Development of a fluorescent-bead-based multiplex immunoassay to determine immunoglobulin G subclass responses to Neisseria meningitidis serogroup A and C polysaccharides.

A fluorescent-particle-based multiplex flow cytometric immunoassay (MIA) for the detection of serum immunoglobulin G (IgG) and two IgG subclasses, IgG1 and IgG2, specific for Neisseria meningitidis serogroup A (MenA) and C (MenC) polysaccharides (PS) was developed. The assay comprised three separate duplex assays, one for the detection of the IgG response to MenA and MenC PS, another for the detection of the IgG1 response to MenA and MenC PS, and a third for the detection of the IgG2 response to MenA and MenC PS. Next, the three separate duplex assays were combined and analyzed as a hexaplex assay. No interference between monoplex, duplex, and hexaplex assays was observed, and the assay was found to have low intra- and interassay variation (<9.0% and <27%, respectively). Comparison of the meningococcal subclass MIA to the in-house enzyme-linked inmmunosorbent assays showed a good correlation (R >or= 0.85) for each of the subclasses. We conclude that the hexaplex meningococcal subclass MIA is an easy and specific assay for the determination of anti-MenA and anti-MenC PS subclass IgG, requiring minimal amounts of serum to study IgG subclass responses to vaccines.