title: Enzyme-linked immunosorbent assay for quantitative determination of capsular polysaccharide production in Streptococcus pneumoniae clinical isolates.
abstract: A simple, specific, sensitive and reproducible ELISA has been developed to quantify the level of CPS (capsular polysaccharide) production in supernatants of Streptococcus pneumoniae cell cultures. CPSs from Strep. pneumoniae have been widely used as vaccine antigens. The quantification method is based on two type-23F serotype-specific polyclonal antibodies: IgG, purified from sera of mice immunized with a pneumococcal type-23F CPS conjugate, used in the coating step, and a serotype-specific rabbit serum as the second antibody. Solutions of purified type-23F CPS were used as standards. The relationship between A(492) and type-23F CPS concentration was linear over the range 1-310 ng/ml (r=0.989), with 1 ng/ml as the lower limit of sensitivity. The specificity of ELISA was assessed because purified type-19F CPS and cell-wall polysaccharide samples were not detected after their evaluation by the ELISA described in the present study. Repeatability and intermediate precision of the assay were good, the coefficients of variation being 3 and 10% respectively. This ELISA allowed selection of an appropriate vaccine strain, for a natural polysaccharide vaccine, among several 23F pneumococcal clinical isolates and constituted a valuable analytical tool for Strep. pneumoniae fermentation and CPS purification follow-up.
authors.affiliation: División de Vacunas, Centro de Ingeniería Genética y Biotecnología (CIGB), Ave. 31, entre 158 y 190, Apartado 6162, CP 10600, La Habana, Cuba. yoelys.cruz@cigb.edu.cu
authors.forename: Yoelys
authors.initials: Y
authors.lastname: Cruz-Leal
authors.forename: Tamara
authors.initials: T
authors.lastname: Menéndez
authors.forename: Edelgis
authors.initials: E
authors.lastname: Coizeau
authors.forename: Raúl Rafael
authors.initials: RR
authors.lastname: Espinosa
authors.forename: Leonardo
authors.initials: L
authors.lastname: Canaan
authors.forename: Francles
authors.initials: F
authors.lastname: Blanco
authors.forename: Tania
authors.initials: T
authors.lastname: Carmenate
authors.forename: Janoi
authors.initials: J
authors.lastname: Chang
authors.forename: Dianelys
authors.initials: D
authors.lastname: Quiñones
authors.forename: Isis
authors.initials: I
authors.lastname: Tamargo
authors.forename: José
authors.initials: J
authors.lastname: Cremata
authors.forename: Vicente
authors.initials: V
authors.lastname: Verez-Bencomo
authors.forename: Gerardo
authors.initials: G
authors.lastname: Guillén
mesh_terms: D000818:Animals; D016667:Bacterial Capsules / Q000096:biosynthesis / Q000737:chemistry*; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008586:Meningitis, Pneumococcal / Q000175:diagnosis*; D051379:Mice; D008807:Mice, Inbred BALB C; D011135:Polysaccharides, Bacterial / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D015203:Reproducibility of Results; D013296:Streptococcus pneumoniae / Q000145:classification* / Q000378:metabolism
chemical_list: D011135:Polysaccharides, Bacterial