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

Abstract

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.

Natural Development of Antibodies against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis Protein Antigens during the First 13 Years of Life

“…This concordance is more pronounced among anti-CbpA, antiPcpA, and anti-PhtD antibodies. We previously showed the absence of significant cross-reactivity between these antibodies using our immunoassay (16). These results suggest that the maturation of immune responses against the pneumococcal antigens studied is synchronous.…”

“…We analyzed as many samples as possible from each study subject on the same plate, and all samples were assayed in duplicate. Further details of the serology protocol have been published elsewhere (16).…”

Detection of antibody responses against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis proteins in children with community-acquired pneumonia: effects of combining pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness

“…The description of the serology protocol employed and the procedures for serum sample analyses have been previously published [7]. Briefly, acute and convalescent serum samples were assayed with a multiplexed bead-based serological test, using Luminex xMAP® Technology, for the investigation of The mean fluorescence intensity (MFI) values provided an indirect measure of the antibody concentrations.…”

“…Acute and convalescent serum samples were assayed on the same plate, and positive and negative controls were analyzed on each plate. Samples were assayed using 1:400 and 1:1,600 dilutions, as these dilutions were in the linear range of the dilution curve, especially the dilution of 1:1,600, and further dilutions were performed if needed [7]. Herein, the results of antibody levels of acute serum samples are reported for the dilution of 1:1,600.…”

Proteomics-driven design of a multiplex bead-based platform to assess natural IgG antibodies to pneumococcal protein antigens in children

“…The use of multiplexed immunoassays to a limited number of pneumococcal surface proteins has recently been reported to evaluate their immunogenicity and for the diagnosis of pneumococcal disease in children which is usually challenging due to limited sensitivity of current diagnostic tests and interference with colonization [9,15,16]. The multiplexed immunoassay described in this report allowed the detection of antibody responses to a much larger and extensive collection of pneumococcal protein antigens than previously described serological assays.…”