Development of an Avian Antitoxin to Type A Botulinum Neurotoxin

  • B. S. Thalley
  • M. B. van Boldrik
  • S. B. Carroll
  • W. Tepp
  • B. R. DasGupta
  • D. C. Stafford


Most commercially available antitoxins and antivenoms are raised in horses and purified by bulk fractionation techniques. These preparations frequently elicit deleterious side effects that compromise their efficacy and the treatment of intoxication or envenomation. Alternatively, the use of hyperimmune immunoglobulin from the egg yolks of laying hens has shown considerable promise as a cost-effective and potentially safer source of antitoxins and antivenoms. To investigate the utility of this system in the development of a botulism antitoxin, laying hens were immunized with purified 150 kDa type A botulinum neurotoxin detoxified with formaldehyde. Antibodies present in egg yolk were then isolated by polyethylene glycol (PEG) fractionation and further purified by affinity chromatography using immobilized toxoid. These antibodies exhibited a high titer of toxoid reactivity as measured by enzyme immunoassay. A mouse lethality/protection assay demonstrated the efficacy of these antibodies in neutralizing type A botulinum neurotoxin, where PEG-fractionated and affinity-purified antibodies neutralized 38 and 340 I.U. of type A botulinum neurotoxin per milligram of protein, respectively. Thus, the avian antibody preparations, even if only PEG-fractionated, are significantly more potent (neutralization titer per mg protein) than conventional equine botulism antiserum preparations. In addition to their superior potency, hyperimmune avian antibodies, whether PEG-fractionated or affinity-purified, are expected to significantly increase antitoxin safety.


Botulinum Neurotoxin Serum Sickness Neutralization Titer Infant Botulism Connaught Laboratory 
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  1. 1.
    Hatheway CL. Toxigenic clostridia. Clinical Microbiol Review 1990; 3: 66–98.Google Scholar
  2. 2.
    Middlebrook JL, Dorland RB. Bacterial toxins: Cellular mechanisms of action. Microbiol Rev 1984; 48: 199–221.PubMedGoogle Scholar
  3. 3.
    World Health Organization. Progress in the characterization of venoms and standardization of antivenoms. WHO Offset Publication No. 58. Geneva, 1981.Google Scholar
  4. 4.
    Botulism Antitoxin, Trivalent (Equine) Types A, B, and E; Connaught Laboratories package insert.Google Scholar
  5. 5.
    Frankovich TL, Amon SS. Clinical trial of botulism immune globulin for infant botulism. West J Med 1991; 154: 103.PubMedGoogle Scholar
  6. 6.
    Schwartz PJ, Amon SS. 1992. Botulism immune globulin for infant botulism arrives–one year and a Gulf War later. West J Med 1992; 156: 197–198.Google Scholar
  7. 7.
    Tacket CO, Shandera WC, Mann J, Hargrett NT, Blake PA. Equine antitoxin use and other factors that predict outcome in type A foodbome botulism. Am J Med 1984; 76: 794–798.PubMedCrossRefGoogle Scholar
  8. 8.
    Merson MH, Hughes JR, Dowell VR, Taylor A, Barker WH, Gangarosa EJ. Current trends in botulism in the United States. J Am Med Assoc 1974; 229: 1305–1308.CrossRefGoogle Scholar
  9. 9.
    Black RE, Gunn RA. Hypersensitivity reactions associated with botulinal antitoxin. Am J Med 1980; 69: 567–570.PubMedCrossRefGoogle Scholar
  10. 10.
    Carroll SB. Antivenoms and methods for making antivenoms. U.S. Patent Application (pending ) 1989.Google Scholar
  11. 11.
    Thalley BS, Carroll SB. Rattlesnake and scorpion antivenoms from the egg yolks of immunized hens. Biotechnology 1990; 8: 934–938.PubMedCrossRefGoogle Scholar
  12. 12.
    DasGupta BR, Sathyamoorthy V. Purification and amino acid composition of type A botulinum neurotoxin. Toxicon 1984; 22: 415–424.PubMedCrossRefGoogle Scholar
  13. 13.
    Singh BR, DasGupta BR. Molecular differences between type A botulinum neurotoxin and its toxoid. Toxicon 1989; 27: 403–410.PubMedCrossRefGoogle Scholar
  14. 14.
    Poison A, von Wechmar MB, van Regenmortel MHV. Isolation of viral IgY antibodies from yolks of immunized hens. Immunol Comm 1980; 9: 475–493.Google Scholar
  15. 15.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265–275.PubMedGoogle Scholar
  16. 16.
    Cardella MA. Botulinum toxoids. In: Lewis KH, Cassel K Jr, eds. Botulism: Proceedings of a symposium PHS Publication No. 999-FPI, Washington, D.C.: Government Printing Office 1964: 113–130.Google Scholar
  17. 17.
    Reed Li, Muench H. A simple method of estimating 50% endpoints. Amer J Hyg 1938; 27: 493–497.Google Scholar
  18. 18.
    The United States Pharmacopeia, Twenty-second Revision. (United States Phannacopeial convention, Inc. Rockville, MD.) 16th edition, 1990: 186.Google Scholar
  19. 19.
    Benson HN, Brumfield HP, Pomeroy BS. Requirement of avian Cl for fixation of guinea pig complement by avian antibody-antigen complexes. J Immunol 1961; 87: 616–622.PubMedGoogle Scholar
  20. 20.
    Rose ME, Orlans E. Fowl antibody IV. The estimation of hemolytic fowl complement. Immunology 1962; 5: 642–648.PubMedGoogle Scholar
  21. 21.
    Sutherland SK. Serum reactions: An analysis of commercial antivenoms and the possible role of anticomplementary activity in de-novo reactions to antivenoms and antitoxins. Med J Aust 1977; 1: 613–615.PubMedGoogle Scholar
  22. 22.
    Barandun S, Kistler P, Jeunet F, Isliker H. Intravenous administration of human gamma-globulin. Vox Sang 1962; 7: 157.PubMedCrossRefGoogle Scholar
  23. 23.
    Jurkovich GJ, Luterman A, McCullar K, Ramenofsky ML, Curren PW. 1988. Complications of Crotalidae antivenin therapy. J Trauma 1988; 28 (7): 1032–1037.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1993

Authors and Affiliations

  • B. S. Thalley
    • 1
  • M. B. van Boldrik
    • 1
  • S. B. Carroll
    • 2
  • W. Tepp
    • 3
  • B. R. DasGupta
    • 3
  • D. C. Stafford
    • 1
  1. 1.Ophidian Pharmaceuticals, Inc.USA
  2. 2.Howard Hughes Medical Institute and University of Wisconsin-MadisonUSA
  3. 3.Food Microbiology and ToxicologyUniversity Of Wisconsin-MadisonUSA

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