Aim: To develop an antigen detection test that would quickly exclude H. ducreyi infection in
individuals with genital ulcers.
Materials and Methods: H. ducreyi strains A54 and A68 were grown on Modified Bieling
(MB) agar plates and in MB broth under microaerophilic conditions. The 58.5 kDa GroEL
Heat Shock Protein (HSP) was extracted from H. ducreyi strain A54 by means of sonication.
The purified HSP was used to raise antibodies in rabbits. HSP determination and separation
was done on SDS PAGE gels and protein was eluted by means of a passive elution process.
Antibody was purified by affinity chromatography and a fraction of the antibody was
conjugated to a chromogen to be used as a detection antibody. An ELISA was developed to
evaluate the antibody response to the HSP. A second ELISA was developed to evaluate test
parameters.
Results: A good immune response was achieved with the crude serum of one of the three
rabbits when tested against the antigen by means of ELISA. However, after purification of the
IgG from the serum of the same rabbit no antigen-antibody binding was observed. Anti-rabbit
IgG was able to recognise the antibodies.
Discussion and Conclusion: While the Fc portion of the purified IgG remained active, the
Fab portion of the antibody had lost biological activity. This loss of biological activity of
antibody can be attributed to the low pH of the elution buffers used during the purification
steps. Alternative antibody purification systems need to be explored. The use of monoclonal
antibodies also needs to be considered. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2010.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9565 |
| Date | January 2010 |
| Creators | Pillay, Mona. |
| Contributors | Sturm, A. Willem. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0025 seconds