title: Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes.
abstract: Serum IgG levels specific for Streptococcus pneumoniae are currently quantified using the ELISA. However, this method has significant limitations in that a separate test is required for each serotype-specific antibody. Here, we describe a rapid and simple method for the simultaneous quantitation of IgG to nine pneumococcal serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F, 23F). Pneumococcal polysaccharides were covalently attached to fluorescent microspheres and these beads were serologically assessed using 89-SF standard serum. The multiplex assay was linear over a 24-fold serum dilution range and comparison of monoplex and nonaplex assays revealed no evidence of bead interference. The nonaplex assay was shown to be specific with <30% heterologous inhibition occurring and assay sensitivity was high with the LOD ranging between 32.3 and 109.7 pg/ml. The assay was shown to have low inter- and intra-assay variability and conjugated microspheres were shown to remain stable over 12 months. When samples were assayed there was a good correlation of the nonaplex assay with the ELISA. Finally, four bead sets coated with meningococcal polysaccharides (serogroups A, C, Y and W135) were added into the nonaplex assay, with no effect on the pneumococcal or meningococcal IgG levels generated. The pneumococcal nonaplex assay will prove to be a valuable tool to aid in the evaluation of pneumococcal capsular polysaccharide-based vaccines.
authors.affiliation: Health Protection Agency, Vaccine Evaluation Department, Manchester Medical Microbiology Partnership, PO Box 209, Clinical Sciences Building, Manchester Royal Infirmary, Manchester, M13 9WZ, United Kingdom. gouri.lal@hpa.org.uk
authors.forename: Gouri
authors.initials: G
authors.lastname: Lal
authors.forename: Paul
authors.initials: P
authors.lastname: Balmer
authors.forename: Elaine
authors.initials: E
authors.lastname: Stanford
authors.forename: Sarah
authors.initials: S
authors.lastname: Martin
authors.forename: Rosalind
authors.initials: R
authors.lastname: Warrington
authors.forename: Ray
authors.initials: R
authors.lastname: Borrow
mesh_terms: D000907:Antibodies, Bacterial / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007163:Immunosorbent Techniques; D008863:Microspheres; D009345:Neisseria meningitidis / Q000276:immunology; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000276:immunology*; D013296:Streptococcus pneumoniae / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D007074:Immunoglobulin G; D011135:Polysaccharides, Bacterial