Method for simultaneous measurement of antibodies to 23 pneumococcal capsular polysaccharides

Abstract

We describe a fluorescent covalent microsphere immunoassay (FCMIA) method for the simultaneous (multiplexed) measurement of immunoglobulin G (IgG) antibodies to 23 pneumococcal capsular polysaccharide (PnPS) serotypes present in the pneumococcal polysaccharide vaccine (PPV23) licensed by the Food and Drug Administration, i.e., PnPSs 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. In addition, the assay incorporates an internal control that allows for contemporaneous evaluation of the effectiveness of pneumococcal cell wall polysaccharide (C-PS) preadsorption and a second control of PnPS 25 (which is not present in any polysaccharide or conjugate vaccine), which can be used to evaluate interassay reproducibility (useful for pre- versus postvaccination studies). The FCMIA was standardized with U.S. reference antipneumococcal serotype standard serum 89S-2. Preadsorption of 89S-2 with each PnPS and C-PS yielded homologous inhibition for serotypes 1, 6B, 9N, 9V, 11A, 12F,14, 15B, 18C, 19A, 19F, 20, 22F, 25, and 33F; heterologous inhibition for serotypes 9V, 10A, 11A, 12F, 15B, 17F, 20, and 23F; and neither homologous nor heterologous inhibition for serotypes 2, 3, 4, and 5. The minimum detectable concentrations for the 24 multiplexed (PnPS and C-PS) FCMIAs ranged from 20 pg/ml for PnPS 3 to 600 pg/ml for PnPS 14. The PnPS FCMIA method has numerous benefits over enzyme-linked immunosorbent assays commonly used to measure anti-PnPS-specific IgG levels, including increased speed, smaller sample volumes, equivalent or better sensitivity, and increased dynamic range.

FIG. 1.

Periodate oxidation of PnPS (A), conjugation with ADH (B), and covalent coupling of PnPS to microsphere (C).

FIG. 2.

4-PL logistic fit of 24 anti-PnPS and anti-C-PS IgG concentrations in U.S. reference pneumococcal antiserum 89S-2 versus MFI measured by FCMIA. Standard curves were prepared after serum preadsorption with 100 μg of C-PS per ml. The C-PS standard curve was obtained from a 25-plex standard curve run without serum preadsorption with C-PS.

FIG. 3.

4-PL logistic fit of anti-PnPS 25 MFI versus dilutions of serum 89S-2 measured by FCMIA.

FIG. 4.

C-PS inhibition of multiplexed 24 anti-PnPS and C-PS IgGs (serum 89S-2). Assays were performed with and without C-PS preadsorption (100 μg/ml), and the results were compared. Inhibition is expressed as the percent reduction in MFI for each analyte when assays with and without C-PS preadsorption were compared.

FIG. 5.

Anti-PnPS IgG inhibition matrix for serum 89S-2. Dark and light shadings represent homologous and heterologous inhibition, respectively, by a specific PnPS according to the criterion described in the text.