Date of Award

1987

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Biological Sciences

Keywords

Biology, Microbiology.

Rights

info:eu-repo/semantics/openAccess

Abstract

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for measuring Staphylococcus aureus alpha toxin. This assay was 500 to 1,000 times more sensitive than the commonly used hemolytic titration assay, and was less variable. The binding of alpha toxin to the adsorbed antibody was most effective after an overnight incubation at 27 C. The toxin was detectable even at a log$\sb2$ 17 dilution of an S. aureus culture supernatant. A modified version of the ELISA, a competitive enzyme- linked immunosorbent assay (CELIA) was developed for detection of immunologically active fragments of alpha toxin. In the CELIA, immunologically reactive fragments bound to the adsorbed antitoxin. However, since these fragments were unable to simultaneously bind the second (enzyme- labelled) antibody, a decrease in absorbance was noted in these wells. Fragmentation analysis of alpha toxin with trypsin, under a variety of conditions, produced a variety of fragments which were recognized by either antibinding, or indirect hemagglutinating antibodies. However, the most striking feature of these results, was a 20,000 Dalton fragment that was recognized by both populations of antibodies. This fragment appeared early in the digestion course and was resistant to further tryptic digestion. A similarly sized fragment also spontaneously appeared in purified alpha toxin preparations. Five monoclonal antibodies (MAb-1 to MAb-5) to alpha toxin were developed. However, when the relative specificities of these antibodies were examined by ELISA, MAb-1 and MAb-2 recognized either the same determinant, or, two determinants that are close together. Monoclonal antibodies 1, 3, 4, and 5, recognized different antigenic determinants. All the monoclonal antibodies protected rabbit erythrocytes from alpha toxin- mediated lysis. When examined in the IHA test MAbs-1, 2, 4, and 5 agglutinated toxoid- coated erythrocytes. This indicated that these antibodies recognized determinants that were outside of the erythrocyte receptor binding site of alpha toxin. Monoclonal antibody- 3, an antibinding antibody, also did not bind to the 9,000 Dalton fragment that contains this binding site. Monoclonal antibody- 3 therefore recognizes an antigenic determinant that is outside of the binding site, but, is still close enough so that the bound antibody molecule sterically hinders simultaneous binding to the receptor. (Abstract shortened with permission of author.)Dept. of Biological Sciences. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1987 .S979. Source: Dissertation Abstracts International, Volume: 48-11, Section: B, page: 3202. Thesis (Ph.D.)--University of Windsor (Canada), 1987.

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