Serological assessment of the early response to eradication therapy using an immunodominant outer membrane protein of Helicobacter pylori

Abstract

Eradication of Helicobacter pylori infection cures gastritis and prevents recurrence of peptic ulcers. Endoscopy is usually used to evaluate the effectiveness of eradication therapy. We designed a new noninvasive assay system for the early evaluation of eradication of H. pylori infection in which a crude H. pylori outer membrane protein preparation (HPOmp) is used as an antigen, and we determined the sensitivity and specificity of the serological assay system. Immunoblot analysis showed that anti-HPOmp antibodies reacted to a protein with a molecular mass of approximately 29 kDa. In those patients who responded to therapy, the anti-HPOmp immunoglobulin G (IgG) titers measured by enzyme-linked immunosorbent assay (ELISA) at 1 month after the end of therapy were significantly lower than those before treatment (34.8% reduction; P < 0.001), and the posttreatment reduction in the antibody titer was significantly greater than that of the titer measured with a commercially available anti-H. pylori IgG ELISA (34.8% versus 16.1%; P < 0.001). When a 25% reduction of anti-HPOmp IgG titer at 1 month after the end of treatment was taken as the cutoff value for H. pylori eradication, the sensitivity and specificity of our new assay were 75% (51 of 68 treatment responders) and 96% (22 of 23 nonresponders), respectively. Our results indicate that the novel serological test with HPOmp might be a clinically useful tool for assessment of eradication of H. pylori.

FIG. 1

SDS-PAGE profiles of HPOmp fraction and immunoreactivity by WB analysis. (A) SDS-PAGE patterns of soluble fraction (lane S) and membrane fractions (lanes 1–6) separated by 25% to 65% (wt/wt) linear SDG. (B and C) Immunoreactivities of protein A-Sepharose purified IgG obtained from an H. pylori-infected patient (B) and anti-urease monoclonal antibody (C) (kindly provided by K. Nagata) against HPOmp protein by WB. Lanes S and 1 to 6 correspond to the same numbers as in panel A. Lane L is N-lauroylsarcosine-treated HPOmp. Molecular mass markers (M) are shown to the left of columns.

FIG. 2

Immunoreactivities detect by WB analysis at pretreatment and at 6 months after treatment for responders. Fraction 5 shown in Fig. 1A was used for WB as HPOmp. Immunoreactivities against HPOmp protein of serum samples obtained from 12 patients before treatment (A) and at 6 months after therapy (B) are shown. The molecular mass markers (M) are shown in the left column.

FIG. 3

Distribution of EU for anti-HPOmp antibody detected in the sera of H. pylori-infected and control individuals. ∗, P < 0.005, compared with the infected group.

FIG. 4

Serial changes in titers of serum IgG antibody against HPOmp protein for responders and nonresponders. Titers obtained before eradication therapy and at 1, 3, 6, and 12 months after the end of therapy are expressed as percentages of the individual pretreatment titers measured by HPOmp ELISA and GAP IgG ELISA. Data are mean percentages of pretreatment titers ± standard deviations. Closed circles, titer measured by HPOmp ELISA; open circles, titer measured by GAP IgG ELISA; solid lines, titer in responders; dotted lines, titer in nonresponders. † and ∗, P < 0.001 compared with the pretreatment titer and with the GAP IgG ELISA titer, respectively.

FIG. 5

Specificity and sensitivity of HPOmp ELISA for assessment of response to therapy at 1 month after the end of therapy. Dotted lines indicate the relative percentages corresponding to 25 and 30% reductions compared to the pretreatment titer. Closed circles, percentages of pretreatment titers for HPOmp ELISA for four individuals who were categorized as responders but showed evidence of reappearance of H. pylori infection at 6 months after therapy.