Development and analytical characterization of a 24-plex microsphere-based assay for the quantitation of the antibodies against pneumococcal vaccine serotypes

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Background Quantitative assessment of pneumococcal vaccine–induced immunity is essential for monitoring vaccine effectiveness. The World Health Organization (WHO) pneumococcal polysaccharide enzyme-linked immunosorbent assay (Pn PS ELISA) remains the reference standard for measuring serotype-specific IgG concentrations; however, it is limited by its low throughput and high resource requirements. Our study aim is to develop and analytically characterize a 24-plex microsphere-based immunoassay for simultaneous quantitation of serotype-specific IgG antibodies against 24 pneumococcal vaccine serotypes. Methods Pneumococcal polysaccharides were conjugated to magnetic microspheres using the DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride) coupling method, and assay performance was evaluated using the WHO reference serum (007sp). Results The 24-plex assay demonstrated assay working ranges with lower limits of quantitation between 0.4 and 6.7 ng/mL and upper limits up to 38,699 ng/mL. Homologous inhibition exceeded 80% across all serotypes, while heterologous inhibition remained below 20% except for structurally related serogroups (6A/6B, 9N/9V, 19A/19F). Intra- and inter-assay precision met the predefined acceptance criterion of coefficients of variation below 20% for all serotypes, and accuracy ranged between 87% and 118%. Concentration-based comparison with the WHO Pn PS ELISA using Deming regression showed slopes close to unity for most serotypes, indicating quantitative consistency between the two assays within the evaluated reference-serum dilution series. Conclusion Our multiplex assay covers 24 pneumococcal vaccine serotypes supporting its analytical performance and comparability with WHO Pn PS ELISA. Our 24-plex assay provides a high-throughput and efficient method for pneumococcal serotype-specific IgG quantification, while further clinical and inter-laboratory evaluation is warranted.