• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of iaiPermissionsJournals.ASM.orgJournalIAI ArticleJournal InfoAuthorsReviewers
Infect Immun. Sep 1997; 65(9): 3743–3752.
PMCID: PMC175534

Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries.


To produce antibodies capable of neutralizing botulinum neurotoxin type A (BoNT/A), the murine humoral immune response to BoNT/A binding domain (H(C)) was characterized at the molecular level by using phage antibody libraries. Mice were immunized with BoNT/A H(C), the spleens were harvested, and single-chain Fv (scFv) phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes. Phage expressing BoNT/A binding scFv were isolated by selection on immobilized BoNT/A and BoNT/A H(C). Twenty-eight unique BoNT/A H(C) binding scFv were identified by enzyme-linked immunosorbent assay and DNA sequencing. Epitope mapping using surface plasmon resonance in a BIAcore revealed that the 28 scFv bound to only 4 nonoverlapping epitopes with equilibrium constants (Kd) ranging from 7.3 x 10(-8) to 1.1 x 10(-9) M. In a mouse hemidiaphragm assay, scFv binding epitopes 1 and 2 significantly prolonged the time to neuroparalysis, 1.5- and 2.7-fold, respectively, compared to toxin control. scFv binding to epitopes 3 and 4 showed no protection against neuroparalysis. A combination of scFv binding epitopes 1 and 2 had an additive effect on time to neuroparalysis, which increased to 3.9-fold compared to the control. The results suggest that there are two "productive" receptor binding sites on H(C) which lead to toxin internalization and toxicity. Blockade of these two epitopes with monoclonal antibodies may provide effective immunoprophylaxis or therapy against BoNT/A intoxication.

Full Text

The Full Text of this article is available as a PDF (199K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Atassi MZ, Dolimbek BZ, Hayakari M, Middlebrook JL, Whitney B, Oshima M. Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855-1296 with antitoxin antibodies from three host species. J Protein Chem. 1996 Oct;15(7):691–700. [PubMed]
  • Black JD, Dolly JO. Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. I. Ultrastructural autoradiographic localization and quantitation of distinct membrane acceptors for types A and B on motor nerves. J Cell Biol. 1986 Aug;103(2):521–534. [PMC free article] [PubMed]
  • Black RE, Gunn RA. Hypersensitivity reactions associated with botulinal antitoxin. Am J Med. 1980 Oct;69(4):567–570. [PubMed]
  • Breitling F, Dübel S, Seehaus T, Klewinghaus I, Little M. A surface expression vector for antibody screening. Gene. 1991 Aug 15;104(2):147–153. [PubMed]
  • Cathala G, Savouret JF, Mendez B, West BL, Karin M, Martial JA, Baxter JD. A method for isolation of intact, translationally active ribonucleic acid. DNA. 1983;2(4):329–335. [PubMed]
  • Chen F, Kuziemko GM, Amersdorfer P, Wong C, Marks JD, Stevens RC. Antibody mapping to domains of botulinum neurotoxin serotype A in the complexed and uncomplexed forms. Infect Immun. 1997 May;65(5):1626–1630. [PMC free article] [PubMed]
  • Chothia C, Lesk AM. Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol. 1987 Aug 20;196(4):901–917. [PubMed]
  • Clackson T, Hoogenboom HR, Griffiths AD, Winter G. Making antibody fragments using phage display libraries. Nature. 1991 Aug 15;352(6336):624–628. [PubMed]
  • Clayton MA, Clayton JM, Brown DR, Middlebrook JL. Protective vaccination with a recombinant fragment of Clostridium botulinum neurotoxin serotype A expressed from a synthetic gene in Escherichia coli. Infect Immun. 1995 Jul;63(7):2738–2742. [PMC free article] [PubMed]
  • Daniels-Holgate PU, Dolly JO. Productive and non-productive binding of botulinum neurotoxin A to motor nerve endings are distinguished by its heavy chain. J Neurosci Res. 1996 May 1;44(3):263–271. [PubMed]
  • De Bellis D, Schwartz I. Regulated expression of foreign genes fused to lac: control by glucose levels in growth medium. Nucleic Acids Res. 1990 Mar 11;18(5):1311–1311. [PMC free article] [PubMed]
  • Deshpande SS, Sheridan RE, Adler M. A study of zinc-dependent metalloendopeptidase inhibitors as pharmacological antagonists in botulinum neurotoxin poisoning. Toxicon. 1995 Apr;33(4):551–557. [PubMed]
  • Dowell VR., Jr Botulism and tetanus: selected epidemiologic and microbiologic aspects. Rev Infect Dis. 1984 Mar-Apr;6 (Suppl 1):S202–S207. [PubMed]
  • Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. [PMC free article] [PubMed]
  • Emanuel P, O'Brien T, Burans J, DasGupta BR, Valdes JJ, Eldefrawi M. Directing antigen specificity towards botulinum neurotoxin with combinatorial phage display libraries. J Immunol Methods. 1996 Jun 21;193(2):189–197. [PubMed]
  • Foote J, Milstein C. Kinetic maturation of an immune response. Nature. 1991 Aug 8;352(6335):530–532. [PubMed]
  • Güssow D, Clackson T. Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res. 1989 May 25;17(10):4000–4000. [PMC free article] [PubMed]
  • Hatheway CH, Snyder JD, Seals JE, Edell TA, Lewis GE., Jr Antitoxin levels in botulism patients treated with trivalent equine botulism antitoxin to toxin types A, B, and E. J Infect Dis. 1984 Sep;150(3):407–412. [PubMed]
  • Hawkins RE, Russell SJ, Winter G. Selection of phage antibodies by binding affinity. Mimicking affinity maturation. J Mol Biol. 1992 Aug 5;226(3):889–896. [PubMed]
  • Hoogenboom HR, Griffiths AD, Johnson KS, Chiswell DJ, Hudson P, Winter G. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 1991 Aug 11;19(15):4133–4137. [PMC free article] [PubMed]
  • Huston JS, George AJ, Adams GP, Stafford WF, Jamar F, Tai MS, McCartney JE, Oppermann H, Heelan BT, Peters AM, et al. Single-chain Fv radioimmunotargeting. Q J Nucl Med. 1996 Sep;40(3):320–333. [PubMed]
  • Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotný J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R, et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879–5883. [PMC free article] [PubMed]
  • Johnsson B, Löfås S, Lindquist G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal Biochem. 1991 Nov 1;198(2):268–277. [PubMed]
  • Karlsson R, Michaelsson A, Mattsson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immunol Methods. 1991 Dec 15;145(1-2):229–240. [PubMed]
  • Lang AB, Cryz SJ, Jr, Schürch U, Ganss MT, Bruderer U. Immunotherapy with human monoclonal antibodies. Fragment A specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin. J Immunol. 1993 Jul 1;151(1):466–472. [PubMed]
  • LaPenotiere HF, Clayton MA, Middlebrook JL. Expression of a large, nontoxic fragment of botulinum neurotoxin serotype A and its use as an immunogen. Toxicon. 1995 Oct;33(10):1383–1386. [PubMed]
  • Marks C, Marks JD. Phage libraries--a new route to clinically useful antibodies. N Engl J Med. 1996 Sep 5;335(10):730–733. [PubMed]
  • Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 1991 Dec 5;222(3):581–597. [PubMed]
  • Marks JD, Tristem M, Karpas A, Winter G. Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes. Eur J Immunol. 1991 Apr;21(4):985–991. [PubMed]
  • McCafferty J, Griffiths AD, Winter G, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990 Dec 6;348(6301):552–554. [PubMed]
  • Middlebrook JL, Brown JE. Immunodiagnosis and immunotherapy of tetanus and botulinum neurotoxins. Curr Top Microbiol Immunol. 1995;195:89–122. [PubMed]
  • Munro S, Pelham HR. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. [PubMed]
  • Schier R, Balint RF, McCall A, Apell G, Larrick JW, Marks JD. Identification of functional and structural amino-acid residues by parsimonious mutagenesis. Gene. 1996 Mar 9;169(2):147–155. [PubMed]
  • Schier R, Bye J, Apell G, McCall A, Adams GP, Malmqvist M, Weiner LM, Marks JD. Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection. J Mol Biol. 1996 Jan 12;255(1):28–43. [PubMed]
  • Schier R, Marks JD, Wolf EJ, Apell G, Wong C, McCartney JE, Bookman MA, Huston JS, Houston LL, Weiner LM, et al. In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library. Immunotechnology. 1995 May;1(1):73–81. [PubMed]
  • Schier R, McCall A, Adams GP, Marshall KW, Merritt H, Yim M, Crawford RS, Weiner LM, Marks C, Marks JD. Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J Mol Biol. 1996 Nov 8;263(4):551–567. [PubMed]
  • Tacket CO, Shandera WX, Mann JM, Hargrett NT, Blake PA. Equine antitoxin use and other factors that predict outcome in type A foodborne botulism. Am J Med. 1984 May;76(5):794–798. [PubMed]
  • Tomlinson IM, Walter G, Jones PT, Dear PH, Sonnhammer EL, Winter G. The imprint of somatic hypermutation on the repertoire of human germline V genes. J Mol Biol. 1996 Mar 15;256(5):813–817. [PubMed]
  • Weber JT, Goodpasture HC, Alexander H, Werner SB, Hatheway CL, Tauxe RV. Wound botulism in a patient with a tooth abscess: case report and review. Clin Infect Dis. 1993 May;16(5):635–639. [PubMed]
  • Williams RS, Tse CK, Dolly JO, Hambleton P, Melling J. Radioiodination of botulinum neurotoxin type A with retention of biological activity and its binding to brain synaptosomes. Eur J Biochem. 1983 Mar 15;131(2):437–445. [PubMed]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • MedGen
    Related information in MedGen
  • Nucleotide
    Published Nucleotide sequences
  • Protein
    Published protein sequences
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...