Disodium p nitrophenyl phosphate substrate

Immulon 2HB microtitre plates (DYNEX) were coated and dried overnight at 37 °C with salmon sperm DNA (100 μg ml⁻¹ in TE buffer). DNA was nitrocellulose filtered for dsDNA or heated for 5 min at 100 °C for ssDNA. After blocking for 60 min at 37 °C (2% BSA, 2% fetal calf serum, 0.1% Tween-20 and 0.02% sodium azide in PBS) sera diluted 1:100 in 50% block were applied (60–120 min at 37 °C) followed by alkaline phosphatase-conjugated goat anti-mouse IgM or IgG (Southern Biotech) diluted in blocking buffer for 60 min at 37 °C. Secondary antibodies were detected by using disodium p-nitrophenyl phosphate substrate (Sigma-Aldrich) and absorbance (OD) read at 405 nm. For detection of total Ig in culture supernatants or sera plates were coated with 10 μg/ml goat anti-mouse IgG (heavy and light chain specific; Southern biotech) in PBS at 4 °C overnight. Plates were blocked with 1% BSA in PBS and incubated with various dilutions of serum sample or cell culture supernatants in PBS. After incubation with 1:1,000 diluted alkaline phosphatase-conjugated IgM, IgG, IgG1 or IgG2c (Southern Biotech) colour was developed as above with disodium p-nitrophenyl phosphate substrate.