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Haemophilus influenzae Protocols pp 243–261Cite as

ELISA

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Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 71))

Abstract

Enzyme linked immunosorbent assay (ELISAs) has been used as a convenient routine immunological assay for many specific antigens to monitor antibody responses to vaccination, and has replaced other methods, e.g., radioimmunoassay (FARR assay) (1,2). For example, ELISA using tetanus toxoid, 1diphtheria toxoid, Haemophilus influenzae Type b (Hib) polysaccharide and Neisseria meningitidis A/C polysaccharide are used to determine serum antibodies following vaccination. In these assays, the antigen, e.g., toxoid (tetanus, diphtheria) or polysaccharide (Hib PRP, Men C) alone or conjugated to poly-L-lysine or methylated human serum albumin (mHSA), is coated onto plastic of 96-well microtiter plates (3-5). By direct or indirect methods the antibodies in serum samples can be detected using enzyme-linked secondary antibody conjugates, e.g., horseradish peroxidase or alkaline phosphatase, and appropriate substrates using color reaction at specific absorbance e.g., ODA405. The principle of the indirect ELISA is shown in Fig. 1. (6). Using dilutions of a standard serum or monoclonal antibody with known concentration of total/ specific antibody or given arbitrary units, it is possible to compare dilutions of unknown samples against a standard curve. In this chapter we demonstrate how to establish an ELISA for antibody to H. influenzae (Hi) inner-core lipopolysaccharide (LPS) epitopes.

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References

  1. Farr R. S. (1958) A quantitative immunochemical measure of the primary interaction between I*BSA and antibody. J. Infect. Dis. 103, 239–262.

    CAS  PubMed  Google Scholar 

  2. Phipps D. C., West J., Eby R., Koster M., Madore D. V., and Quataert S. A. (1990) An ELISA employing a Haemophilus influenzae type b oligosaccharidehuman serum albumin conjugate correlates with radio-antigen binding assay. J. Immunol. Meth. 135, 121–128.

    Article  CAS  Google Scholar 

  3. Gray B. M. (1979) ELISA methodology for polysaccharide antigens: protein coupling of polysaccharides for adsorption to plastic tubes. J. Immunol. Meth. 28, 187–192

    Article  CAS  Google Scholar 

  4. Carlone G. M., Frasch C. E., Siber G. R., et al. Multicenter comparison of levels of antibody to the Neisseria meningitidis Group A capsular polysaccharide measured by using an enzyme-linked immunosorbent assay. J. Clin. Microbiol. 30, 154–159.

    Google Scholar 

  5. Gheesling L. L., Carlone G. M., Pais L. B., et al. (1994) Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardised enzyme-linked immunosorbent assay. J. Clin. Microbiol. 32, 1475–1482.

    CAS  PubMed  Google Scholar 

  6. Wood H. C. and Wreghitt T. G. (1990) Techniques, in ELISA in the Clinical Microbiology Laboratory (Wreghitt T. G., and Morgan-Capner P., eds.), Laverham Press, Whaddon, Salisbury (Public Health Laboratory Service series).

    Google Scholar 

  7. Plested J. S., Gidney M. A. J., Coull P. A., et al. (2000) Enzyme linked immunosorbent assay (ELISA) for the detection of serum antibodies to the innercore lipopolysaccharide of Neisseria meningitidis Group B. J. Immunol. Methods 237, 73–84.

    Article  CAS  PubMed  Google Scholar 

  8. Abdillahi H. and J. T. Poolman. (1988) Typing of group-B Neisseria meningitidis with monoclonal antibodies in the whole-cell ELISA. J. Med. Microbiol. 26, 177–180.

    CAS  PubMed  Google Scholar 

  9. Pollard A. J., Galassini R., Rouppe van der Voort E. M., et al. (1999) Humoral immune responses to Neisseria meningitidis in children. Infect. Immumol. 67, 2441–2451.

    CAS  Google Scholar 

  10. Plested J. S., Makepeace K., Jennings M. P., et al. (1999) Conservation and accessibility of an inner-core lipopolysaccharide epitope of Neisseria meningitidis. Infect. Immunol. 67, 5417–5426.

    CAS  Google Scholar 

  11. Gidney M. A. J., Lacelle S., Cox A. D., Martin A., Moxon E. R., and Richards J. J. Production and characterisation of a monoclonal antibody (L6A9) against innercore epitope in Haemophilus influenzae lipopolysaccharide. (in preparation).

    Google Scholar 

  12. Estabrook M. M, Baker C. J., and Griffis J. M. (1993) The immune response of children to meningococcal lipooligosaccharides during disseminated disease is directed primarily against two monoclonal antibody-defined epitopes. J. Infect. Dis. 167, 966–970.

    CAS  PubMed  Google Scholar 

  13. Goldblatt D., Vaz A. R., and Miller E. (1998) Antibody avidity as a surrogate marker of successful priming by Haemophilus influenzae type b conjugate vaccines following infant immunization. J. Infect. Dis. 117, 1112–1115.

    Article  Google Scholar 

  14. Anttila M., Voutilainen M., Jantti V., et al. (1999) Contribution of serotypespecific IgG concentration, IgG subclasses and relative antibody avidity to opsonophagocytic activity against Streptococcus pneumoniae. Clin. Exp. Immunol. 118, 402–407.

    Article  CAS  PubMed  Google Scholar 

  15. Granoff D. M., Maslanka S. E., Carlone G. M., et al. (1998) A modified enzyme-linked immunosorbent assay for measurement of antibody responses to meningococcal C polysaccharide that correlate with bactericidal responses. Clin. Diagn. Lab. Immunol. 5, 479–485

    CAS  PubMed  Google Scholar 

  16. Hood D. W., Cox A. D., Wakaarhuk W. W., et al. (2001) Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H. influenzae strain Rd (RM118). Glycobiol. 11, 957–967.

    Article  CAS  Google Scholar 

  17. Mansson M., Bauer S. H., Hood D. W., et al. (2001) A new structural type for Haemophilus influenzae lipopolysaccharide from nontypeable Haemophilus influenzae strain 486. Eur. J. Biochem. 268, 2148–2159.

    Article  CAS  PubMed  Google Scholar 

  18. Risberg A., Masoud H., Martin A., et al., (1999) Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule-deficient strain of Haemophilus influenzae Rd. Eur. J. Biochem. 261, 171–180.

    Article  CAS  PubMed  Google Scholar 

  19. Anderson P., Johnston R., and Smith D. (1972) Human serum activities against Haemophilus influenzae type b. J. Clin. Invest. 51, 31–38.

    Article  CAS  PubMed  Google Scholar 

  20. Westphal O., and Jann. J. K. (1965) Bacterial lipopolysaccharides extraction with water-phenol and further applications of the procedure. Methods Carbohydr. Chem. 5, 83–91.

    CAS  Google Scholar 

  21. Masoud H., Moxon E. R., Martin A., et al. (1997) Structure of the variable and conserved lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae serotype b Eagan. Biochemistry 36, 2091–2103.

    Article  CAS  PubMed  Google Scholar 

  22. Shulman M., Wilde C. D., and Kohler G. (1978) A better cell line for making hybridomas secreting specific antibodies. Nature 276, 269–270.

    Article  CAS  PubMed  Google Scholar 

  23. Carlin N. I., Gidney M. A., Lindberg A. A., and Bundle D. R. (1986) Characterisation of Shigella flexneri-specific murine monoclonal antibodies by chemically defined glycoconjugates. J. Immunol. 137, 2361–2366.

    CAS  PubMed  Google Scholar 

  24. Ito J. I., Wunderlich A. C., Lyons J., et al. (1980) Role for magnesium in the nzyme-linked immunosorbent assay for lipopolysaccharides of rough Escherichia coli strain J15 and Neisseria gonorrhoeae. J. Infect. Dis. 142, 532–537.

    CAS  PubMed  Google Scholar 

  25. Law B. J. and Marks M. I. (1985) Age-related prevelance of human serum IgG and IgM antibody to the core glycolipid of Escherichia coli strain J5, as measured by ELISA. J. Infect. Dis. 151, 988–994.

    CAS  PubMed  Google Scholar 

  26. Freudenberg M. A., Fomsgaard A., Mitov I., and Galanos C. (1989) ELISA for antibodies to lipid A, lipopolysaccharides and other hydrophilic antigens. Infection 17, 322–328.

    Article  CAS  PubMed  Google Scholar 

  27. Kuhn H. M., Brade L., Appelmelk B. J., et al. (1992) Characterisation of the epitope specificity of murine monoclonal antibodies directed against lipid A. Infect. Immunol. 60, 2201–2210.

    CAS  Google Scholar 

  28. Petrov A. B., Semenov B. F., Vrtanyan Y. P., et al. (1992) Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes. Infect. Immunol. 60, 3897–3903.

    CAS  Google Scholar 

  29. Appelmelk B. J., Verweij-Van Vaught A. M., MacLaren D. M., and Thijs L. G. (1985) An enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies to different parts of the gram-negative lipopolysaccharide core region. J. Immunol. Meth. 82, 199–207.

    Article  CAS  Google Scholar 

  30. Kirkland T. M., Colwell D. E., Michalek S. M., et al. (1986) Analysis of the fine specificity and cross-reactivity of monoclonal anti-lipid A antibodies. J. Immunol. 137, 3614–3619.

    CAS  PubMed  Google Scholar 

  31. Stoll C., Schedel I., and Peest D. (1985) Serum antibodies against common antigens of bacterial lipopolysaccharides in healthy adults and in patients with multiple myeloma. Infection 13, 115–119.

    Article  CAS  PubMed  Google Scholar 

  32. Erich T., Schellekends J., Bouter A., et al. (1989) Binding characteristics and cross-reactivity of three different antilipid A monoclonal antibodies. J. Immunol. 143, 4053–4060.

    CAS  PubMed  Google Scholar 

  33. Lagace J., Arsenault S., and Cohen E. H. (1994) Alcian blue-treated polystyrene microtitre plates for use in an ELISA to measure antibodies agains synthetic peptides. J. Immunol. Meth. 175, 131–135.

    Article  CAS  Google Scholar 

  34. Hardy E., Ohlin M., and Llano M. (1994) Enhanced ELISA sensitivity using TCA for efficient coating of biologically active lipopolysaccharides for lipid A to the solid phase. J. Immunol. Meth. 176, 111–116.

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Pediatrics, University of Oxford, Oxford, UK

    Joyce S. Plested & Philip A. Coull

  2. Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada

    Margaret Anne J. Gidney

Authors
  1. Joyce S. Plested
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  2. Philip A. Coull
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  3. Margaret Anne J. Gidney
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Editor information

Editors and Affiliations

  1. Department of Pediatrics, John Radcliffe Hospital, Headington, Oxford, UK

    Mark A. Herbert , Derek W. Hood  & E. Richard Moxon ,  & 

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© 2003 Humana Press Inc.

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Plested, J.S., Coull, P.A., Gidney, M.A.J. (2003). ELISA. In: Herbert, M.A., Hood, D.W., Moxon, E.R. (eds) Haemophilus influenzae Protocols. Methods in Molecular Medicine™, vol 71. Humana Press. https://doi.org/10.1385/1-59259-321-6:243

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  • DOI: https://doi.org/10.1385/1-59259-321-6:243

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-928-5

  • Online ISBN: 978-1-59259-321-7

  • eBook Packages: Springer Protocols

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