Simultaneous quantitative measurement of IgG antibodies to Streptococcus pneumoniae serotypes by microsphere photometry*1

Rationale We developed a quantitative immunoassay using microsphere photometry to enable simultaneous, quantitative measurement of IgG antibodies to 24 Streptococcus pneumoniae serotypes as an aid in evaluating the immune response to pneumococcal vaccination in children and adults. Methods Pneumococcal polysaccharide antigens (24 serotypes, ATCC, Rockville, MD) were coupled covalently to polystyrene microspheres (Luminex Corp., Austin, TX). Each polysaccharide was coupled to a different fluorescent microsphere, and the microspheres were mixed to create a single immunosorbent reagent. This reagent was used to capture antibodies to S. pneumoniae in patient's sera, standards and controls. After incubating with the immunosorbent, bound antibodies were detected with R-phycoerythrin conjugated, goat anti-human IgG antibody using a flow microfluorometer (Luminex Corp.). The concentrations of IgG antibodies to each serotype were determined from standard curves using a primary reference material (10 serotypes, US Reference Pneumococcal Antiserum, FDA, Bethesda, MD)or a secondary reference material (14 serotypes, in-house preparation). Testing for antibodies to all 24 serotypes was completed in 2 hours. Results Measurements of anti-pneumococcal antibodies in sera from 80 healthy adults agreed with published data for the 10 standardized serotypes. The immunoassay detected concentrations ranging from 0.005 to 7.8 micrograms/ml. Interassay reproducibility across different serotypes ranged from 4.8 to 11.9%. Conclusions The microsphere immunoassay we describe is accurate and reproducible, and is suitable for clinical evaluation of patients treated with pneumococcal vaccines. Quantitative results are available for all serotypes in current vaccines.