Abstract
The measurement of chicken and human antibodies to Clostridium botulinum neurotoxins A, B, and E was accomplished by affinity isolation of complexes containing these antibodies. By this approach, a mixture of toxin with the test antibody, fluoresceinated antibody, and enzyme (Russell's viper venom factor X activator)-labeled antibody is allowed to form a complex in solution phase. This complex is then bound to a matrix containing antifluorescein antibody. All components not bound to the matrix are washed off, and the complex is isolated intact by elution with fluorescein, which competes with the complex for binding to the antifluorescein matrix. The eluted complex is then bound to a matrix which specifically binds the test antibody (anti-chicken immunoglobulin Y [IgY] or anti-human IgG), and the bound complex is measured by using the enzyme label. Using this approach, we were able to measure as little as 1 ng of specific antibody per ml from affinity-isolated, monospecific chicken antibody preparations and to measure antibody specifically from IgY fractions of monospecific chicken antibody preparations. Human antibodies from subjects immunized with pentavalent toxoid preparations were detectable at dilutions as great as 24,300-fold, and undiluted serum from most control subjects showed no measurable antibody. Antibody was also measured in 65 subjects who were receiving preparations of neurotoxin A (BOTOX) for the treatment of spastic disorders. Eighteen of them had toxin-specific antibody reactive with toxin B, and two of them had toxin-specific antibody reactive with toxin A. The two patients having antibody to toxin A were refractory to treatment with this toxin. This approach of isolation of hapten-labeled immune complexes under nondenaturing conditions with hapten is broadly applicable to the specific measurement of antibodies present at very low concentrations in serum.
Full Text
The Full Text of this article is available as a PDF (99.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Doellgast G. J., Beard G. A., Bottoms J. D., Cheng T., Roh B. H., Roman M. G., Hall P. A., Triscott M. X. Enzyme-linked immunosorbent assay and enzyme-linked coagulation assay for detection of Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes with dual-label antibodies. J Clin Microbiol. 1994 Jan;32(1):105–111. doi: 10.1128/jcm.32.1.105-111.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Doellgast G. J., Triscott M. X., Beard G. A., Bottoms J. D., Cheng T., Roh B. H., Roman M. G., Hall P. A., Brown J. E. Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay. J Clin Microbiol. 1993 Sep;31(9):2402–2409. doi: 10.1128/jcm.31.9.2402-2409.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Doellgast G. J., Triscott M. X., Beard G. A., Bottoms J. D. Enzyme-linked immunosorbent assay-enzyme-linked coagulation assay for detection of antibodies to Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes. J Clin Microbiol. 1994 Mar;32(3):851–853. doi: 10.1128/jcm.32.3.851-853.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Epperson L. W. Therapeutic uses of botulinum toxin (Botox). Ala Med. 1995 Nov;65(5-7):49–50. [PubMed] [Google Scholar]
- Giladi N., Meer J., Kidan C., Greenberg E., Gross B., Honigman S. Interventional neurology: botulinum toxin as a potent symptomatic treatment in neurology. Isr J Med Sci. 1994 Nov;30(11):816–819. [PubMed] [Google Scholar]
- Greene P. E., Fahn S. Use of botulinum toxin type F injections to treat torticollis in patients with immunity to botulinum toxin type A. Mov Disord. 1993 Oct;8(4):479–483. doi: 10.1002/mds.870080411. [DOI] [PubMed] [Google Scholar]
- Habermann E., Bernáth S. Preparation, measurement and possible use of human antitoxin against Cl. botulinum A, B, and E toxins. Med Microbiol Immunol. 1975 Jul 2;161(3):203–210. doi: 10.1007/BF02121011. [DOI] [PubMed] [Google Scholar]
- Schwarz P. J., Arnon S. S. Botulism immune globulin for infant botulism arrives--one year and a Gulf War later. West J Med. 1992 Feb;156(2):197–198. [PMC free article] [PubMed] [Google Scholar]
- Shone C., Appleton N., Wilton-Smith P., Hambleton P., Modi N., Gatley S., Melling J. In vitro assays for botulinum toxin and antitoxins. Dev Biol Stand. 1986;64:141–145. [PubMed] [Google Scholar]
- Siegel L. S. Human immune response to botulinum pentavalent (ABCDE) toxoid determined by a neutralization test and by an enzyme-linked immunosorbent assay. J Clin Microbiol. 1988 Nov;26(11):2351–2356. doi: 10.1128/jcm.26.11.2351-2356.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Voss E. W., Jr, Eschenfeldt W., Root R. T. Fluorescein: a complete antigenic group. Immunochemistry. 1976 May;13(5):447–453. doi: 10.1016/0019-2791(76)90382-7. [DOI] [PubMed] [Google Scholar]
- Voss E. W., Jr Kinetic measurements of molecular interactions by spectrofluorometry. J Mol Recognit. 1993 Jun;6(2):51–58. doi: 10.1002/jmr.300060203. [DOI] [PubMed] [Google Scholar]