Evaluation of Antigen-Specific IgG1 Antibodies

Serum was harvested from blood collected by lateral tail vein sampling or cardiac puncture postmortem. OVA-specific and NP-specific IgG1 ELISA was performed as described (Brooks et al., 2020 (link); Tan et al., 2020 (link)). Briefly, ELISA plates were coated with NP conjugates (NP1-RSA, NP25-BSA, NP10-BSA all from LGC Biosearch; NP3-OVA, NP7-OVA, NP9-OVA and NP19-OVA were conjugated in-house as described above), OVA (Sigma Aldrich), BSA (Research Products International) or HEL antigen (10μg/mL; Sigma Aldrich), samples were added, and HRP-labeled anti-IgG1 antibodies (Southern Biotech) were used to detect plate-bound IgG1. Plates were developed with slow kinetic form TMB (Sigma Aldrich) and stopped with 1N sulfuric acid. Absorbance was measured at 450nm using spectrophotometer (SpectraMax M5, Molecular Devices). Relative titers were interpolated from standard curves generated using samples with known high titers of antigen-specific antibody.