A Multiplexed Fluorescent Microsphere Immunoassay for Antibodies to Pneumococcal Capsular Polysaccharides

Abstract

We developed a multiplexed indirect immunofluorescent assay for antibodies to pneumococcal polysaccharides (PnPs) based on the Luminex multiple analyte profiling system (Luminex, Austin, TX). The assay simultaneously determines serum IgG concentrations to 14 PnPs serotypes: 1, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 12F; 14, 18C, 19F, and 23F. To assess the specificity of the multiplexed assay for each individual serotype, inhibition-of-binding studies were conducted using adult serum samples obtained after pneumococcal vaccination. Except for the closely related serotypes 9V and 9N, we demonstrated inhibition by homologous serotypes of more than 95% and inhibition by heterologous serotypes of less than 15% for all 14 PnPs serotypes. There was, however, high heterologous inhibition of 50% or greater with some serotypes. These cross-reacting antibodies could not be removed by preabsorption with pneumococcal C-polysaccharide but were removed by additional preabsorption with serotype 22F polysaccharide. The multiplexed Luminex assay showed good overall agreement with a well-established enzyme-linked immunosorbent assay that is currently recommended for evaluation of pneumococcal vaccine immunogenicity.

Diagnosis of Invasive Pneumococcal Infection by Serotype-Specific Urinary Antigen Detection

“…First, performing multiple ELISAs on each sample is relatively labor intensive, although some automation of the process would be relatively straightforward, and the assays could potentially be "multiplexed" using coated-bead suspension array technology as described previously for the assay of antibodies against pneumococcal capsular polysaccharides (17). Second, previous studies have demonstrated that detectible pneumococcal C polysaccharide is present in the urine of healthy infants with upper respiratory tract colonization (4,5,8).…”

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“…Another innovative triplex assay was created by American researchers for the detection of Corynebacterium diphtheriae, Clostridium tetani and Haemophilus influenzae type b [122]. The assay incorporates three antigens: tetanus and diphtheria toxoids and the capsular polysaccharide of H. influenzae conjugated to human serum albumin (HbO-HA).…”

“…Second, there is a need to exclude antibodies specific to soluble pneumococcal cell wall polysaccharides and polysaccharide C typical for non-capsular strains of S. pneumoniae, because they do not confer immunity against this pathogen. Therefore, solution used to dilute the serum should contain absorbents -soluble polysaccharides of the cell wall, including polysaccharide C and serotype 22F polysaccharide [117,122]. The same modifications are introduced to reference ELISAs, improving specificity of the assay and revealing its actual sensitivity.…”

“…Differences between ELISA and MIA results suggest that the established protective threshold of 0.35 µg/ml should be reconsidered for the use in MIA and probably analyzed for each serotype separately [124]. A similar study was conducted a few years earlier in the USA (Table 2) [122]. An immunoassay was designed to simultaneously detect antibodies to 14 pneumococcal polysaccharide serotypes.…”

Measurement of Pneumococcal Polysaccharide Antibodies

“…Although the Luminex method has been approved for the measurement of specific anti-pneumococcal serotype IgG antibodies, the correlation of this test with the standard ELISA test is poor and its use as s a diagnostic tool for SAD still needs further study. Recent studies designed to validate the Luminex assay for anti-pneumococcal IgG antibodies have shown different degrees of variability when compared to the ELISA method [4]. Results obtained in a recent comprehensive comOther studies claim that although interlaboratory analytical variability between assays exists, this variability has a relatively minor impact on classifying patients as "protected" or "nonprotected" when using established diagnostic algorithms [6,7].…”