Enzyme-Linked Immunosorbent Assay for Measuring Antibodies to Pneumococcal Polysaccharides for the PNEUMOVAX 23 Vaccine: Assay Operating Characteristics and Correlation to the WHO International Assay

Abstract

The Merck pneumococcal (Pn) enzyme-linked immunosorbent assays (ELISAs) for measuring antibodies to 12 serotypes (serotypes 1, 3, 4, 6B, 7F, 8, 9V, 12F, 14, 18C, 19F, and 23F) were validated in 1999. Merck Laboratories developed the Pn assays using 10 g/ml C polysaccharide, 100 g/ml Pn polysaccharide (PnPs) 25, and 100 g/ml PnPs 72 for preadsorption of samples, standards, and controls in order to improve the specificity to the Pn serotypes in the vaccine. The Pn assays utilize postimmunization sera obtained from subjects immunized with PNEUMOVAX 23 as standards for measuring immunoglobulin G concentrations in sera obtained from vaccine clinical trials with adults and infants. This material was calibrated to the Pn reference standard serum, 89SF, subjected to the Merck Pn ELISA adsorbants. Comparisons were made between the Merck Pn assay and the international Pn assay, showing moderate agreement between the two assay formats. This work describes the test procedures and operating characteristics of the Merck Pn assays and the results of experiments performed to compare the Merck Pn ELISAs to the international Pn ELISAs.PNEUMOVAX 23 (pneumococcal [Pn] polyvalent vaccine), which was licensed in 1983, is a sterile, liquid vaccine for intramuscular or subcutaneous injection. It consists of a mixture of highly purified capsular polysaccharides from 23 of the most prevalent or invasive serotypes of Streptococcus pneumoniae, including the six serotypes (serotypes 6B, 9V, 14, 19A, 19F, and 23F) that most frequently cause invasive drug-resistant Pn infections among children and adults in the United States (1). Bacterial capsular polysaccharides (Ps) induce antibodies primarily by T-cell-independent mechanisms (15); therefore, the antibody response to most Pn capsular polysaccharide types is generally poor or inconsistent in children Ͻ2 years of age, whose immune systems are immature and unable to respond effectively to the polysaccharide antigens.The immunogenicity of PNEUMOVAX 23 in early clinical trials was determined using a radioimmunoassay measuring antibodies to the Pn polysaccharides (PnPs) (14). However, due to the practicality of supporting large-scale testing, the enzyme-linked immunosorbent assay (ELISA) became the preferred method for estimating antibody concentrations. In this respect, the first-generation ELISA showed a poor correlation of antibody concentration with the efficacy of the vaccines. This observation was attributed to an overestimation of the serotype-specific PnPs antibody concentrations due to the presence of contaminating C Ps in the antigen preparations used in the ELISA (4, 6). Subsequently, a second-generation ELISA was developed by taking steps to neutralize C Ps antibodies in the serum prior to ELISA measurements. These Pn assays, utilizing the steps with preadsorption of test sera with C Ps, were compared in a multicenter study (11). The study compared results among 12 laboratories, with each laboratory testing the same series of 48 Pn paired adult serum specimens from 24 individuals (q...

Development and Characterization of a Multiplex Bead-Based Immunoassay To Quantify Pneumococcal Capsular Polysaccharide-Specific Antibodies

“…In short, the MBIA data presented here met the WHO qualification guidelines, with 75 to 92% of the serum panel showing strong agreement to preestablished WHO values and falling within the acceptable 40% error range. Many other assay protocols published in the literature have failed to meet the qualification criteria for the same Pn Sts evaluated in this study (1,2,6,19).…”

Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the Staphylococcus aureus Iron Surface Determinant B Antigen

“…Specificity experiments demonstrated that the assay is more than 97% specific (Fig. 5) and an E. coli adsorbent or non-IsdB antigen adsorbent is not required as in the case for Streptococcus pneumoniae serologic assays (28,42,46).…”

Development of a Multi-Plex Electrochemiluminescent Assay for the Detection of Serum Antibody Responses to Meningococcal Conjugate Vaccines

“…To best evaluate and optimize the assay, we used standard reference sera [15,17] as well as evaluating serum antibody responses to licensed vaccines in infant rhesus monkeys, a clinically relevant animal model for human infant vaccination. Both CDC 1992 and 89SF reference sera have been used to evaluate and standardize antibody based assays [22,23].…”