RESUMEN
We recently described the use of ion exchange chromatography for analysis and the industrial scale preparation of pools of oligosaccharides of intermediate chain length from polysaccharides of Haemophilus influenzae type b (Hib) and Neisseria meningitidis groups A and C. These negatively charged "sized" oligosaccharides are activated and conjugated to the carrier protein (CRM197) to prepare the corresponding glycoconjugate vaccines. Characterization and accurate determination of the degree of polymerization (DP) of the pool of oligosaccharides is essential for the consistent production of these conjugate vaccines. This paper describes the colorimetric assays used for determination of the average DP of the Hib and meningococcal oligosaccharides, and the qualification of these assays achieved by size characterization of the respective oligosaccharides by use of physicochemical methods, including liquid chromatography, mass spectrometry (ionspray) and NMR spectroscopy.
Asunto(s)
Cápsulas Bacterianas/química , Vacunas Bacterianas/metabolismo , Oligosacáridos/aislamiento & purificación , Cápsulas Bacterianas/inmunología , Proteínas de la Membrana Bacteriana Externa/análisis , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Vacunas Bacterianas/uso terapéutico , Cromatografía Liquida , Colorimetría , Vacunas contra Haemophilus/análisis , Vacunas contra Haemophilus/inmunología , Vacunas contra Haemophilus/aislamiento & purificación , Haemophilus influenzae tipo b/inmunología , Humanos , Espectrometría de Masas , Meningitis/prevención & control , Meningitis Meningocócica/prevención & control , Peso Molecular , Neisseria meningitidis/inmunología , Resonancia Magnética Nuclear Biomolecular , Oligosacáridos/análisis , Oligosacáridos/inmunología , Polisacáridos Bacterianos/análisis , Polisacáridos Bacterianos/inmunología , Polisacáridos Bacterianos/aislamiento & purificación , Vacunas Conjugadas/análisis , Vacunas Conjugadas/inmunología , Vacunas Conjugadas/aislamiento & purificaciónRESUMEN
We have developed a chromatographic method suitable for the fractionation of polysaccharides having a negatively charged group. The method permits the removal of all those polysaccharide fragments having a short sequence and which are likely unsuitable for conjugate vaccine construction. The selected polysaccharide fragments can be used to produce glycoconjugate vaccines containing a restricted saccharide polydispersion. We have applied this chromatographic method to three different antigens, Haemophilus influenzae type b and Neisseria meningitidis group A and group C polysaccharides. The method is easily adapted for manufacturing purposes.
Asunto(s)
Antígenos Bacterianos/química , Cápsulas Bacterianas/química , Vacunas contra Haemophilus/química , Polisacáridos Bacterianos/química , Vacunas Conjugadas/química , Cromatografía por Intercambio Iónico , Ensayo de Inmunoadsorción Enzimática , Hidrólisis , Espectrometría de Masas , Neisseria meningitidis , UltrafiltraciónRESUMEN
The possibility of using a recombinant fragment of the CagA (128 kDa protein) for the diagnosis of Helicobacter pylori infection was evaluated. Following cloning of the gene coding for the CagA, a recombinant fragment of it was expressed in Escherichia coli, purified and used in Western blot and an EIA to screen sera from 82 patients with gastroduodenal disease who underwent endoscopic examination. In Western blot, good correlation was found between the serological data obtained with the recombinant antigen and those obtained using non-purified extracts of Helicobacter pylori. The EIA using the antigen showed a sensitivity of 96.2% and a specificity of 96.6% compared with Western blot. These data indicate that the recombinant protein is a reliable antigen for detection of infections with Helicobacter pylori strains that are associated with disease. The EIA assay described may be used in follow-up of the progression of the illness and the results of therapy.