Goat anti mouse iga antibodies

MultiScreen-HA filter plates (Merck Millipore) were coated by incubation overnight at 4 °C with 2 μg/ml goat anti-mouse IgM/G/A (H+L) antibodies (Merck Millipore) or 10 μg/ml goat anti-mouse IgA antibodies (Southern Biotech). Plates were washed before adding 1 × 10⁴ or 1 × 10⁵ red cell-depleted spleen or bone marrow cells per well in 200 μl Iscove's modified Dulbecco's medium supplemented with 10% FCS and incubated at 37 °C for 19 h. The plates were washed before incubation with secondary antibodies diluted in block (PBS with 1% FCS (Gibco), 0.05% Tween 20 (Sigma) and 0.6% skim milk powder (Devondale, Brunswick, VIC, Australia)): goat anti-mouse IgG1/IgG2a/IgG2b/IgG3 antibodies conjugated to HRP or goat anti-mouse IgM antibodies conjugated to HRP or goat anti-mouse IgA antibodies conjugated to biotin (all Southern Biotech). For biotinylated antibodies, plates were washed and incubated with streptavidin-HRP (Southern Biotech) diluted in blocking buffer. After further washing, 100 μl of substrate solution (250 μg/ml 3-amino-9-ethylcarbazole (Sigma-Aldrich) in 0.05 M sodium acetate (pH 5.0) and 0.03% H₂O₂) was added to each well. ELISPOTs were counted on an ELISPOT reader (Autoimmun Diagnostika GMBH, Strasburg, Germany).