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.
Schlottmann
Sonela A
SA
Vaccine and Biologics Research, Merck Research Laboratories, WYN-2 466, Devon Park Dr., Wayne, PA 19087-8630, USA.
Jain
Neil
N
Chirmule
Narendra
N
Esser
Mark T
MT
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007118:Immunoassay / Q000379:methods / Q000592:standards; D007074:Immunoglobulin G; D008863:Microspheres; D009025:Morpholines; D022242:Pneumococcal Vaccines / Q000737:chemistry / Q000302:isolation & purification; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D012015:Reference Standards; D013296:Streptococcus pneumoniae / Q000737:chemistry*
C467606:4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D007074:Immunoglobulin G; D009025:Morpholines; D022242:Pneumococcal Vaccines; D011135:Polysaccharides, Bacterial