Goat anti mouse igg alkaline phosphatase

125Tg/B6 and B6 NFATc2^+/+ or NFATc2^−/− mice were immunized with 50 µg beef insulin in CFA subcutaneously at the base of the tail. Sera were obtained pre-immunization and 2 weeks post-immunization from each mouse. ELISA was used to detect insulin-specific antibodies, as has been previously described (Kendall et al., 2004 (link)). Briefly, the O.D. 405 nm of wells incubated with >100-fold excess insulin in solution was subtracted from the O.D. 405 nm of uninhibited parallel wells to calculate insulin-specific binding. Goat anti-mouse IgM-alkaline phosphatase or goat anti-mouse IgG-alkaline phosphatase (Southern Biotech) were used to detect serum antibody bound to the insulin-coated ELISA plate.

Anti-MTg autoantibodies were determined by ELISA, as described in detail previously (36 (link)). Sera from individual mice were diluted 1/50 or 1/100 and plated in duplicate on MTg-coated ELISA plates. Anti-Ro and anti-La Abs were quantified by ELISA. Briefly, plates were coated overnight with 5 μg/ml anti-Ro Ab (Sigma-Aldrich, St. Louis, MO) or anti-La Ab (Abcam, Cambridge, MA) diluted in PBS. After washing with PBS with 0.5% Tween and blocking with 0.2% BSA in PBS, serum was diluted and incubated on plates overnight. Bound Ig was detected with goat anti-mouse IgG–alkaline phosphatase (0.2 μg/ml; Southern-Biotech, Birmingham, AL) diluted in blocking buffer. Plates were developed with SIGMAFAST p-Nitrophenyl phosphate Tablets (Sigma-Aldrich), and specific absorbance was measured at 405 nm using a Molecular Devices SpectraMax micro-plate reader.

Concentrations of total IgG in the serum were determined using U-bottom vinyl plates (Costar) that were coated with goat anti-mouse Ig (H+L) (SouthernBiotech), and bound Abs were detected with goat anti-mouse IgG-alkaline phosphatase (SouthernBiotech). Purified mouse IgG (SouthernBiotech) was used as a standard. To detect rheumatoid factor, plates were coated with Purified mouse IgG1, lambda (BD Pharmingen), and bound Abs were detected with rat anti-mouse kappa light chain-biotin (SouthernBiotech) followed by streptavidin-alkaline phosphatase. Anti-type II collagen, anti-CCP and anti-dsDNA titers were determined using the anti-mouse Type II Collagen IgG (Chondrex, Inc.), QUANTA Lite CCP3 IgG (INOVA Diagnostics, Inc.), or mouse anti-dsDNA total Ig (Alpha Diagnostic International) ELISA kits, respectively, according to the manufacturer’s instructions.