Proteomics-driven design of a multiplex bead-based platform to assess natural IgG antibodies to pneumococcal protein antigens in children.

Pneumococcal surface proteins are potential candidates for the development of protein-based vaccines and serological assays. The objective of the study was to develop a multiple bead-based immunoassay using Luminex xMAP® technology for the quantitation of natural antibodies against Streptococcus pneumoniae proteins and the characterization of the acute serum response following pneumococcal pneumonia in children. Sixty-four recombinantly produced pneumococcal proteins, which were selected based on their proteomic experimental identification by "shaving" live cells with trypsin followed by LC/MS/MS analysis, were coupled to fluorescent SeroMAP® beads and anti-pneumococcal specific IgG levels were determined in sera. Multiplex assay was validated through comparison of IgG levels to 14 randomly chosen pneumococcal antigens by using multiplex and singleplex assays. Acute serum IgG levels against RrgB were significantly lower in children ≤ 4 years old with pneumococcal pneumonia than those in controls. In addition, there was a small trend toward slightly lower antibody levels for PrsA, RrgC and RrgB in pneumonia patients of the all age group.