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Abstract
Purpose
Serotyping pneumococcal isolates is important to monitor efficacy of pneumococcal vaccines. Because of difficulties of typing pnueumocci, a multiplex bead-based (multibead) serotyping assay was recently introduced. The aim of this study is to establish a new multibead serotyping assay and to apply this method to analyze clinical isolates of pneumococci in Korea.
Methods
To establish the multibead serotyping assay, six key reagents were transferred from University of Alabama at Birmingham (UAB) to Ewha Center for Vaccine Evaluation and Study (ECVES): bead set coated with polysaccharide and monoclonal antibody pool were used in one multiplex inhibition—type immunoassay and 2 bead sets coated DNA probe and 2 primer pools were used in two multiplex PCR—based assays. After multibead serotyping assay was set up, 75 test samples of pneumococci were analyzed whether ECVES is able to identify serotype correctly. After confirming the performance, serotyping assay was applied to identify serotypes of 528 clinical isolates of pneumococci collected from 3 different hospitals.
Results
After establishment of the multibead pneumococcal serotyping assay system at ECVES, 75 test samples were analyzed. There was no discrepancy of serotypes of 75 test samples between the results assigned at UAB and those at ECVES. The serotypes of 528 pneumococci isolated from patients or healthy subjects were determined in 94.3% of isolates (498/528).
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Fig. 1.
Scheme of multibead serotyping assay procedure. (A) Multibead serotyping assay using monocional antibodies (Reaction A). In this assay, the bacterial lysates and a mixture of color—coded beads coupled to reference capsular polysaccharides (PSs) are incubated with a mixture of monocional antibodies that bind the immobilized capsular PSs. If a capsular P5 is in the sample, the PS will inhibit the binding of the monoclonal antibody to the corresponding PS—coated bead. The amount of the monoclonal antibody bound to the beads is determined using a fluorescently labeled anti—mouse immunoglobulin secondary antibody. (B) Multibead serotyping assay using wzy PCR (Reaction B and C). The assay is designed to detect pneumococcal serotypes by identifying wzy gene. wzy from a pneumococcal lysate is PCR amplified with a mixture of PCR primers and the resulting PCR product is identified by hybridizing it to luminex beads which are conjugated with a serotype specific probe.
Fig. 2.
Specificity of multibead serotyping assay. (A) Normalized fluorescence signals of the beads coated with pneumococcal polysaccharides obtained with the panel of a test sample by use of the multiplexed inhibition type immunoassay with monoclonal antibodies (Reaction A). The numbers on the X axis represents the numbers assigned to the bead regions in the test. (B and C) Geometric mean signal of the beads coated with probes obtained with the panel of each sample by use of the PCR-based multiplexed assay (Reaction B and C). The numbers on the X axis represents the numbers assigned to the bead regions in the test.
Table 1.
Bead Regions and Serotype Specificity in Reaction A, B and C
Table 2.
Serotyping Results Deduced bv Multibead Serotyping Assav 10f 528 Clinical Isolates
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