The develolpment of a rapid diagnostic test for the detection of haemophilus ducrey.

Abstract

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.