Method Description

IgG antibodies to Streptococcus pneumoniae serotypes are measured by microsphere photometry (also known as bead-based multi-plex immunoassay) using pneumococcal polysaccharides conjugated to MagPlex microspheres.

This assay detects IgG antibodies to different serotypes of S pneumoniae. The serotype-specific antibodies bound in the first stage of the assay are detected with R-phycoerythrin-conjugated, anti-human IgG antibody in the second stage of the assay.

Pneumococcal polysaccharides are modified with 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and conjugated to magnetic carboxylated polystyrene microparticles (hereinafter referred to as magnetic beads) that have been dyed into spectrally distinct regions to be identified by the Luminex FlexMap 3D (FM3D) instrument. A mixture of 24 different beads (23 coupled to a different pneumococcal polysaccharide and 1 that is not coupled, to serve as a check for non-specific binding) is prepared in 1% BSA in PBS.

Standards and controls are prepared from pools of patient serum that have previously detected antibodies for the 23 serotypes of S pneumoniae. A dilution series of standard, controls, and patient sera are added to a microwell plate.

An equal amount of the bead mixture is added and allowed to incubate. The plate is washed using 1% BSA in PBS on a magnetic plate washer to remove unbound material. A substrate containing a reporter molecule, R-phycoerythrin conjugated anti-human IgG antibody, is added to the plate. After incubation, the plate is read on the Luminex FM3D where the magnetic beads are differentiated and quantitated by 2 separate lasers. The red diode laser (635 nm) excites the fluorescent dyes contained within the beads, and the green laser (532 nm) excites the reporter molecule bound to the bead surface. Data reduction software is used to quantify the amount of IgG antibody in unknown samples by correlation to a standard curve using a 5-Parameter Logistic (PL) curve fit.(Unpublished Mayo method)