Immunoblot assay for serodiagnosis of Helicobacter pylori infections

Abstract

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 assaypositive 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.

Helicobacter pylori Infection in Children in Estonia: Decreasing Seroprevalence During the 11‐Year Period of Profound Socioeconomic Changes

“…Borderline results were confirmed by immunoblot analysis. Acid-glycine-extracted cell surface proteins of the H. pylori strain NCTC 11637 [14] were used as the antigen for both the ELISA and for the immunoblot assay. Each ELISA plate and each immunoblot assay contained serum samples from Group 1991 and Group 2002 proportionately to their overall amounts.…”

“…Relative molecular masses (M r ) of the separated proteins were estimated by using precision marker proteins (Bio-Rad, Richmond, USA). The immunoblot assay was performed as described previously [14]. Sera staining with a high M r protein (120 kDa CagA) alone or with the 87, 33 and 30 kDa proteins were scored as positive [14].…”

Combined serum IgG response toHelicobacter pyloriVacA and CagA predicts gastric cancer

“…Immunoblot analysis for the detection of antibodies was performed as described by Nilsson et al (1997). SDS-PAGE was performed using Protean II Cell Vertical Electrophoresis equipment (Bio-Rad, Richmond, CA).…”

Is the Association Between Helicobacter pylori and Gastric Cancer Confined to CagA‐Positive Strains?

“…We used a slightly modified version of the immunoblot method described by Nilsson et al [22] to determine the presence of antibodies to CagA. We used 10% separating gels, each gel was loaded with 150 µg of antigen, and the proteins were transferred to nitrocellulose membranes.…”