Elisa 96 well plates

IgG serum antibody titers were measured by using ELISA for tetanus toxoid (TT); diphtheria toxoid (DT); pertussis toxoid (PT); Haemophilus influenzae type b-polysaccharide (Hib); pneumococcal polysaccharide serotypes (pn) 1, 14, 23, and 26; and poliovirus serotypes 1, 2, and 3. For TT and DT (both obtained from Statens Serum Institut, Copenhagen, Denmark), and PT (Sigma) and Hib (HbO-HA, polysaccharide conjugated to human serum albumin, obtained from NIBSC, South Mimms, UK), ELISA 96-well plates (Greiner Bio-One GmbH) were coated with 5-μg/ml antigen. For antibodies against poliovirus, a commercial ELISA was used according to the instructions of the manufacturer (Demeditec Diagnostics GmbH, Kiel, Germany). The following WHO standards were used for calibration: TE-3 for TT, 10/262 for DT, 06/140 for pertussis, 09/222 for Hib, and 82/585 for poliovirus (NIBSC, South Mimms, UK). Protective antibody concentrations were defined as ≥ 0.1 IU/ml for TT and DT, ≥ 24 IU/ml for pertussis, ≥ 1 μg/ml for Hib, ≥ 10 U/ml for polio, and ≥ 0.35 μg/ml for pneumococcal polysaccharides. A positive response was defined as ≥ 4 times the minimum level of detection in the pre-vaccination sample (d+0) and ≥ 100% increase between the pre-vaccination sample (day 0) and the post-vaccination samples.