title: Measurement of antibodies to pneumococcal, meningococcal and haemophilus polysaccharides, and tetanus and diphtheria toxoids using a 19-plexed assay.
abstract: The measurement of antibody responses to vaccination is useful in the assessment of immune status in suspected immune deficiency. Previous reliance on enzyme-linked immunoabsorbent assays (ELISA) has been cumbersome, time-consuming and expensive. The availability of flow cytometry systems has led to the development of multiplexed assays enabling simultaneous measurement of antibodies to several antigens. We optimized a flow cytometric bead-based assay to measure IgG and IgM concentrations in serum to 19 antigens contained in groups of bacterial subunit vaccines: pneumococcal vaccines, meningococcal vaccines, Haemophilus influenzae b (Hib), and tetanus and diphtheria toxoid vaccines. 89-SF was employed as the standard serum. The assay was used to determine specific antibody levels in serum from 193 healthy adult donors. IgG and pneumococcal IgM antibody concentrations were measurable across 3 log10 ranges encompassing the threshold protective IgG antibody levels for each antigen. There was little interference between antibody measurements by the 19-plexed assay compared with monoplexed assays, and a lack of cross-reactive IgG antibody, but evidence for cross-reacting IgM antibody for 3/19 pneumococcal antigens. 90th centile values for 15/19 IgG concentrations and 12/12 IgM concentrations of the 193 adult sera were within these ranges and percentages of sera containing protective IgG antibody levels varied from 4% to 95% depending on antigen. This multiplexed assay can simultaneously measure antibody levels to 19 bacterial vaccine antigens. It is suitable for use in standard clinical practice to assess the in vivo immune response to test vaccinations and measure absolute antibody levels to these antigens.
authors.affiliation: Medical Research Council Centre for Immune Regulation and Clinical Immunology Service, Institute of Biomedical Research, School of Immunity and Infection, College of Medicine and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Alison M E
authors.initials: AM
authors.lastname: Whitelegg
authors.forename: Jane
authors.initials: J
authors.lastname: Birtwistle
authors.forename: Alex
authors.initials: A
authors.lastname: Richter
authors.forename: John P
authors.initials: JP
authors.lastname: Campbell
authors.forename: James E
authors.initials: JE
authors.lastname: Turner
authors.forename: Tarana M
authors.initials: TM
authors.lastname: Ahmed
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Giles
authors.forename: Mark
authors.initials: M
authors.lastname: Fellows
authors.forename: Tim
authors.initials: T
authors.lastname: Plant
authors.forename: Alastair J
authors.initials: AJ
authors.lastname: Ferraro
authors.forename: Mark
authors.initials: M
authors.lastname: Cobbold
authors.forename: Mark T
authors.initials: MT
authors.lastname: Drayson
authors.forename: Calman A
authors.initials: CA
authors.lastname: MacLennan
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D004168:Diphtheria Toxoid / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods / Q000592:standards; D018073:Haemophilus Vaccines / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*; D007075:Immunoglobulin M / Q000097:blood / Q000276:immunology*; D022401:Meningococcal Vaccines / Q000276:immunology*; D008875:Middle Aged; D022242:Pneumococcal Vaccines / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013745:Tetanus Toxoid / Q000276:immunology*; D055815:Young Adult
chemical_list: D004168:Diphtheria Toxoid; D018073:Haemophilus Vaccines; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D022401:Meningococcal Vaccines; D022242:Pneumococcal Vaccines; D013745:Tetanus Toxoid