Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides

Abstract

The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

Evaluation of a Validated Luminex-Based Multiplex Immunoassay for Measuring Immunoglobulin G Antibodies in Serum to Pneumococcal Capsular Polysaccharides

“…This result is likely due to the improved specificity of the dLIA method over the ELISA. The specificity of the dLIA platform is described in another paper ( 18 ).…”

“…This feature of the dLIA platform might lead to improved correlation between IgG and OPA assay measurements in adult clinical studies. Note that some level of IgG cross-reactivity was observed between the structurally related serotypes, 6A and 6B, as well as between 19A and 19F, but no interference was observed in the multiplex assay ( 18 ). Also, the dLIA has a greater dynamic range than the ELISA.…”

“…The dLIA procedure was performed as described elsewhere in this issue ( 18 ) using the international reference standard serum, 007sp, to calculate all serotype-specific IgG antibody concentrations.…”

“…The dLIA platform has been validated for the 13 PnPS serotypes found in 13vPnC. Both platforms quantify serotype-specific serum IgG antibodies against an international reference standard with known IgG concentrations ( 18 ). However, unlike the ELISA procedure, which relies on passive adsorption of PnPS to polystyrene assay plates, the dLIA procedure uses a standard chemical process to couple poly- l -lysine (PLL)-conjugated PnPS to fluorescent Luminex microspheres.…”

Simultaneous Detection of Bluetongue Virus Serotypes Using xMAP Technology

“…The Luminex technology was originally developed for the detection of multiple analytes, such as cytokines and B-cells [ 25 ], however, xMAP multiplex technology has evolved to provide a diagnostic tool in clinical and veterinary fields. xMAP assays have been used in the detection of both proteins [ 26 , 27 ] and nucleic acid [ 28 , 29 ]. In the latter, a PCR step is initially performed to amplify and label the target sequence, which is then hybridized with a polystyrene microsphere via a coupled complimentary oligo.…”