Total serum Ig was measured by sandwich ELISA. High-binding assay plates were coated overnight with 50 µl capture antibody (Bethyl Labs) diluted in carbonate buffer. Plates were washed with wash buffer (0.1% Tween20 in PBS) and blocked for 2 h at 37°C with 50 µl of 5% BSA and 0.05% sodium azide in PBS. After washing, 50-µl mouse serum samples (diluted in 1% BSA and 0.1% Tween20 in PBS) were added and incubated for 2 h at 37°C. Plates were washed and incubated with alkaline phosphatase–conjugated detection antibodies (Bethyl Labs) for 1 h. Plates were washed and incubated with 100 µl phosphatase substrate solution (Sigma) for 10–15 min, and absorbance was measured at 405 nm.
Polyreactivity was assessed by performing ELISA as previously described (Gitlin et al., 2016 (link)) using cardiolipin (Sigma), LPS (Sigma), human insulin (Sigma), double-stranded DNA (Sigma), and KLH (Sigma).
SRBC-specific IgM, IgG3, and IgG1 in the sera were measured by flow cytometry–based MFIs (median fluorescence intensity) of anti-IgM (Clone II/41; eBioscience), anti-IgG3 (Clone R40-82; BD PharMingen), and anti-IgG1 (A85-1; BD Biosciences) binding to SRBC-bound serum antibodies using a method described previously (McAllister et al., 2017 (link)).
Polyreactivity was assessed by performing ELISA as previously described (Gitlin et al., 2016 (link)) using cardiolipin (Sigma), LPS (Sigma), human insulin (Sigma), double-stranded DNA (Sigma), and KLH (Sigma).
SRBC-specific IgM, IgG3, and IgG1 in the sera were measured by flow cytometry–based MFIs (median fluorescence intensity) of anti-IgM (Clone II/41; eBioscience), anti-IgG3 (Clone R40-82; BD PharMingen), and anti-IgG1 (A85-1; BD Biosciences) binding to SRBC-bound serum antibodies using a method described previously (McAllister et al., 2017 (link)).
