Optimization and application of a multiplex bead-based assay to quantify serotype-specific IgG against Streptococcus pneumoniae polysaccharides: response to the booster vaccine after immunization with the pneumococcal 7-valent conjugate vaccine

Abstract

We describe the optimization and application of a multiplex bead-based assay (Luminex) to quantify antibodies against polysaccharides of 13 pneumococcal serotypes. In the optimized multiplex immunoassay (MIA), intravenous immune globulin was introduced as an in-house reference serum, and nonspecific reacting antibodies were adsorbed with the commercial product pneumococcal C polysaccharides Multi. The antibody concentrations were assessed in 188 serum samples obtained pre- and post-booster vaccination at 11 months after administration of a primary series of the pneumococcal seven-valent conjugate vaccine (PCV-7) at 2, 3, and 4 months of age. The results of the MIA were compared with those of the ELISA for the serotypes included in the seven-valent conjugated polysaccharide vaccine and for a non-vaccine serotype, serotype 6A. The geometric mean concentrations of the antibodies determined by MIA were slightly higher than those determined by ELISA. The correlations between the assays were good, with R(2) values ranging from 0.84 to 0.91 for all serotypes except serotype 19F, for which R(2) was 0.70. The concentrations of antibody against serotype 6A increased after the administration of PCV-7 due to cross-reactivity with serotype 6B. The differences between the results obtained by ELISA and MIA suggest that the internationally established protective threshold of 0.35 microg/ml should be reevaluated for use in the MIA and may need to be amended separately for each serotype.

FIG. 1.

Similarities of the curves obtained by MIA with serial dilutions of the lot 89S and IVIG reference sera for serotypes 6A, 6B, and 14. The median fluorescent intensities are plotted against the IgG concentration. Similar curves were obtained for the other serotypes.

FIG. 2.

Correlation between IgG concentrations obtained by serotype 14- and serotype 23F-specific MIAs by using adsorption with CWPS Multi (x axis) and CWPS plus serotype 22F capsular polysaccharide (y axis). The dotted lines indicate the perfect correlation. The IgG concentrations are log transformed.

FIG. 3.

High degree of correlations between the IgG concentrations obtained by ELISA and MIA by using infant sera obtained pre- and post-booster vaccination with PCV-7. The dotted lines indicate the perfect correlation. The IgG concentrations are log transformed.

FIG. 4.

Visualization of the larger dynamic range of the serotype 14-specific MIA compared to that of the serotype 14-specific ELISA. Serial dilutions of the lot 89S reference serum were made and tested in both assays. The MFI (left y axis) and optical density (OD; right y axis) are plotted against the IgG concentration. By the MIA, the dynamic range was 0.1 to 90 ng/ml, whereas the ELISA allowed assessment of the IgG concentration only over a range of 0.1 to 30 ng/ml.

FIG. 5.

Influence of the IgG concentration range on the correlations between the results of the MIA and ELISA. The IgG concentrations were obtained for serotype 4 pre-booster vaccination (A), post-booster vaccination (B), and pre- and post-booster vaccination combined (C). The IgG concentrations are log transformed.

FIG. 6.

IgG concentrations and GMCs for 13 different serotypes obtained by MIA of sera taken from infants 1 month post-booster vaccination with PCV-7. Each colored dot represents an individual serum sample. GMCs are indicted by the black dashes in the colored dots. The horizontal dashed line indicates the protective concentration of 0.35 μg/ml. The serotypes that are depicted within the box with a dashed border are the serotypes used to compare MIA and ELISA in this study.

FIG. 7.

Relative cumulative distribution curves of the pre- and post-booster vaccination IgG concentrations for four serotypes obtained by MIA and ELISA. The vertical dotted line indicates the protective concentration of 0.35 μg/ml. The percentage of subjects (y axis) is plotted against the IgG concentration (x axis). The graph for serotype 4 is illustrative for the other serotypes, for which the results are not depicted here.