Rapid Multiplex Assay for Serotyping Pneumococci with Monoclonal and Polyclonal Antibodies

Abstract

We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotypespecific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy.Streptococcus pneumoniae is a well-known human pathogen and a major etiologic agent for pneumonia, meningitis, otitis media, and sepsis, primarily among young children and older adults (3). S. pneumoniae is divided into 90 serotypes, based on its expression of serologically distinct carbohydrate capsules (6). Antibodies to a capsular polysaccharide (PS) provide protection against pneumococci expressing the same capsular serotype. Currently available pneumococcal vaccines contain a mixture of capsular PS of multiple serotypes. One pneumococcal vaccine (called PS vaccine) contains capsular PS from 23 commonly found serotypes (17). The most recently developed type of vaccine (called conjugate vaccine) contains capsular PS from 7 to 11 serotypes that are conjugated to a protein molecule (26). A 7-valent conjugate vaccine was introduced in 2000 for routine immunization in the United States and has reduced the incidence of invasive pneumococcal diseases in children (25).Accurate, efficient serotyping of pneumococcal isolates is important for measuring the efficacy of pneumococcal vaccines. Since the vaccine-induced antibodies provide serotypespecific protection, following the introduction of a new pneumococcal vaccine, pneumococci expressing the serotypes included in the vaccine would become less common while the prevalence of the pneumococci expressing nonvaccine serotypes would stay the same. As a result, a reduction in vaccine serotypes reflects the vaccine efficacy. In some cases, pneumococci expressing the non...

Biochemical, Genetic, and Serological Characterization of Two Capsule Subtypes among Streptococcus pneumoniae Serotype 20 Strains

“…Murine mAb were generated by immunization with serotype 20 PS purchased from ATCC, as described (16). These antibodies include IgM mAbs (Hyp20M1, Hyp20M3, and Hyp20M4) and IgG mAbs (Hyp20G1, Hyp20G2, Hyp20G3, Hyp20G4, Hyp20G5, and Hyp20G6).…”

Spectrum of Pneumococcal Serotype 11A Variants Results from Incomplete Loss of Capsule O -Acetylation

“…Murine hybridomas Hyp11AM1 and Hyp11AG2 were produced using a previously described method (9) and selected for binding to serotype 11A capsule PS. Hyp11AM1 produces IgM monoclonal antibody (MAb) and Hyp11AG2 produces IgG MAb.…”

Discovery of a New Capsular Serotype (6C) within Serogroup 6 ofStreptococcus pneumoniae

“…Since vaccine-induced immunoprotection is serotype specific, serotyping pneumococcal isolates from patients is an important tool for monitoring the effectiveness of pneumococcal vaccines (3). Because the classical quellung reaction with rabbit antisera is tedious to perform (13), we have developed a new serotyping system based on mouse monoclonal antibodies (mAbs) and a multiplexed immunoassay (27). While validating the new system, we found that a minor fraction of the isolates determined to be serotype 6A by quellung reaction bound to one 6A-specific mAb (Hyp6AG1) but not to the other (Hyp6AM3), whereas the majority of the serotype 6A isolates bound to both mAbs (12).…”