The production and characterisation of rabbit antiserum and murine monoclonal antibodies to Haemophilus ducreyi

Abstract

Summary. Rabbit antiserum and murine monoclonal antibodies were raised against a strain of Haemophilus ducreyi. The antiserum gave high immunofluorescence titres and strong dot blot reactions with all H. ducreyi strains tested and the only cross reaction was with Bordetella pertussis. Three monoclonal antibodies, all of isotype IgG2a, also gave high immunofluorescence titres with H . ducreyi but did not cross react with any other species tested. Immunoblotting showed the monoclonal antibodies to react with a single polypeptide band of mol. wt 29000 in the outermembrane fraction of H . ducreyi. These antibodies have potential for use as diagnostic reagents and for investigating the pathogenicity of H. ducreyi. Introdnc tionHaemphilus ducreyi is the causative organism of the sexually transmitted disease chancroid, the commonest cause of genital ulcers in the tropics. However, in recent years there have been increasing numbers of outbreaks in the more temperate regions of Europe and North America. 92 H . ducreyi is a fastidious organism and, despite improved culture methods, isolation rates from genital ulcers and buboes can still be lower than 60%. In many countries where facilities for culture are often not available, diagnosis of chancroid continues to be based on clinical features and the exclusion of other causes of genital ulcers. This form of diagnosis is prone to e r r~r .~ Direct microscopy of gram-stained smears is also insensitive; the typical "shoal of fish" morphology of H . ducreyi is often not seen, and other contaminating gram-negative bacilli may look ~i m i l a r .~?~To overcome these problems, several groups of workers have attempted to develop immunological methods of diagnosis. Denys et al. ' raised an antiserum for use in an immunofluorescence assay for H. ducreyi. This antiserum, however, had to be absorbed extensively with other Haemophilus spp. to eliminate cross-reactivity. Even after absorption it gave a moderate fluorescence with several other Haemophilus spp., in particular H . parainfluenzae. In 1984, Hansen and Loftus6 raised monoclonal

Use of electroporation to construct isogenic mutants of Haemophilus ducreyi

“…While this particular approach is limited somewhat by the necessity of first having to clone the H. ducreyi gene encoding the antigen of interest, the existence of a large number of MAbs reactive with different surface antigens of this pathogen (11,15,28,32) indicates that antibody probes that will facilitate such molecular cloning efforts are now available. More importantly, it should now be possible to test any putative H. ducreyi virulence determinant if probes for the determinant are available.…”

Chancroid and Haemophilus ducreyi: an update.

“…It is also possible that the nature of the antigen or the immunization protocol may quantitatively or qualitatively affect the production of antibody to H. ducreyi. Finn et al (58) prepared polyclonal antisera to H. ducreyi in rabbits by an immunization protocol, which involved subcutaneous inoculation of washed, live bacteria together with Freund's incomplete adjuvant, followed 2 weeks later by an i.m. injection of washed bacteria and then three daily intravenous inoculations of washed bacteria.…”

Characterization of a novel lipoprotein expressed by Haemophilus ducreyi

“…In Western blot (immunoblot) analyses, the absorbed rabbit antiserum binds to a 28K or 28.5K antigen in each of 450 isolates of H. ducreyi tested (25). A monoclonal antibody (MAb) also binds to either the 28K or 28.5K antigen in all 450 isolates, indicating that these proteins are antigenically related (8,25). Taken together, the data suggest that the 28K and 28.5K proteins may be useful as a serodiagnostic reagent for chancroid or as a target antigen for detection of H. ducreyi.…”