In this study, IVIG was introduced as an in-house reference serum for the MIA. to the pertussis vaccine in the Dutch NIP (study ISRCTN97785537) (1). All children in the study were immunized four times with the combination product, which consisted of the diphtheria-attenuated pertussis-tetanus vaccine, inactivated ROBO1 polio vaccine, and type b vaccine (Pediacel; Sanofi-Pasteur, Lyon, France) and PCV-7 (Wyeth Vaccines) in 2007. Calibration sera. The calibration panel used for the pneumococcal ELISA consisted of 12 serum samples and was supplied by the National Institute for Biological Standards and Control (NIBSC; Hertfordshire, United Kingdom). The concentrations of IgG antibodies against the seven serotypes included in PCV-7 for this panel were assessed by MIA and ELISA and were compared with the IgG concentrations published elsewhere (http://www.vaccine.uab.edu/qc3.pdf). Coupling of polysaccharides to beads. The coupling of the polysaccharides to carboxylated microspheres was performed as described previously (11, 16, 20). Briefly, purified capsular polysaccharides were conjugated to poly-l-lysine. Acetyllovastatin The conjugates were coupled to carboxylated beads (Bio-Rad Laboratories, Hercules, CA). All capsular polysaccharides except polysaccharide 6A were obtained from the American Type Culture Collection (ATCC; Manassas, VA); polysaccharide 6A was kindly provided by Wyeth Vaccines. The same procedure was also used to create CWPS-specific beads by using the CWPS Multi preparation (Statens Serum Institute, Copenhagen, Denmark). Of the CWPS Multi polysaccharides, 2.5 mg was used for the coupling of the polysaccharide to poly-l-lysine. The coupling of the conjugate to the beads was Acetyllovastatin performed by use of a 1.5-h incubation. MIA. MIA was performed as described previously (16, 20) but with minor modifications. Sera were diluted and incubated for 1 h or overnight in adsorbent buffer containing 15 g/ml CWPS Multi and 5% antibody-depleted human serum (ADHS; Valley Biomedical, Winchester, VA) in phosphate-buffered saline (pH 7.2). A 10% (wt/vol) solution of IVIG (lyophilized IVIG; Sanquin, Amsterdam, Netherlands) was used as an in-house reference serum. IVIG contains purified IgG from a pool of at least 1,000 plasma samples obtained from blood donors from the Dutch population. The donors were not Acetyllovastatin immunized with a pneumococcal vaccine. A total of 3,000 beads of each serotype-specific bead Acetyllovastatin set were used per well. Analysis of the beads was performed on a BioPlex 100 apparatus (Bio-Rad) and by use of the BioPlex software package (version 4.1.1; Bio-Rad). ELISA. ELISA was performed according to the WHO guidelines for the ELISA for the quantitation of serotype-specific IgG (www.vaccine.uab.edu/WHO2.pdf). The concentrations of antibodies against serotype 4, 6A, 6B, 9V, 14, 18C, 19F, and 23F polysaccharides were assessed. Cross-reactivity. Sera that reacted with both serotype 6A- and serotype 6B-specific beads were used for assessment of possible cross-reactivity between serotypes 6A and 6B. In the first step, 500 l of serum (diluted 1,000, 2,000, or 5,000 times) was incubated with 1.25 105 CWPS Multi-coupled Luminex beads overnight at room temperature. Subsequently, the mixture was centrifuged at 14,000 to remove the CWPS antibodies bound to the CWPS-coupled beads, the supernatant was mixed with 1.25 105 beads coupled with either serotype 6A or serotype 6B polysaccharides, and the mixture was incubated for 2 h at room temperature. The mixtures were centrifuged at 14,000 axis divided by the change in the axis of the trend line. The concentrations of the calibration sera obtained by the ELISA could be evaluated by using the WHO guidelines (www.vaccine.uab.edu). RESULTS Evaluation of IVIG as an in-house reference serum. In this study, IVIG was introduced as an in-house reference serum for the MIA. IVIG is a pool.