NP-specific IgG1 Antibody Quantification

NP-specific IgG1 antibody were measured similar to what was described previously (Cho et al., 2018 (link)). In brief, 96-well plates were first coated overnight at 4°C with NP₂₇-BSA (Biosearch Technologies), followed by blockade of non-specific binding by incubation with blocking buffer (1% of BSA in PBS) for 1 h at room temperature. Mouse serum was diluted to 10⁻⁴ of the original concentration in blocking buffer and then added to the plates, followed by incubation for 1 h at room temperature. Plates were washed with washing buffer (0.05% Tween-20 in PBS) three times. Bound antibodies were detected by HRP-conjugated anti-mouse IgG1 antibodies (5300-05; 1:1,000; Southern Biotech). The reactions were developed by incubation for 15 min at room temperature with TMB substrate (BioLegend) and were stopped by the addition of 2N H₂SO₄. Absorbance was measured by a microreader (Bio-Rad) at 450 nm.