Approach to Validating an Opsonophagocytic Assay for Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae (pneumococcus) polysaccharide serotype-specific antibodies that have opsonophagocytic activity are considered a primary mechanism of host defense against pneumococcal disease. In vitro opsonophagocytic assays (OPAs) with antibody and complement to mediate opsonophagocytic killing of bacteria have been designed and developed as an adjunct to the standardized serum immunoglobulin G antipneumococcal capsular polysaccharide enzyme immunoassay to assess the effectiveness of pneumococcal vaccines. OPA presents challenges for assay standardization and assay precision due to the multiple biologically active and labile components involved in the assay, including human polymorphonuclear leukocytes or cultured effector cells, bacteria, and complement. Control of these biologically labile components is critical for consistent assay performance. An approach to validating the performance of the assay in accordance with International Conference for Harmonization guidelines, including its specificity, intermediate precision, accuracy, linearity, and robustness, is presented. Furthermore, we established parameters for universal reagents and standardization of the use of these reagents to ensure the interlaboratory reproducibility and validation of new methodologies.

Post Hoc Analysis of a Randomized Double-Blind Trial of the Correlation of Functional and Binding Antibody Responses Elicited by 13-Valent and 7-Valent Pneumococcal Conjugate Vaccines and Association with Nasopharyngeal Colonization

“…Validated microcolony OPA assays were used to measure the 13 pneumococcal serotypes contained in the PCV13. These assays were based on previously described methods (2,3). OPA assay titers were defined as the interpolated reciprocal serum dilution that results in complement-mediated killing of 50% of the bacteria in the assay.…”

Multilaboratory Comparison of Streptococcus pneumoniae Opsonophagocytic Killing Assays and Their Level of Agreement for the Determination of Functional Antibody Activity in Human Reference Sera

“…OPA reflects in vivo mechanisms of defense against pneumococcal infection and is recognized as being increasingly important for regulatory purposes, especially for evaluating the serotypes in extended formulations that are not present in PCV7. Several modifications to the OPA first described by Romero-Steiner et al (19) have been described (9,11). Perhaps the most significant modification has been the capacity to measure functional antibodies in multiplex formats to evaluate functional responses to four or more serotypes and reduce the number of opsonophagocytic assays for the evaluation of multivalent vaccines (1-3, 14, 15).…”

Relationship between IgG titers and opsonocytophagocytic activity of anti-pneumococcal antibodies after immunization with the 7-valent conjugate vaccine in allogeneic stem cell transplant

“…12 Briefly, heatinactivated human serum samples were serially diluted and incubated with the S. pneumoniae OPA bacterial test strains (B2000 CFU per well) and 12.5% of baby rabbit complement in a 96-well microtitre plate. The plates were shaken for 30 min at 37 1C (opsonization step).…”