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. 1986 Nov;66(2):481–489.

Studies on the specificity of antibodies to ovalbumin in normal human serum: technical considerations in the use of ELISA methods.

P J Kilshaw, F J McEwan, K C Baker, A J Cant
PMCID: PMC1542523  PMID: 3815902

Abstract

As part of a study designed to reveal information about molecular features of allergenic food proteins after absorption from the gut the specificity of antibodies in normal human serum to hen's egg ovalbumin was investigated using ELISA techniques. Preliminary investigations with monoclonal antibodies and hyperimmune rabbit antiserum specific for ovalbumin in its native and denatured form established that the molecule underwent an extensive conformational change on adsorption to polyvinyl chloride microtitre plates. The native conformation could be retained by using antibodies to couple the protein to the surface. Serum from 90% of healthy adult human donors contained IgG antibodies to ovalbumin. In nearly all cases the antibodies were specific predominantly for the native molecule and could not be absorbed with denatured ovalbumin or peptides prepared from it by cleavage with cyanogen bromide or trypsin. Antibodies to denatured ovalbumin were detected in most sera but at very low levels and were preferentially absorbed by the homologous antigen; peptides and native ovalbumin showing variable absorptive activity. Thus, although ovalbumin is ingested largely in a denatured form, the serum antibody response is stimulated mainly by topographic epitopes of the native molecule.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cant A., Marsden R. A., Kilshaw P. J. Egg and cows' milk hypersensitivity in exclusively breast fed infants with eczema, and detection of egg protein in breast milk. Br Med J (Clin Res Ed) 1985 Oct 5;291(6500):932–935. doi: 10.1136/bmj.291.6500.932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cantarero L. A., Butler J. E., Osborne J. W. The adsorptive characteristics of proteins for polystyrene and their significance in solid-phase imunoassays. Anal Biochem. 1980 Jul 1;105(2):375–382. doi: 10.1016/0003-2697(80)90473-x. [DOI] [PubMed] [Google Scholar]
  3. Dierks S. E., Butler J. E., Richerson H. B. Altered recognition of surface-adsorbed compared to antigen-bound antibodies in the ELISA. Mol Immunol. 1986 Apr;23(4):403–411. doi: 10.1016/0161-5890(86)90138-0. [DOI] [PubMed] [Google Scholar]
  4. Elsayed S., Hammer A. S., Kalvenes M. B., Florvaag E., Apold J., Vik H. Antigenic and allergenic determinants of ovalbumin. I. Peptide mapping, cleavage at the methionyl peptide bonds and enzymic hydrolysis of native and carboxymethyl OA. Int Arch Allergy Appl Immunol. 1986;79(1):101–107. [PubMed] [Google Scholar]
  5. Finn R., Harvey M. M., Johnson P. M., Verbov J. L., Barnes R. M. Serum IgG antibodies to gliadin and other dietary antigens in adults with atopic eczema. Clin Exp Dermatol. 1985 May;10(3):222–228. doi: 10.1111/j.1365-2230.1985.tb00561.x. [DOI] [PubMed] [Google Scholar]
  6. Friguet B., Djavadi-Ohaniance L., Goldberg M. E. Some monoclonal antibodies raised with a native protein bind preferentially to the denatured antigen. Mol Immunol. 1984 Jul;21(7):673–677. doi: 10.1016/0161-5890(84)90053-1. [DOI] [PubMed] [Google Scholar]
  7. Gardner M. L. Intestinal assimilation of intact peptides and proteins from the diet--a neglected field? Biol Rev Camb Philos Soc. 1984 Aug;59(3):289–331. doi: 10.1111/j.1469-185x.1984.tb00708.x. [DOI] [PubMed] [Google Scholar]
  8. Hoffman D. R. Immunochemical identification of the allergens in egg white. J Allergy Clin Immunol. 1983 May;71(5):481–486. doi: 10.1016/0091-6749(83)90465-7. [DOI] [PubMed] [Google Scholar]
  9. Holt P. G., Sedgwick J. D., Stewart G. A., O'Leary C., Krska K. ELISA plaque assay for the detection of antibody secreting cells: observations on the nature of the solid phase and on variations in plaque diameter. J Immunol Methods. 1984 Nov 16;74(1):1–7. doi: 10.1016/0022-1759(84)90360-0. [DOI] [PubMed] [Google Scholar]
  10. Husby S., Oxelius V. A., Teisner B., Jensenius J. C., Svehag S. E. Humoral immunity to dietary antigens in healthy adults. Occurrence, isotype and IgG subclass distribution of serum antibodies to protein antigens. Int Arch Allergy Appl Immunol. 1985;77(4):416–422. doi: 10.1159/000233819. [DOI] [PubMed] [Google Scholar]
  11. Kilshaw P. J., Cant A. J. The passage of maternal dietary proteins into human breast milk. Int Arch Allergy Appl Immunol. 1984;75(1):8–15. doi: 10.1159/000233582. [DOI] [PubMed] [Google Scholar]
  12. Klausner R. D., Kempf C., Weinstein J. N., Blumenthal R., Van Renswoude J. The folding of ovalbumin. Renaturation in vitro versus biosynthesis in vitro. Biochem J. 1983 Jun 15;212(3):801–810. doi: 10.1042/bj2120801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Langeland T. A clinical and immunological study of allergy to hen's egg white. II. Antigens in hen's egg white studied by crossed immunoelectrophoresis (CIE). Allergy. 1982 Jul;37(5):323–333. doi: 10.1111/j.1398-9995.1982.tb01918.x. [DOI] [PubMed] [Google Scholar]
  14. Layton G. T., Stanworth D. R. The quantitation of IgG4 antibodies to three common food allergens by ELISA with monoclonal anti-IgG4. J Immunol Methods. 1984 Oct 26;73(2):347–356. doi: 10.1016/0022-1759(84)90410-1. [DOI] [PubMed] [Google Scholar]
  15. Nisbet A. D., Saundry R. H., Moir A. J., Fothergill L. A., Fothergill J. E. The complete amino-acid sequence of hen ovalbumin. Eur J Biochem. 1981 Apr;115(2):335–345. doi: 10.1111/j.1432-1033.1981.tb05243.x. [DOI] [PubMed] [Google Scholar]
  16. Scott H., Fausa O., Ek J., Brandtzaeg P. Immune response patterns in coeliac disease. Serum antibodies to dietary antigens measured by an enzyme linked immunosorbent assay (ELISA). Clin Exp Immunol. 1984 Jul;57(1):25–32. [PMC free article] [PubMed] [Google Scholar]
  17. Sedgwick J. D., Holt P. G. The ELISA-plaque assay for the detection and enumeration of antibody-secreting cells. An overview. J Immunol Methods. 1986 Feb 27;87(1):37–44. doi: 10.1016/0022-1759(86)90341-8. [DOI] [PubMed] [Google Scholar]
  18. Vaidya H. C., Dietzler D. N., Ladenson J. H. Inadequacy of traditional ELISA for screening hybridoma supernatants for murine monoclonal antibodies. Hybridoma. 1985 Fall;4(3):271–276. doi: 10.1089/hyb.1985.4.271. [DOI] [PubMed] [Google Scholar]
  19. Voller A., Bidwell D. E., Bartlett A. Enzyme immunoassays in diagnostic medicine. Theory and practice. Bull World Health Organ. 1976;53(1):55–65. [PMC free article] [PubMed] [Google Scholar]
  20. Yokota Y., Matsushima A., Inada Y. Isolation of peptides with immunoreactivity from ovalbumin by trypsin digestion. FEBS Lett. 1982 May 17;141(2):225–228. doi: 10.1016/0014-5793(82)80053-7. [DOI] [PubMed] [Google Scholar]

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