Characterization of antibodies against Helicobacter pylori

Sammanfattning: H. pylori is associated with the development of peptic ulcer, gastritis, and gastric cancer. Eradication of H. pylori by antibiotic treatment gives a healing of peptic ulcer and gastritis. However, due to drug resistance, there is a great need to explore alternative ways to eliminate bacterial infections.The goals of the present study were to characterize MAbs against surface components of H. pylori, and to investigate the functions of the bacterial surface components.Murine MAbs against surface components of H. pylori were produced by immunization of mice followed by hybridoma formation. One of the MAbs of the IgG1 subclass, was specific for both the spiral and coccoid forms of H. pylori. It reacted with a 28 kDa protein that was present in all the 5 strains of H. pylori tested. The MAb based indirect immunofluorescence microscopy on formalinfixed antral and corpus biopsy specimens from H. pylori associated gastritis patients showed that 9 of 9 antral and 5 of 6 corpus specimens harbored the coccoid form of H. pylori.An ScFv-phage which was derived from an M13 phage and mRNA of hybridomas secreting H. pylori MAbs, reacted with a 30 kDa H. pylori surface antigen. By means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro the recombinant phage exhibited a bacteriocidal effect. When H. pylori was pretreated with the phage before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced. The parental MAbs were of the IgG1 subclass. The antigen was identified as a urease associated 30 kDa protein. The MAb decreased viability of the spiral form in broth culture, and reduced intracellular ATP in both spiral and coccoid forms of H. pylori.One MAb, 5D6 of the IgG2a subclass, was specific to a 56 kDa H. pylori protein. Immunofluorescence microscopy showed that this protein was located on the surface of both the spiral and coccoid forms of H. pylori. The MAb also bound to cells of the basal third of the oxyntic mucosa of rat stomachs and these cells were identified as gastric ECL cells. Sera of H. pylori-positive patients were investigated for ECL cell (auto)antibodies by means of ELISA using purified rat ECL cells as antigen. Ten (25%) of 40 sera from patients with atrophic corpus predominant gastritis scored positive; 2 (8%) of 26 sera from the patients with duodenal ulcer scored positive; only 1 serum (6%) from 16 healthy subjects was slightly positive. Four positive clones were obtained from a DNA hybridization of H. pylori and human gastric mucosa.The present results show that the MAb based immunochemistry provides a rapid and specific detection of both the spiral and coccoid forms. Binding of a urease associated 30 kDa protein by an ScFv-phage, or by its parental MAbs, decreased the viability of H. pylori. The protein may be considered as a candidate for a future vaccine. An antigenic mimicry which was found in surface of H. pylori and gastric ECL cells suggests a pathogenic role in gastritis.