SummaryHelicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value.

Preclinical mouse infection models are widely used for Helicobacter vaccine development, but how well such models mimic important aspects of human infections is unknown. A comparison of Helicobacter pylori immunoproteomes of infected mice with previously reported patient data reveals a high agreement in the antigens recognized, suggesting that H. pylori in vivo protein composition and recognition by the host immune system are comparable in mice and humans. Murine Helicobacter models may thus be valid to screen antigens for human vaccination.

Complement-binding antibodies (2 examples of anti-Jka and 2 anti-kell) which inhibit agglutination of enzyme-treated cells have been investigated in the sera of 4 patients. The sera containing anti-Jka also contained rhesus antibodies (c + E and D + C) which could not be readily identified by an enzyme technique due to failure of the Jka+ cells of the corresponding Rh phenotype to react. The inhibition of the enzyme test reactivity was revealed using a control reagent added to each test after the initial examination for agglutination. Addition of ethylenediaminetetraacetic acid to the sera prior to testing resulted in identifiable reactivity with panel red cells. Failure to recognise this phenomenon in sera containing antibodies reacting only by an enzyme technique could result in misidentification, delay in providing compatible blood and transfusion of incompatible blood. Routine control of enzyme tests to detect false-negative results is recommended.

Two patients' sera inhibited agglutination of enzyme-treated red blood cells by antibodies. This blocking effect was detected by using a mixture of antibodies as an enzyme test reagent for the routine control of a two-stage papain test. The blocking factor appeared to be IgG and its effect was complement dependent. It blocked reactions with red blood cells treated with papain, bromelin, or ficin. Reactions with both IgM and IgG antibodies of various specificities were blocked. The presence of the blocking factor in a patient's serum may result in failure to detect clinically significant antibodies unless a control system is used which will confirm that the cells in each test can be agglutinated by enzyme reacting antibodies.