Goat anti mouse igg3 hrp

For basal serum Ab (IgM/IgG1/IgG2b/IgG2c/IgG3/IgA) measurement, microtiter plates were coated with goat anti-mouse Ig (5 μg/ml, Southern Biotech) overnight at 4 °C. For NP-specific Ab measurement, NP(27)-BSA (Biosearch Technology) or NP(9)-BSA (high affinity) (Biosearch Technology) was used as the capture antigen. Then, nonspecific binding was blocked with 0.5% BSA in PBS for 2 h at 37 °C. Diluted serum samples were incubated in plates for 1 h at 37 °C. Plates were incubated for 1 h with goat anti-mouse IgA-HRP, goat anti-mouse IgM-HRP, goat anti-mouse IgG1-HRP, goat anti-mouse IgG2b-HRP, goat anti-mouse IgG2c-HRP, and goat anti-mouse IgG3-HRP (all from Southern Biotech) and then for 15–30 mins with 100 µl/well TMB (BioLegend) substrate solution, followed by incubation with 50 µL 2 N H2SO4 to stop the reaction. Absorbance values were read at 450 nm using a microplate reader (Cytation5, BioTek).

An ELISA assay was performed as described previously (27 (link)). In brief, we coated the plates with NP2-BSA (2.5 mg/ml) or NP30-BSA (2.5 mg/ml) at 4°C overnight, washed the plates with PBST for three times, and blocked them with 2% BSA in PBST for 2 h at room temperature. Then, the serum from the mice which were immunized with NP-KLH on day 7, 14 and 21, or NP-Ficoll on day 7 were diluted with 1% BSA in PBS, added into the plates, and incubated at room temperature for 2 h. Then the plates were washed with PBST for 6 times and incubated with goat anti-mouse IgG1-HRP, goat anti-mouse IgG3-HRP, or goat anti-mouse IgM-HRP (Southern Biotech) for 1 h at room temperature. Finally, the plates were washed with PBST for 6 times, developed with TMB (Vector Laboratories), stopped, and read at 450 and 570 nm using a BioTek plate reader.

Spike protein (1 μg ml⁻¹) diluted in coating buffer [28.5 mM Na₂CO₃ and 71.4 mM NaHCO₃ (pH 9.6)] was used to coat 96-well plates for 2 hours at 37°C. Wells were washed and then blocked with 2% BSA in PBS-T for 2 hours at 37°C. Mouse sera (serially diluted 10-fold in PBS-T containing 1% BSA) were incubated in the wells for 1 hour at 37°C and then washed with PBS-T. Goat anti-mouse IgG-HRP (GenScript, catalog no. A00160), goat anti-mouse IgG1-HRP (SouthernBiotech, catalog no. 1073), goat anti-mouse IgG2a-HRP (SouthernBiotech, catalog no. 1083), goat anti-mouse IgG2b-HRP (SouthernBiotech, catalog no. 1093), goat anti-mouse IgG2c-HRP (SouthernBiotech, catalog no. 1077), or goat anti-mouse IgG3-HRP (SouthernBiotech, catalog no. 1103) was added. Wells were washed again with PBS-T before the addition of tetramethylbenzidine solution (Thermo Fisher Scientific, catalog no. J60461). Antibody titers were defined as the reciprocal serum dilution at which the absorbance at 450 nm exceeded background by greater than 0.5 absorbance units.