A novel chemistry for conjugating pneumococcal polysaccharides to Luminex microspheres

Here we describe a novel method to conjugate pneumococcal polysaccharides (PnPS) to Luminex microspheres for use in serological assays. 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium (DMTMM) modification of PnPS and conjugation to carboxyl functional groups on Luminex microspheres (COOH–DMTMM method) was shown to be a reproducible chemistry that efficiently conjugated PnPS to Luminex microspheres without affecting the antigenicity of a broad set of PnPS. The COOH–DMTMM method was compared to three other methods for robustness, reproducibility and effect on PnPS antigenicity in a multiplexed assay format. The other methods examined included adsorption of the unmodified PnPS to Luminex microspheres, oxidation of the PnPS to conjugate them to amino-modified microspheres using carbodiimide chemistry and poly-l-lysine modification of the PnPS before conjugating to carboxy Luminex microspheres using carbodiimide chemistry. Of the four methods, the COOH–DMTMM chemistry was shown to be a robust methodology, producing stable PnPS coupled microspheres with a 4-log dynamic range and low cross-reactivity when used in a PnPS-specific IgG serology assay. This novel chemistry should be useful for developing serological assays to measure antibodies to polysaccharides for use in vaccine and epidemiology studies.