Immulon 4HBX high binding plates (ThermoFisher, Rockford, IL) were used for ELISA assays. For ANA ELISAs, plates were coated with a 1:10 dilution of poly-L-lysine (Sigma-Aldrich, St. Louis, MO), followed by coating with salmon sperm dsDNA (Sigma-Aldrich, St. Louis, MO) or Smith/Ribonucleoprotein SmRNP (Arotec Diagnostics, Wellington, New Zealand). Plates were blocked with 4% non-fat dry milk in PBS and coated with diluted serum samples with serial double dilution. Detection of antibody subtypes was performed using the following combinations of primary and secondary detection antibodies: primary- anti-IgG-biotin (Jackson ImmunoResearch, West Grove, PA), anti-IgG2b-biotin (Southern Biotech, Birmingham, AL), anti-IgG2c-alkaline phosphatase (Southern Biotech, Birmingham, AL) and secondary- streptavidin-alkaline phosphatase (Vector Laboratories, Burlingame, CA). Plates were developed with PNPP (p-nitrophenyl phosphate, disodium salt) (ThermoFisher, Rockford, IL) substrate and quantitation was performed as previously described [50 (link)]. Anti-DNA or anti-SmRNP IgG-subclass titers were determined by calculating arbitrary units by using serial dilution of the serum from a positive animal as a standard, and were expressed as units per volume.