Monoclonal Antibodies Against the Capsular Polysaccharides A, C, Y, W, and X of Neisseria meningitidis: A Platform for the Quality Control of Meningococcal Vaccines

  • Elizabeth González
  • Fátima Reyes
  • Oscar Otero
  • Frank Camacho
  • Maribel Cuello
  • Fidel Ramírez
  • Reinaldo AcevedoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1969)


Vaccination has reduced morbidity and mortality of many diseases that previously caused devastating epidemics and deaths globally. Vaccines as a biological product may contain microorganisms or their derivatives. This aspect together with the fact that they are administered to healthy individuals (mainly children) means that approximately 70% of vaccines development time is dedicated to quality control. Monoclonal antibodies (MAbs) have become essential analytical tools for application in ELISAs, Western and Dot blotting, immunoprecipitation, and flow cytometric assays that ensure the quality control of vaccines. The aim of this work is to present a review of the methods used to obtain a platform of MAbs against Neisseria meningitidis polysaccharide antigens to use as an analytical tool for quality control of anti-meningococcal polysaccharide (Ps) vaccines. The MAbs obtained are used in five sandwich ELISAs developed for Ps quantification. The assays showed good reproducibility and repeatability, with quantitation and detection limits below 1 ng/mL. Dot Blot, as the Identity test of the Ps vaccine, was carried out to positively identify licensed and experimental vaccines. All assays described are suitable for the screening of multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability.

Key words

Monoclonal antibody Analytical tool Polysaccharide quantitation ELISA and Identity Test 


  1. 1.
    Greenwood B (2014) The contribution of vaccination to global health: past, present and future. Philos Trans R Soc Lond Ser B Biol Sci 369(1645):20130433CrossRefGoogle Scholar
  2. 2.
    Bustreo F, Okwo-Bele JM, Kamara L (2015) World Health Organization perspectives on the contribution of the global alliance for vaccines and immunization on reducing child mortality. Arch Dis Child 100(Suppl 1):s34–s37CrossRefGoogle Scholar
  3. 3.
    WHO. Web page: Immunization, vaccines and biologicals
  4. 4.
    US Pharmacopeia (2007) Guideline for Submitting Requests for Revision to USP-NF. V3.1 April.VACCINESGoogle Scholar
  5. 5.
    WHO. Web page Vaccine and immunization quality and safety
  6. 6.
    CECMED (2000) Requisitos para la liberación de lotes de vacunas. Regulación No.19-2000. CECMED, La HabanaGoogle Scholar
  7. 7.
    European Pharmacopoeia 5.0. 01/2005:20701. 2.7.1. Immunochemical methods. p 1879Google Scholar
  8. 8.
    Ochoa RF (2013) Técnicas inmunoenzimáticas en el desarrollo clínico de vacunas, Primera Edición. Finlay Ediciones, Havana, CubaGoogle Scholar
  9. 9.
    Saydam M, Rigsby P, Mawas F (2014) A novel enzyme-linked immuno-sorbent assay (ELISA) for the quantification of total and free polysaccharide in Haemophilus influenzae B Tetanus toxoid conjugate vaccines in monovalent and combined vaccine formulations. Biologicals 42:29–33CrossRefGoogle Scholar
  10. 10.
    Saleem M, Kamal M (2008) Monoclonal antibodies in clinical diagnosis: a brief review application. Afr J Biotechnol 7:923–925Google Scholar
  11. 11.
    Payne WJ, Marshall DL, Shockley RK, Martin WJ (1988) Clinical laboratory applications of monoclonal antibodies. Clin Microbiol Rev 1:313–329CrossRefGoogle Scholar
  12. 12.
    Falero G, Rodríguez BL, Rodríguez I, Campos J, Ledon T, Valle E, Silva Y, Marrero K, Suzarte E, Valmaseda T, Moreno A, Fando R (2003) Production and characterization of monoclonal antibodies to E1 Tor toxin co-regulated pilus of Vibrio cholerae. Hybrid Hybridomics 22(5):315–320CrossRefGoogle Scholar
  13. 13.
    Barberá R, Domínguez F, Otero O, Gutiérrez M, Falero G, Reyes G, Sotolongo F, Sierra G (2011) Development and characterization of murine monoclonal antibody specific for the P1.4 PorA proteins from strain B: 4:P1.(7b).4. of Neisseria meningitidis. Vaccimonitor 20:6–10Google Scholar
  14. 14.
    Waliza A, Shyamasree G (2013) Monoclonal antibodies: a tool in clinical research. Indian J Clin Med 4:9–21CrossRefGoogle Scholar
  15. 15.
    Hnasko RM, Stanker LH (2015) Hybridoma technology. In: ELISA: methods and protocols, Methods in molecular biology, vol 1318. Springer Science+Business Media, New YorkCrossRefGoogle Scholar
  16. 16.
    Ward PA et al (1999) Monoclonal antibody production. A report of the committee on methods of producing monoclonal antibodies. Institute for laboratory animal research. National Research CouncilGoogle Scholar
  17. 17.
    Reyes F, Amin N, Otero O, Aguilar A, Cuello M, Valdés Y, García LG, Cardoso D, Camacho F (2013) Four monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, C, Y and W135: its application in identity tests. Biologicals 41(4):275–278CrossRefGoogle Scholar
  18. 18.
    Yokoyama WM (2001) Monoclonal antibody supernatant and ascites fluid production. Curr Protoc Immunol 40:2.6.1–2.6.9Google Scholar
  19. 19.
    Fitzgerald J, Leonard P, Darcy E, O’Kennedy R (2011) Immunoaffinity chromatography. Methods Mol Biol 681:35–59CrossRefGoogle Scholar
  20. 20.
    ImmunoReagents Inc. (2015) Antibody purification methods. IRU - ImmunoReagents University, Raleigh, NCGoogle Scholar
  21. 21.
    Ezzatifar F, Majidi J, Baradaran B, Maleki LA, Abdolalizadeh J, Yousefi M (2015) Large scale generation and characterization of anti-human IgA monoclonal antibody in ascitic fluid of Balb/c mice. Adv Pharm Bull 5(1):97–102PubMedPubMedCentralGoogle Scholar
  22. 22.
    Amersham Pharmacia Biotech (2001) Affinity chromatography. Principles and methods. Handbooks. Editon AC 18-1022-29. Amersham Pharmacia Biotech, Little ChalfontGoogle Scholar
  23. 23.
    Beatty JD, Beatty BG, Vlahos WG (1987) Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods 100:173–179CrossRefGoogle Scholar
  24. 24.
    Johnson M (2013) Mouse antibody isotypes/classes and subclasses. Mater methods 3:152 (Labome.
  25. 25.
    Clark M Antibody-antigen reactions. Department of pathology. University of Cambridge, CambridgeGoogle Scholar
  26. 26.
    Frank SA (2002) Specificity and cross-reactivity. In: Immunology and evolution of infectious disease. Princeton University Press, Princeton, NJGoogle Scholar
  27. 27.
    Agnememel A, Traincard F, Dartevelle S, Mulard L, Mahamane AE, Oukem-Boyer OO, Denizon M, Kacou-N Douba A, Dosso M, Gake B, Lombart JP, Taha MK (2015) Development and evaluation of a dipstick diagnostic test for Neisseria meningitidis serogroup X. Clin Microbiol 53:449–454CrossRefGoogle Scholar
  28. 28.
    Wedege E, Høiby EA, Rosenqvist E, Frøholm LO (1990) Serotyping and subtyping of Neisseria meningitidis isolates by co-agglutination, dot-blotting and ELISA. J Med Microbiol 31:195–201CrossRefGoogle Scholar
  29. 29.
    Ortika BD, Habib M, Dunne EM, Porter BD, Satzke C (2013) Production of latex agglutination reagents for pneumococcal serotyping. BMC Res Notes 6:49CrossRefGoogle Scholar
  30. 30.
    Cook MC, Gibeault S, Filippenko V, Ye Q, Wang J, Kunkel JP (2013) Serogroup quantitation of multivalent polysaccharide and polysaccharide conjugate meningococcal vaccines from China. Biologicals 41:261–268CrossRefGoogle Scholar
  31. 31.
    Cook MC, Bliu A, Kunkel JP (2013) Quantitation of serogroups in multivalent polysaccharide-based meningococcal vaccines: optimisation of hydrolysis conditions and chromatographic methods. Vaccine 31:3702–3711CrossRefGoogle Scholar
  32. 32.
    Reyes F, Otero O, Cuello M, Amin N, García L, Cardoso D, Camacho F (2014) Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroups A, C, W and Y in multivalent vaccines. J Immunol Methods 407:58–62CrossRefGoogle Scholar
  33. 33.
    Chen PS, Toribara TY, Warner H (1956) Microdetermination of phosphorus. Anal Chem 28:1756–1758CrossRefGoogle Scholar
  34. 34.
    Svennerholm L (1957) Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim Biophys Acta 24:604–611CrossRefGoogle Scholar
  35. 35.
    Lamb DH, Lei QP, Hakim N, Rizzo S, Cash P (2005) Determination of meningococcal polysaccharides by capillary zone electrophoresis. Anal Biochem 338:263–269CrossRefGoogle Scholar
  36. 36.
    Kao G, Tsai CM (2004) Quantification of O-acetyl, N-acetyl and phosphate groups and determination of the extent of O-acetylation in bacterial vaccine polysaccharides by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD). Vaccine 22:335–344CrossRefGoogle Scholar
  37. 37.
    Crowther JR (2009) The ELISA guidebook, Methods in molecular biology, vol 516. Humana Press, New YorkGoogle Scholar
  38. 38.
    Plikaytis BD, Carlone GM, Edmonds P, Mayer LW (1986) Robust estimation of standard curves for protein molecular weight and linear-duplex DNA base-pair number after gel electrophoresis. Anal Biochem 152:346–364CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Elizabeth González
    • 1
  • Fátima Reyes
    • 1
  • Oscar Otero
    • 1
  • Frank Camacho
    • 1
  • Maribel Cuello
    • 1
  • Fidel Ramírez
    • 1
  • Reinaldo Acevedo
    • 1
    Email author
  1. 1.Monoclonal Antibodies Laboratory, Department of Biological Evaluation, Research AreaFinlay Institute of VaccineWest HavanaCuba

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