Determination of avidity of IgG against protein antigens from Streptococcus pneumoniae : assay development and preliminary application in clinical settings

Abstract

The measurement of antibody levels is a common test for the diagnosis of Streptococcus pneumoniae infection in research. However, the quality of antibody response, reflected by avidity, has not been adequately evaluated. We aimed to evaluate the role of avidity of IgG against eight pneumococcal proteins in etiologic diagnosis. Eight pneumococcal proteins (Ply, CbpA, PspA1 and 2, PcpA, PhtD, StkP-C, and PcsB-N) were used to develop a multiplex bead-based avidity immunoassay. The assay was tested for effects of the chaotropic agent, multiplexing, and repeatability. The developed assay was applied to paired samples from children with or without pneumococcal disease (n = 38 for each group), determined by either serology, polymerase chain reaction (PCR), or blood culture. We found a good correlation between singleplex and multiplex assays, with r ≥ 0.94.The assay was reproducible, with mean inter-assay variation ≤ 9% and intra-assay variation < 6%. Children with pneumococcal disease had lower median avidity indexes in the acute phase of disease for PspA1 and 2 (p = 0.042), PcpA (p = 0.002), PhtD (p = 0.014), and StkP-C (p < 0.001). When the use of IgG avidity as a diagnostic tool for pneumococcal infection was evaluated, the highest discriminative power was found for StkP-C, followed by PcpA (area under the curve [95% confidence interval, CI]: 0.868 [0.759–0.977] and 0.743 [0.607–879], respectively). The developed assay was robust and had no deleterious influence from multiplexing. Children with pneumococcal disease had lower median avidity against five pneumococcal proteins in the acute phase of disease compared to children without disease.

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