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J Immunol Methods. 2014 Mar;405:130-43. doi: 10.1016/j.jim.2014.02.002. Epub 2014 Feb 11.

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

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Federal University of Bahia School of Medicine, Salvador, Bahia, Brazil. Electronic address:
Federal University of Bahia School of Medicine, Salvador, Bahia, Brazil.
National Institute for Health and Welfare, Helsinki, Finland.
DST/NRF Vaccine Preventable Diseases, Respiratory and Meningeal Pathogens Research Unit, University of the Witwatersrand, Johannesburg, South Africa.
Valneva Austria, GmbH, Campus Vienna Biocenter 3, Vienna, Austria.
Pathology Department, Federal University of Bahia School of Medicine and Centro de Pesquisa Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil.
Department of Pediatrics, Federal University of Bahia School of Medicine, Salvador, Bahia, Brazil.


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.


Antibody; Etiological diagnosis; Multiplex; Recombinant proteins; Serology; Validation

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