The plasma harvested from HIS mice (7 days after the first and second immunization; 10 days after the third immunization) and B cell clone culture supernatants were screened by ELISA for the presence of total human IgM, total human IgG and antigenspecific antibodies. Measurement of total IgM and IgG was performed by coating 96-well plates either with AffiniPure F(ab') 2 fragment goat anti-human IgM (Fc5m-specific, Jackson Immu-noResearch) or AffiniPure goat anti-human IgG (Fcc fragmentspecific; Jackson ImmunoResearch). Control human serum protein calibrator (Dako) with known IgM (0.8 mg/ml) and IgG (10.4 mg/ml) concentrations was used as a standard to be compared to the samples. For the detection of antigen-specific antibodies, 96-well plates were coated either with tetanus vaccine (Nederlands Vaccin Instituut) or Engerix B (GlaxoSmithKline) (106 diluted in PBS) for 1 h at 37uC or overnight at 4uC. Alternatively, Ridascreen Tetanus IgG ELISA plates (Biopharm)