Hrp conjugated rabbit anti mouse ig antibody

Twenty-one days post-challenge (p.c.), animals were sacrificed by cervical dislocation, the spleens were removed and weighed, and single-cell suspensions were prepared. Serial dilutions of isolated spleen cells were seeded onto M. dunni cells, and cells were incubated under standard tissue culture conditions for 3 days. When cells reached ~100% confluence, they were fixed with ethanol and labeled with F-MuLV Env-specific monoclonal antibody MAb 720 (32 (link)) and then with a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig antibody (Dako, Hamburg, Germany). The assay was developed using aminoethylcarbazole (Sigma-Aldrich, Deisenhofen, Germany) as substrate to detect foci. The resulting foci were counted, and infectious centers (IC)/10⁸ spleen cells were calculated.

Non-modified FCS, citrullinated-FCS (Cit-FCS) and Ca-FCS were coated overnight at a concentration of 10 µg/ml (diluted in pH 9.6 0.1 M carbonate-bicarbonate buffer) on Nunc Maxisorp plates (Thermo Scientific). The plates were washed with PBS/0.05% Tween (Sigma) and subsequently blocked for 6 hours at 4°C with 100 µl of PBS/1% BSA (Sigma). After washing, the wells were incubated with 50 µl serum 1/50 diluted in PBS/1% BSA/0.05% Tween. The ELISA plates were incubated overnight on ice. Total Ig, IgG1, and IgG2a were detected using HRP-conjugated rabbit anti-mouse Ig antibody (Dako), HRP-conjugated goat anti-mouse IgG2a, HRP-conjugated goat anti-mouse IgG2c, HRP-conjugated goat anti-mouse IgG1 (all from Southern Biotec) or HRP-conjugated rabbit anti-human IgG (Dako). HRP enzyme activity was visualized using ABTS. As a standard, serial dilutions of a pooled serum sample from mice with CIA were used.