title: Quantification of capsular polysaccharide of Streptococcus pneumoniae serotype 14 in culture broth samples.
abstract: Streptococcus pneumoniae is a major cause of mortality in underdeveloped countries, where more than one million people die from pneumococcal disease every year. Vaccines are the most efficient method for preventing the infection and are based on the capsular polysaccharide (PS) protection. The serotype 14 is the most frequent in pediatric infections worldwide. This study aimed to establish a quantification protocol for PS present in culture broth samples of S. pneumoniae serotype 14 (PS14) and use this protocol for selection of the best PS14 producer strain. Phenol-sulfuric, HPSEC, competitive ELISA, and sandwich ELISA methods were tested for PS14 quantification. Sandwich ELISA was the method with the best reproducibility and sensitivity and the least susceptible to interferences. The quantification limit and detection limit of this method were 0.99 and 0.57 ng/mL, respectively. Statistical analysis was performed to calculate the coefficient of variation (CV) intraassay (1-3% intraplate and 2-6% interplate) and interassay (11-15%) and the reproducibility in different days (CV<20%). The sandwich ELISA allows us to select, among six strains evaluated, the strain 5287 as the best PS14 producer (11.68 mg PS14/biomass) and it was shown to be the best choice for measurement of pneumococcal polysaccharides in culture broth samples.
authors.affiliation: Laboratório de Bioprocessos, Centro de Biotecnologia, Instituto Butantan, 05503-900 São Paulo, SP, Brazil.
authors.forename: Verônica M R
authors.initials: VM
authors.lastname: Gogola
authors.forename: Talita S
authors.initials: TS
authors.lastname: Carmo
authors.forename: Carolina S F
authors.initials: CS
authors.lastname: Geraldo
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Figueiredo
authors.forename: Viviane M
authors.initials: VM
authors.lastname: Gonçalves
mesh_terms: D016667:Bacterial Capsules / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003470:Culture Media; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D019800:Phenol; D012703:Serotyping; D013045:Species Specificity; D013296:Streptococcus pneumoniae / Q000737:chemistry* / Q000145:classification / Q000254:growth & development / Q000472:pathogenicity; D013464:Sulfuric Acids
chemical_list: D003470:Culture Media; D013464:Sulfuric Acids; C056696:pneumococcal polysaccharide, type 14; D019800:Phenol; C033158:sulfuric acid