A fluorescent multiplexed bead-based immunoassay (FMIA) for quantitation of IgG against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis protein antigens

• Published in: Journal of immunological methods 2014-03, Vol.405, p.130-143
• Main Authors: Andrade, Dafne C., Borges, Igor C., Laitinen, Hanna, Ekström, Nina, Adrian, Peter V., Meinke, Andreas, Barral, Aldina, Nascimento-Carvalho, Cristiana M., Käyhty, Helena
• Format: Article
• Language: English
• Subjects: Acute Disease; Antibodies, Bacterial - blood; Antibodies, Bacterial - immunology; Antibody; Antigens, Bacterial - immunology; Bacterial Proteins - immunology; Child; Child, Preschool; Community-Acquired Infections - blood; Community-Acquired Infections - diagnosis; Community-Acquired Infections - immunology; Etiological diagnosis; Fluorescence; Haemophilus influenzae - immunology; Humans; Immunoassay - methods; Immunoglobulin G - blood; Immunoglobulin G - immunology; Infant; Microspheres; Moraxella (Branhamella) catarrhalis - immunology; Multiplex; Otitis Media - blood; Otitis Media - diagnosis; Otitis Media - microbiology; Pneumonia - blood; Pneumonia - diagnosis; Pneumonia - immunology; Recombinant proteins; Reproducibility of Results; Sensitivity and Specificity; Serology; Streptococcus pneumoniae - immunology; Validation
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2014.02.002

Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are pathogens commonly associated with infectious diseases in childhood. This study aimed to develop a fluorescent multiplexed bead-based immunoassay (FMIA) using recombinant proteins for the quantitation of serum IgG antibodies against these bacteria. Eight pneumococcal proteins (Ply, CbpA, PspA1, PspA2, PcpA, PhtD, SP1732-3 and SP2216-1), 3 proteins of H. influenzae (NTHi Protein D, NTHi0371-1, NTHi0830), and 5 proteins of M. catarrhalis (MC Omp CD, MC_RH4_2506, MC_RH4_1701, MC_RH4_3729-1, MC_RH4_4730) were used to develop the FMIA. Optimal coupling concentrations for each protein, comparison of singleplex and multiplex assays, specificity, reproducibility, and correlation to ELISA for six pneumococcal antigens were determined for validation. FMIA was then used to analyze acute and convalescent paired serum samples of 50 children with non-severe pneumonia. The coupling concentrations varied for different antigens, ranging from 1.6 to 32μg of protein/million beads. Correlation between singleplexed and multiplexed assays was excellent, with R≥0.987. The FMIA was specific, reaching >92% homologous inhibition for all specificities; heterologous inhibition ≥20% was found only in six cases. The assay was repeatable, with averages of intra-assay variation ≤10.5%, day-to-day variation ≤9.7% and variation between technicians ≤9.1%. Comparison with ELISA for pneumococcal antigens demonstrated good correlation with R ranging from 0.854 (PspA2) to 0.976 (PcpA). The samples from children showed a wide range of antibody concentrations and increases in convalescent samples. In conclusion, the FMIA was sensitive, specific, and repeatable, using small amounts of recombinant proteins and sera to detect antibodies against S. pneumoniae, H. influenzae and M. catarrhalis. The methodology would be suitable for studies investigating etiological diagnosis and in experimental vaccine studies. •The assay was specific, accomplishing homologous inhibition for all coupled beads.•The assay had a good correlation with ELISA for all tested pneumococcal antigens.•The developed assay was repeatable in the intra-assay and day-to-day comparisons.•The assay demonstrated a high throughput using small volumes of serum samples.•The optimal coupling concentration should be individually determined for each antigen.