Immunoblot assay for serodiagnosis of Helicobacter pylori infections

An immunoblot assay for the serological diagnosis of Helicobacter pylori infection was evaluated. Serum samples from patients whose gastric biopsy specimens were known to be positive or negative for H. pylori on culture were used to establish interpretive criteria for the immunoblot assay. A panel of sera from patients with diseases other than H. pylori infection and sera from healthy blood donors were included to validate these criteria. All sera were initially assessed in an enzyme immunoassay (Ge-EIA), based on acid glycine-extracted cell surface proteins of H. pylori NCTC 11637. The same antigen extract was used in the immunoblot assay. In addition, the Ge-EIA and the immunoblot assay were compared with a commercially available EIA (Seradyn, Color Vue Pylori). Bands of 110/120 kDa and/or two of five low-molecular-mass proteins (26, 29, 30, 31, and 33 kDa, in any combination) showed a strong correlation with the H. pylori culture-positive patients (97.5%) compared to the correlation obtained with the EIA results (Ge-EIA, 87.5%; Seradyn EIA, 92.5%), and the antibody responses to these proteins were considered specific reactions. In 37 of 40 serum samples from culture-negative patients and also in sera from patients with other disorders, a moderate antibody reactivity to the medium-size proteins (43 to 66 kDa) was observed, and these were considered not valuable for a specific immunoblot assay. Among sera from culture-positive patients, 39 of 40 serum samples were defined to be immunoblot positive, and from among sera from culture-negative patients, 3 of 40 serum samples were defined to be immunoblot positive. The use of sera from patients with negative cultures for H. pylori as negative controls may decrease the sensitivity due to sampling error and false-negative culture results. Immunoblot assay-positive results were detected among 10% of sera from patients with other diseases, whereas they were detected among 42.5% of sera by the Ge-EIA and 47.5% of sera by the Seradyn-EIA. The higher number of EIA-positive sera in this group reflects a possible cross-reactivity (false-positive EIA result). Of the blood donors, representing asymptomatic but possibly colonized subjects, 24% were immunoblot positive. In conclusion, our data indicate that immunoblotting is more sensitive as well as more specific than EIA. Moreover, it permits detection of antibody responses to specific antigens, e.g., the cytotoxin-associated CagA protein, which may have pathological implications.