Antigenic diversity in Haemophilus ducreyi as shown by western blot (immunoblot) analysis

Abstract

The antigenic diversity within a panel of 63 Haemophilus ducreyi isolates was examined by Western blot (immunoblot) analysis with a pool of 238 well-characterized human antisera. When a serum pool adsorbed on a mixture of Haemophilus influenzae, H. parainfluenzae, and H. parahaemolyticus was used, the immunoprotiles suggested that prominent antigenic proteins involved in the human immune response have apparent molecular masses of 63, 42, 34 to 30, and 28.5 to 28 kDa. Preliminary subcellular localization revealed that these antigens are associated with the cellular membrane. Two subsets of antigens were discriminated by detergent extraction. There was no evidence that the antigen composition is altered by changing the growth conditions. With a serum pool adsorbed on the Haemophilus spp. mixture supplemented with Actinobacillus actinomycetemcomitans, Pasteurella ureae, Neisseria gonorrhoeae, and Escherichia coli, antigenic determinants more specific for H. ducreyi were identified. An immunodominant 28.5to 28-kDa protein was expressed by all H. ducreyi isolates. In the range from 34 to 30 kDa, 56 isolates revealed a dominant protein with variable molecular mass. By using both proteins (28.5 to 28 kDa and 34 to 30 kDa) as immunotypic markers, seven different immunopatterns were identified. Antigenic diversity among isolates from different geographical origins as well as from a single area was observed.Haemophilus ducreyi is a poorly known and fastidious microorganism causing chancroid, a sexually transmitted disease (STD) that is characterized by genital ulcers and by abcedation of the inguinal lymph nodes (6). Recently, chancroid has received more interest because it is emerging as the major cause of genital ulcer disease in several parts of the developing world, and it is reappearing in poor minority populations in Europe and the United States (10,14,16). In addition, several reports suggest that genital ulcer disease, in particular chancroid, is a risk factor for the sexual transmission of the human immunodeficiency virus (9). Little is known about the antigenic composition of H. ducreyi, and the nature of the immune response upon infection has been poorly studied. Serum immunoglobulin G (IgG) and IgM antibodies have been detected in patients with a clinical diagnosis of chancroid both by dot immunobinding and by an enzyme immunoassay (8,13). Western blot analysis of sera from normal and immunized rabbits has shown that rabbits immunized with H. ducreyi respond with a humoral immune response in which multiple antigenic polypeptides are detected. The most prominent antigens are reported to have apparent molecular masses of 79, 62, 55, 49, and 26 kDa (7) and of 67, 42, 22.5, and 20 kDa (12). However, the specificity of the antigens towards H. ducreyi has not been demonstrated.In the present study, the antigenic diversity occurring within a panel of H. ducreyi isolates was examined by Western blot (immunoblot) analysis. Prominent proteins involved in the human immune response and H. ducreyispecific antigens were iden...

Antigen detection and immunological typing of Haemophilus ducreyi with a specific rabbit polyclonal serum

“…Immunological typing of H. ducreyi with the adsorbed rabbit serum. The antigens reacting with the adsorbed polyclonal rabbit serum were identified by Western blot analysis and were compared with those reacting with an adsorbed pool of well-characterized sera from patients with cultureproven chancroid (16). Figure 1 compares the different immunoprofiles that were observed with either serum among 450 H. ducreyi isolates.…”

“…Before each injection as well as at week 10, 1 ml of venous blood was collected and the titer of serum antibodies reacting with crude H. ducreyi cocktail antigen was determined. The titer was defined to be the inversion of the highest dilution positive by ELISA (11) and Western blot (16). At the third week, this titer leveled off around 50,000.…”

“…Western blot analysis. Western blot analysis was as described previously (16). The rabbit serum dilution was 1/20,000, and bound antibody was detected with a 1/5,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (GIBCO).…”

“…In spite of the low specificity * Corresponding author. observed with the anti-29-kDa MAb, antibodies raised against the 29-kDa antigen seem to be most suitable, as a 29-kDa species-specific antigen was identified recently by Western blot (immunoblot) analysis of H. ducreyi isolates with a pool of well-defined human antisera (16).…”

“…Crude antigen for enzyme-linked immunosorbent assay (ELISA) was obtained by diluting the lysate with distilled water to a protein concentration of 10 mg/ml. Sodium dodecyl sulfate (SDS) (GIBCO) was added to a final concentration of 1% (wt/vol) (16). The protein content was determined as described by Bradford (1).…”