELISA analyses of mouse IgM, IgG, IgA, IgE, and IgG3 in sera from unimmunized and immunized mice were achieved using HRP-conjugated secondary antibodies against each mouse antibody isotype (SouthernBiotech) and the 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate system (Sigma). For ELISPOT assays, MultiScreen-IP filter plates (EMD Millipore) were coated with TNP₃₃-BSA. Splenocytes and bone marrow cells were serially diluted across the plate and subsequently incubated for 16 h at 37 °C. HRP-conjugated goat anti-mouse IgM or IgG antibodies (SouthernBiotech) diluted in blocking buffer (10% FBS in PBS) were added. The spots were detected using TMB substrate for ELISPOT (Mabtech) and scanned and counted with an ImmunoSpot Analyzer (Cellular Technology Ltd.).

Plates were coated with 100 ng/well of SARS-CoV-2 RBD protein (RayBiotech, Norcross, GA, USA) overnight at 4 °C. Plates were washed twice with PBS-T (0.05% Tween 20; Sigma-Aldrich, St. Louis, MO, USA) and blocked with PBS containing 8% FBS (HyClone, Logan, UT, USA) for 2 h at room temperature (RT). Serially diluted (4-fold, starting with 1:40) sera samples were added and incubated for 1 h at RT. This was followed by a 1 h incubation with HRP-conjugated goat anti-mouse IgM or IgG antibodies (Southern Biotech, Birmingham, AL, USA). 3,3′,5,5′ Tetramethylbenzidine (TMB, BD Biosciences, San Jose, CA, USA) was added to the well for 5 min. Then, stop solution was added (Thermo Fisher Scientific, Waltham, MA, USA), and the absorbance was read at 450 nm by a BioTek Cytation 7 plate reader. Binding endpoint titers were determined using a cutoff value determined by the mean of the negative controls (noninfected control-fed or AHCC-fed samples) + 3 x standard deviation (SD).