Antigenicity of Native and Modified Kunitz Soybean Trypsin Inhibitors

  • David L. Brandon
  • Sakhina Haque
  • Mendel Friedman
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 199)


Food provides a continuous antigenic stimulus to the immune system and the antigenicity of processed food proteins should be considered in toxicological evaluations. The antigenicity of the Kunitz trypsin inhibitor was studied using antibodies prepared by inoculating rabbits with native, heat-denatured, and N-acetylcysteine-treated Kunitz soybean trypsin inhibitors. Immunochemical studies using a competitive solid-phase enzyme immunoassay and two groups of sera revealed two patterns of antigenicity. Antibodies elicited with the denatured inhibitor were specific for the denatured conformation of the protein. In contrast, native inhibitor elicited antibodies that selectively recognized determinants in both native and heat-treated protein, but that did not bind trypsin inhibitors treated with N-acetylcysteine. These results imply that: (1) the disulfide bonds must be intact to maintain the native antigenic conformation and (2) the cysteine treatment may suppress allergic manifestations of soybean trypsin inhibitors and possibly other food proteins. These studies were extended by analyzing a panel of monoclonal antibodies prepared against native Kunitz trypsin inhibitor. The inhibitor has at least two distinct antigenic sites (epitopes), one of which is retained under denaturing conditions. The measurement of native Kunitz trypsin inhibitor in food samples by immunoassay appears practical. The relevance of these findings to food processing, food safety, and health is also discussed.


Celiac Disease Trypsin Inhibitor Assay Plate Food Protein Soybean Trypsin Inhibitor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aioti, F. and Paganelli, R. (1983). Food allergy and gastrointestinal diseases. Ann. Allergy. 51, 275–280.Google Scholar
  2. Allison, R. G. (ed.) (1982). “An Evaluation of the Potential for Dietary Protein to Contribute to Systemic Diseases”, FASEB, Rockville, MD, 1–53.Google Scholar
  3. Atassi, M.Z. (1985). Surface-simulation synthesis of the substrate-binding site of an enzyme. Demonstration with trypsin. Biochem. J., 226. 477–485.Google Scholar
  4. Barratt, M.E.J., Strachan, P. J., and Porter, P. (1978). Antibody mechanisms implicated in digestive disturbances following ingestion of soya protein in calves and piglets. Clin. Exp. Immunol., 31, 305–312.Google Scholar
  5. Barratt, M. E. J., Strachan, P. J., and Porter, P. (1979). Immunologically mediated nutritional disturbances associated with soya-protein antigens. Proc. Nutr. Soc., 38. 143–150.CrossRefGoogle Scholar
  6. Boekman, D. E. and Winborn, W. B. (1966). Light and electron microscopy of intestinal ferritin absorption. Observations in sensitized and non-sensitized hamsters. Anat. Rec. 155, 603–622.CrossRefGoogle Scholar
  7. Brandon, D. L. (1984). Interactions of diet and immunity. In: “Nutritional and Toxicological Aspects of Food Safety”, M. Friedman, ed., Plenum Press, New York, pp. 65–90.CrossRefGoogle Scholar
  8. Catsimpoolas, N. and Leuthner, E. (1969). Immunochemical methods for detection and quantitation of Kunitz soybean trypsin inhibitor. Anal. Biochem., 31. 437–447.CrossRefGoogle Scholar
  9. Catsimpoolas, N., Rogers, D. A., and Meyer, E. W. (1969). Immunochemical and disc electrophoresis study of soybean trypsin inhibitor SBTIA-2. Cereal Chem., 46, 136–144.Google Scholar
  10. Cole, S. G. and Kagnoff, M. F. (1985). Celiac disease. Ann. Rev. Nutr., 5, 241–266.CrossRefGoogle Scholar
  11. Concon, J. M., Newburg, D. S., and Eades, S. N. (1983). Lectins in wheat gluten proteins. J. Agric. Food Chem., 31, 939–941.CrossRefGoogle Scholar
  12. Cunningham-Rundles, C. (1983). Isolation and analysis of anti-idiotypic antibodies from IgA-deficient sera. Ann. N.Y. Acad. Sci., 409. 469–477.CrossRefGoogle Scholar
  13. Cunningham-Rundles, C., Brandeis, W. E., Good, R. A., and Day, N. K. (1978). Milk precipitins, circulating immune complexes and IgA deficiency. Adv. Exp. Med. Biol., 107, 523–530.CrossRefGoogle Scholar
  14. Doi, T., Kanatsu, K., Sekita, K., Yoshida, H., Nagai, H., and Hamashima, Y. (1984). Detection of IgA class circulating immune complexes bound to anti-C3d antibody in patients with IgA nephropathy. J. Immunol. Methods. 69, 95–104.CrossRefGoogle Scholar
  15. Emancipator, S. N. and Gallo, G. R. (1983). IgA-immune complex renal disease induced by mucosal immunization. Ann. N.Y. Acad. Sci., 409. 171–175.CrossRefGoogle Scholar
  16. Freed, R. C. and Ryan, D. S. (1978 a). Changes in Kunitz trypsin inhibitor during germination of soybeans: an immunoelectrophoresis assay system. J. Food Sci., 43, 1316–1319.CrossRefGoogle Scholar
  17. Freed, R. C. and Ryan, D. S. (1978 b). Note on modification of the Kunitz soybean trypsin inhibitor during seed germination. Cereal Chem., 55, 534–538.Google Scholar
  18. Friedman, M. and Gumbmann, M. R. (1986). Nutritional improvement of legumes through disulfide interchange. This volume.Google Scholar
  19. Friedman, M., Grosjean, O. K., and Zahnley, J. C. (1982). Inactivation of soya bean trypsin inhibitor by thiols. J. Sci. Food Agric. 33, 165–168.CrossRefGoogle Scholar
  20. Gallagher, P. J. and Gibney, M. J. (1983). Immunological aspects of atherosclerosis: Role of dietary protein, In: “Current Topics in Nutrition and Disease”, M. J. Gibney and D. Kritschevsky, eds., Alan R. Liss, New York, Vol 8, pp. 149–168.Google Scholar
  21. Haeney, M.R., Goodwin, B.J., Barratt, M.E.J., Mike, N., and Asquith, P. (1982). Soy protein antibodies in man: their occurrence and possible relevance in celiac disease. J. Clin. Pathol., 35, 319–322.CrossRefGoogle Scholar
  22. Heppell, L.M.J., Pederson, H.E., and Sissons, J.W. (1985). Potential allergenicity of soya-based infant formulas. Proceedings 13th Int. Congr. Nutr., Brighton, U.K., 190.Google Scholar
  23. Herion, P., Siberdt, D., Francotte, M., Urbain, J., and Bollen, A. (1984). Monoclonal antibodies against plasma protease inhibitors: II. Production and characterization of 25 monoclonal antibodies against human α1-antitrypsin. Correlation between antigenic structure and functional sites. Biosci. Rep., 4, 139–148.CrossRefGoogle Scholar
  24. Horisberger, M. and Tacchini-Vonlanthen, M. (1983 a). Ultrastructural localization of Kunitz inhibitors on thin sections of Glycine max (soybean) cv. Maple Arrow by the gold method. Histochemistry. 77, 37–50.CrossRefGoogle Scholar
  25. Horisberger, M. and Tacchini-Vonlanthen, M. (1983 b). Ultrastructural localization of Bowman-Birk inhibitor on thin sections of Glycine max (soybean) cv. Maple Arrow by the gold method. Histochemistry. 77, 313–321.CrossRefGoogle Scholar
  26. Kagnoff, M. F. (1981). Immunology of the digestive system. In: “Physiology of the Gastrontestinal Tract”, L.R. Johnson, ed., Raven Press, New York, 1337–1359.Google Scholar
  27. Kato, Y., Matsuda, T., Watanabe, K., and Nakamura, R. (1983). Immunochemical studies on the denaturation of ovalbumin stored with glucose. J. Food Sci., 48, 769–772.CrossRefGoogle Scholar
  28. McGhee, J. R. and Mestecky, J. (eds.) (1983)., “The Secretory Immune System”. Ann. N.Y. Acad. Sci., 409. 1–869.Google Scholar
  29. Moroz, L. A. and Yang, W. H. (1980). A specific allergen in food anaphylaxis. New Engl. J. Med., 302, 1126–1128.CrossRefGoogle Scholar
  30. Morton, J. I. and Deutsch, H. F. (1961). Immunochemical studies of modified ovomucoids. Arch. Biochem. Biophys., 93, 661–665.CrossRefGoogle Scholar
  31. Oi, V. T. and Herzenberg, L. A., (1980). Immunoglobulin-producing hybrid cell lines. In: “Selected Methods in Cellular Immunology”, B. Mishell and S. Shiigi, eds., W.H. Freeman, San Francisco, CA., 351–372.Google Scholar
  32. Peeters, H. (ed.) (1979). “Peptides of Biological Fluids”, Pergamon Press, Oxford, England, Vol. 26, 1–720.Google Scholar
  33. Rackis, J. J., Wolf, W. J., and Baker, E. C. (1986s). Protease inhibitors in plant foods: content and inactivation. This volumeGoogle Scholar
  34. Regnier, F. (1983). High-performance liquid chromatography of biopolymers. Science. 222, 245–252.CrossRefGoogle Scholar
  35. Rossebo, L. and Nordal, J. (1971). Soybean trypsin inhibitor: neutralization of its inhibitory effect upon the casein precipitating reaction of trypsin by rabbit antiserum. Z. Lebensmitt. Untersuch. Forsch., 147, 335–338.CrossRefGoogle Scholar
  36. Scott, H., Brandtzaeg, P., Thorsby, E., Baklien, K., Fausa, O., and Ek, J. (1983). Mucosal and systemic immune response patterns in celiac disease. Ann. Allergy, 51, 233–239.Google Scholar
  37. Seegraber, F. J. and Morrill, J. L. (1982). Effect of soy protein on calves’ intestinal absorptive ability and morphology determined by scanning electron microscopy. J. Dairy Sci., 65., 1962–1970.CrossRefGoogle Scholar
  38. Sissons, J. W., Smith, R. H., and Hewitt, D. (1982). Prediction of the suitability of soya-bean products for feeding to preruminant calves by an in vitro immunochemical method. Brit. J. Nutr., 47., 311–318.CrossRefGoogle Scholar
  39. Srihara, P. (1984). Processing to reduce the antigenicity of soybean products for preruminant calf diets. Dissertation, University of Guelph, Guelph, Ontario, 1–141.Google Scholar
  40. Streicher, H. Z., Berkower, I. J., Busch, M., Gurd, F.R.N., and Berzofsky, J. A. (1984). Antigen conformation determines processing requirements for T cell activation. Proc. Natl. Acad. Sci. USA. 81, 6831–6835.CrossRefGoogle Scholar
  41. Toms, G. C. (1981). Lectins in Leguminosae. In; “Advances in Legume Systematics,” R. M. Polhill and P. H. Raven, eds., Royal Botanic Gardens, Kew, 561–577.Google Scholar
  42. Van der Woude, F. J., Hoedemaeker, P. J., van der Giessen, M., de Graeff, P. A., de Monchy, J, The, T. H., and van der Hem, G. K. (1983). Do food antigens play a role in the pathogenesis of some cases of human glomerulonephritis? Clin. Exp. Immunol., 51, 587–594.Google Scholar
  43. Walker, W. A. and Bloch, K. J. (1983). Intestinal uptake of macromolecules: In. vitro and in vivo studies. Ann. N.Y. Acad. Sci., 409. 593–601.CrossRefGoogle Scholar
  44. Wofsy, L. (1983). Methods and applications of hapten-sandwich labeling. Meth. Enzymol., 92, 471–488.Google Scholar

Copyright information

© Plenum Press, New York 1986

Authors and Affiliations

  • David L. Brandon
    • 1
  • Sakhina Haque
    • 1
  • Mendel Friedman
    • 1
  1. 1.Western Regional Research Center, Agricultural Research ServiceU.S. Department of AgricultureBerkeleyUSA

Personalised recommendations