Plates were coated with anti-human IgG (Dako) at 5 mg/ml in PBS or Tetanus vaccine (Dutch vaccine institute, The Netherlands) diluted 1:10 for 1 hr at 37uC or o/n at 4uC and washed in ELISA wash buffer (PBS, 0.5% Tween-20). 4% milk in PBS was used as blocking agent, before serial dilution of cell culture supernatants and enzyme-conjugated detection Abs were added (dilutions 1:2500 for HRP-conjugated anti-IgG (Jackson Immu-noResearch Laboratories) and 1:250 for AP conjugated anti-IgG (DAKO)). TMB substrate/stop solution (Biosource) or Alkaline Phosphatase Substrate (Sigma-Aldrich) was used for development of the ELISAs. To detect Tetanus Toxin we used the ELISA Ridascreen Tetanus (r-biopharm AG, Darmstadt, Germany).