Alkaline phosphatase conjugated goat antimouse secondary antibody

Serum IgG autoantibodies against double-stranded DNA at 7 to 10 months of age were measured by ELISA. Plates were coated with 0.1 mg/mL salmon sperm DNA (ThermoFisher Scientific, Waltham, MA) in PBS in a warm room overnight and rinsed with PBS on the following day. Plates were blocked with 1% BSA in PBS for 1 hour at 37°C. Samples were added at a 1:100 dilution and 6 subsequent two-fold dilutions to 1:3200 and incubated for 2 hours at 37°C. Next, samples were incubated with an alkaline phosphatase conjugated goat antimouse secondary antibody (Southern Biotech) and incubated for 1 hour at 37°C. Plates were then washed, developed with phosphatase substrate (Sigma Aldrich) in 1M diethanolamine, pH 9.8 (Sigma Aldrich), and read at 405 nm using an Infinite F50 microplate reader (Tecan Group, Switzerland). Absorbance is reported as (optical density of sample/optical density of positive control 20-week old MRL/lpr serum on the same plate) × 100. Proteinuria was assessed semiquantitatively via Uristix where +1 is 30 mg/dL, +2 is 100 mg/dL, +3 is 300 mg/dL, and +4 is 2000 mg/dL (Siemens Healthcare, Tarrytown, NY). Spleens were dissected and weighed, and the organ weight was normalized to body weight for assessment of splenomegaly.