Article
A fluorescent multiplexed bead-based immunoassay (FMIA) for quantitation of IgG against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis protein antigens.
Registro en:
ANDRADE, D. C. et al. A fluorescent multiplexed bead-based immunoassay (FMIA) for quantitation of IgG against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis protein antigens. Journal of Immunological Methods, v. 405, p. 130-143, 2014.
1872-7905
10.1016/j.jim.2014.02.002
Autor
Andrade, Dafne Carvalho
Borges, Igor Campos
Laitinen, Hanna
Ekström, Nina
Adrian, Peter V
Meinke, Andreas
Barral, Aldina Maria Prado
Carvalho, Cristiana Maria Costa Nascimento
Käyhty, Helena
Resumen
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 32 μg of protein/million
beads. Correlation between singleplexed and multiplexed assays was excellent, with R ≥ 0.987.
The FMIA was specific, reaching N92% 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 detec
Materias
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