Use of a novel enzyme immunoassay based on detection of circulating antigen in serum for diagnosis of Helicobacter pylori infection

Abstract

Recently, noninvasive diagnostic tests for Helicobacter pylori infection have gained in significance. We have developed a sensitive and specific noninvasive immunoassay based on the detection of an H. pylori circulating antigen (HpCA) in sera from H. pylori-infected individuals. Monospecific antibody and Western blot analyses were used to demonstrate the presence of the target antigen in H. pylori cell lysate and serum samples. A novel enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HpCA in serum. Endoscopic biopsy specimens from the gastric antra of 221 individuals (143 males and 78 females) with dyspeptic symptoms were evaluated for H. pylori infection, with culture used as a "gold standard" for diagnosis. The target H. pylori antigen was identified at 58 kDa. HpCA has been detected by ELISA with high degrees of sensitivity, specificity, and efficiency (>90%), and ELISA results show no significant difference (P > 0.05) from results of H. pylori culture of gastric biopsy specimens. The test's positive and negative predictive values were also high (95 and 86%, respectively). In conclusion, a sensitive and specific immunoassay was developed for the detection of HpCA in human serum. This test can be applied for noninvasive laboratory and field diagnoses of H. pylori infection.

FIG. 1.

Western blot analysis of H. pylori cell lysate and the 58-kDa purified antigen. Lane 1, immunostaining with nonimmunized-rabbit serum; lane 2, immunostaining with polyclonal rabbit IgG to H. pylori cell lysate; lane 3, immunostaining with rabbit IgG-monospecific antibody to the 58-kDa purified antigen. A highly reactive band was identified at 58 kDa by using rabbit IgG anti-H. pylori lysate and the anti-58-kDa antigen. Molecular mass markers (bands are not shown but are indicated by arrows) include phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), glutamate dehydrogenase (55.0 kDa), ovalbumin (42.7 kDa), aldolase (40 kDa), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5 kDa).

FIG. 2.

Western blot analysis of serum samples from H. pylori-infected and noninfected individuals with antisera monospecific to the 58-kDa antigen. A single band was identified at 58 kDa in serum samples from infected individuals only, in addition to a degradation product of 42 kDa in some of these samples. Lane 1, H. pylori cell lysate as a positive control; lanes 2 to 4, serum samples from three noninfected healthy individuals; lanes 5 to 10, serum samples from six individuals infected with H. pylori. Molecular mass bands are not shown but are indicated by arrows.