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ISSN 1025-028X (Print)

2013, Number 3

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VacciMonitor 2013; 22 (3)

SDS-PAGE and densitometric analysis to determine concentration of lipopolysaccharide from Neisseria meningitidis serogroups A, W135 and X

Cuello M, Cabrera O, Rodríguez Y, Thurheim G, Norheim G, Cabrera RA, Álvarez M, Álvarez M, Blain K, Naess L, Rosenqvist E, García L

Language: Spanish
References: 19
Page: 36-42
PDF size: 253.87 Kb.


ABSTRACT

For several years, Finlay Institute, in collaboration with the "Norwegian Institute of Public Health", is working in a project to obtain a vaccinal candidate from outer membrane vesicles (OMV) of N. meningitis serogroups A, W135 and X. To establish the quality specifications dealing with lipopolysaccharides (LPS), main cause of the pirogenicity of vaccines, is a necessity in this project. This work aims at establishing the conditions of SDS-PAGE to be used as a method for the quality control of the OMV as active pharmaceutical ingredient obtained from N. meningitidis serogroups A, W135 and X. The immunotypes of LPS were determined in studies as well as their concentrations as contaminants of OMV. SDS-PAGE gels at 15% and LPS samples from different serogroups were used; these gels were tinted with silver, specific for LPS, and the results were analyzed in a densitometer GS-800 (Bio-Rad) controlled by the program "Quantity One". As a result, LPS profile curves for each serogroup were obtained and the concentration of residual LPS in the OMV of different serogroups of N. meningitidis was quantified. As conclusion, the methodology to determine the concentration of LPS from N. meningitidis serogroups A, X and W135 in samples of OMV with SDS-PAGE was established, in addition the established quality specifications for this parameter were confirmed.


REFERENCES

  1. Rosenstein NE, Perkins BA, Stephens DS, Popoviv T, Hughes JM. menigococcal Disease. N Engl J Med 2001;344(18):1378-85.

  2. Cadoz M. Potential and limitations of polysaccharide vaccine in infancy. Vaccine 1998; 6:1391-6.

  3. Martínez AI, Domínguez D, Oviedo M, Minguell S, Jansa JM, Codina G, et al. Changes in the evolution of meningococcal disease, 2001-2008, Catalonia (Spain). Vaccine 2009;27:3496-8.

  4. WHO. Meningococcal disease, African meningitidis belt, epidemic season 2006. Wkly Epidemiol Rec 2006;81:119-20.

  5. Campa C, Sierra VG, Gutiérrez MM, Biset G, García LG, Puentes G, et al. Method of producing Neisseria meningitidis B vaccine, and vaccine produced by method. US patent 5,597,572. 1997.

  6. Gill CJ, Baxter R, Anemona A, Ciavarro GL, M Dull P. Persistence of immune responses after a single dose of Novartis meningococcal serogroup A, C, W-135 and Y CRM-197 conjugate vaccine (Menveo®) or Menactra® among healthy adolescents. Hum Vaccin 2010; 6(11):881-7.

  7. Achtman M. Global epidemiology of meningococcal disease. In: Cartwringt K, ed. Meningococcal disease. Chichester, UK: John Wiley & Sons; 1995. p. 159-75.

  8. Norheim G, Tunheim G, Naess LM, kristiensen PA, Caugant DA, Rosenqvist E. An outer membrane vesicle vaccine for prevention of serofroup A and W135 meningococcal disease in the African meningitis Belt. Scand. J Immunol 2012;76:99-107.

  9. Acevedo R, Zayas C, Fernández S, Cedre B, Valmaseda T, Cuello M, et al. Vaccine potential of outer membrane vesicles from Neisseria meningitidis serogroup X. In: Abstract 18th International Pathogenic Neisseria Conference. Worzburg, Germany: University of Worzburg Printed. 2012. p. 245.

  10. Osborn MJ. Studies on the gramnegative cell in the lipopolysaccharides of Salmonella typhimurium. Proc Natl Acad Sci USA; 1963;50:499-506.

  11. Walters M, Milton D, Larsson L. Airbone environmental endotoxin: A Cross-validation of sampling and analysis techniques. Appl Environ Micriobiol 1994; 60:996-1005.

  12. Balboa JA, Estrada J, Nápoles LD, González H, Hernández D, Aranguren Y, et al. Purificación de lipopolisacárido de Neisseria meningitidis a partir de una fracción colateral del proceso de producción de VA-MENGOC-BC®. VacciMonitor 2008;17(1):17-26.

  13. Lowry OH, Rosebrough NJ, Farr AL, Randall R. Protein measurement with the Folin phenol reagent. Biol Chem 1951; 193: 265-75.

  14. Wedege E. Immunoblot analysis of sera from patients and vaccinees. In: Pollard A, Maiden MCJ, editors. Methods in molecular medicine, Meningococcal vaccines. Methods and Protocols. Totowa, NJ: Human Press; 2001. p. 275-88.

  15. Laemmli UK. Cleavage of structural proteins during the assambly of the head of bacteriophage T4. Nature 1970;227:680-5.

  16. Jennings MP, Srikhanta YN, Moxon ER, Kramer M, Poolman JT, Kuipers B, et al. The genetic basis of the phase variation repertoire of lipopolysaccharide immunotypes in Neisseria meningitidis. Microbiology 1999;145:3013-21.

  17. Scholten RJ, Kuipers B, Valkenburg HA, Dankert J, Zollinger WD, Poolman JT. Lipo-oligosaccharide immunotyping of Neisseria meningitidis by a whole-cell ELISA with monoclonal antibodies. J Med Microbiol 1994;41:236-43.

  18. Cabrera O, Cuello M, Soto CR, Martínez ME, del Campo JM, Pérez O, et al. New method for obtaining conjugated Vaccines. Vaccine 2006; 24(Supl. 2):76-8.

  19. Pérez O, Lastre M, Cabrera O, del Campo J, Bracho G, Cuello M, et al. New Vaccines Require Potent Adjuvants like AFPL1and AFCo1. Scandinavian Journal of Immunology 2007;66: 271-7.




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