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Updated: Dec 1, 2025
Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens
Published on: April 5, 2019
Assignment of opsonic values to pneumococcal reference serum 007sp and a second pneumococcal OPA calibration serum
Robert L Burton1, Han Wool Kim2, Soyoung Lee2
1Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
Insights
Standardizing opsonophagocytic assay (OPA) data with reference serum 007sp reduced inter-laboratory variation for pneumococcal conjugate vaccines (PCVs). This standardization is crucial for developing next-generation PCVs and ensuring reliable immunogenicity assessments.
Area of Science:
- Immunology
- Vaccinology
Background:
- Pneumococcal conjugate vaccines (PCVs) are vital for reducing Streptococcus pneumoniae disease burden.
- New PCV iterations require immunogenicity assessment, ideally measuring functional antibodies.
- Opsonophagocytic assays (OPAs) measure functional antibodies but exhibit inter-laboratory variability.
Purpose of the Study:
- To assess the effectiveness of standardizing multiplexed OPA data using reference serum 007sp for additional pneumococcal serotypes.
- To establish consensus values for 007sp for 11 new serotypes and create a second calibration serum panel (Ewha Panel B).
Main Methods:
- Five laboratories performed multiplexed OPAs on a panel of sera for 11 Streptococcus pneumoniae serotypes.
- Reference serum 007sp was tested in each run for data standardization.
- Results were analyzed using a mixed effects ANOVA model to quantify inter-laboratory variation reduction.
Main Results:
- Standardization significantly reduced inter-laboratory variation for serotypes 2 (40%), 8 (45%), and 11A (40%).
- Modest reductions in variability were observed for serotypes 12F (14%), 17F (19%), and 20B (24%).
- The impact of standardization was diminished for some sera collected after extended post-vaccination intervals.
Conclusions:
- Standardization using 007sp effectively reduces inter-laboratory variability for several pneumococcal serotypes.
- Consensus values for 007sp and the Ewha Panel B will aid in the development of next-generation PCVs.
- Further refinement of standardization methods may be needed to account for variations in post-vaccination antibody responses.
Abstract:
Pneumococcal conjugate vaccines (PCVs) have been effective in reducing the disease burden caused by Streptococcus pneumoniae. The first licensed PCV (PCV7) was composed of capsular polysaccharides from seven serotypes. This was followed by PCV10, then PCV13, and currently there are a number of higher valency vaccines in development. As part of licensure, new vaccine iterations require assessment of immunogenicity. Since some antibodies can be non-functional, measuring functional antibodies is desirable. To meet this need, opsonophagocytic assays (OPAs) have been developed. Previous studies have shown there can be significant variations in OPA results from different laboratories. We have previously shown that standardizing OPA data using reference serum 007sp can decrease this variation. To extend this approach to additional serotypes, a panel of sera was tested by five laboratories using a multiplexed OPA for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20B, 22F, and 33F. Each sample was tested in five runs with 007sp tested three times in each run. Results were analyzed using a mixed effects ANOVA model. Standardization of the results significantly decreased the inter-laboratory variation for some serotypes. For serotypes 2, 8, and 11A, the variability was reduced by 40%, 45%, and 40%, respectively. For serotypes 12F, 17F, and 20B, the reductions were more modest (14%, 19%, and 24%, respectively). Standardization had little effect for the remaining serotypes. In many cases, the impact of normalization was blunted by the results from five sera that were collected after an extended post-vaccination interval. We have previously reported consensus values for 007sp for 13 serotypes, as well as the creation of a calibration serum panel ("Ewha Panel A"). Here, we report consensus values for 11 additional serotypes for 007sp and the creation of a second serum panel ("Ewha Panel B"). These consensus values will facilitate the development of next-generation PCVs.
