title: Quantitative determination of pneumococcal capsular polysaccharide serotype 14 using a modification of phenol-sulfuric acid method.
abstract: The capsular polysaccharide of Streptococcus pneumoniae, serotype 14, is part of every pneumococcal vaccine presently in the market or under development. A strategy for the quantitative determination of this polysaccharide by the phenol-sulfuric acid method is described. The modality of acid addition is shown to be the critical step for obtaining reproducible test results between different technicians. Raising the incubation temperature above 80 degrees C increased the consistency of the method by more than 60% regardless of the acid addition modality, but at the expense of some loss of sensitivity. Incubation at 110 degrees C was found necessary to obtain reproducible results within 3% for this technique, which was used to follow the enrichment of the polysaccharide during the last steps of purification. A model mixture of the component polysaccharide sugars provided an adequate and economic standard to construct the calibration curve for this assay, with absorbance reading either in the reaction tubes or in a microplate. A similar procedure may be applied to the determination of other bacterial polysaccharides as well.
authors.affiliation: Departamento de Desarrollo Biotecnológico y Producción, Instituto de Higiene, Universidad de la República, Alfredo Navarro 3051, C.P. 11600 Montevideo, Uruguay.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Cuesta
authors.forename: Norma
authors.initials: N
authors.lastname: Suarez
authors.forename: Maria I
authors.initials: MI
authors.lastname: Bessio
authors.forename: Fernando
authors.initials: F
authors.lastname: Ferreira
authors.forename: Hugo
authors.initials: H
authors.lastname: Massaldi
mesh_terms: D016667:Bacterial Capsules / Q000032:analysis*; D001428:Bacterial Vaccines; D019800:Phenol / Q000737:chemistry; D015203:Reproducibility of Results; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000145:classification* / Q000302:isolation & purification; D013464:Sulfuric Acids / Q000737:chemistry; D013696:Temperature
chemical_list: D001428:Bacterial Vaccines; D013464:Sulfuric Acids; D019800:Phenol