Quantifying Mouse AAT Levels in Lupus

Mouse AAT levels in NZM2410 lupus mouse serum were detected by ELISA as previously described.²⁵ (link) Briefly, pooled B6 (adult male) mouse serum was used as a standard, in which mouse AAT concentration was defined as one relative unit. Samples (sera from lupus mice) and standards were diluted and incubated in a microtiter plate (Immulon 4, Dynex Technologies) in Voller’s buffer overnight at 4°C. The plate was blocked with 3% BSA (Sigma) for 1 hr at 37°C. Goat anti-mouse AAT antibody (1:2,000 dilution, MyBiosource, San Diego, CA) was added and incubated for 1 hr at 37°C. Then, horseradish peroxidase (HRP)-conjugated anti-goat-IgG antibody (1:2,500 dilution, R&D Systems, Minneapolis, MN) was added and incubated for 1 hr at 37°C. The plate was washed with PBS-Tween 20 between reactions. After adding substrate (O-Phenyldiamine, Sigma-Aldrich, MO), the plate was read at 490 nm on an MRX microplate reader (Dynex Technologies). The optical density (OD) reading of each sample was used to calculate relative unit based on the standard curve.