The aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecal Helicobacter pylori antigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity for H. pylori antigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form of H. pylori, and fecal samples from H. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41 H. pylori clinical isolates and in fecal samples from seven H. pylori-positive subjects. Several kinds of Helicobacter species (Helicobacter felis, Helicobacter hepaticus, Helicobacter mustelae, and Helicobacter cinaedi) except H. pylori, major bacteria in feces (Campylobacter jejuni, Bacteroides vulgatus, Bifidobacterium breve, Bifidobacterium infantis, and Escherichia coli), and fecal samples from six H. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis of H. pylori infection.Helicobacter pylori causes gastritis and peptic ulcers, and its association with stomach cancer has been studied recently. H. pylori infection can be diagnosed by tests requiring endoscopic biopsy of the gastric mucosa (culture, histology, and the rapid urease test) and by noninvasive tests (serology and the urea breath test) (17). H. pylori is difficult to culture from a fecal sample, because H. pylori outside the stomach probably converts to the nonculturable coccoid form (3, 9).Recently, enzyme immunoassays (EIAs) for the direct detection of the H. pylori antigens in feces have been developed.

Recently, we reported the production of three new monoclonal antibodies with high specificity for a Helicobacter pylori antigen suitable for diagnosis of H. pylori infection. The aim of the present study was to identify the antigen recognized by these monoclonal antibodies concerning both H. pylori and the feces of human subjects infected with H. pylori. The cellular antigen was purified from an H. pylori cell extract by immunoaffinity column chromatography with the monoclonal antibody as a ligand. The amino-terminal amino acid sequences (eight residues) of the purified antigen and H. pylori catalase were the same. The molecular weights of native and subunit, specific catalase activity, and UV and visible spectra of the purified antigen were in good agreement with those of H. pylori catalase. The human fecal antigens were purified from two fecal samples of two H. pylori-positive subjects by ammonium sulfate precipitation, CM-Sephadex C 50 chromatography, and the same immunoaffinity chromatography used for the H. pylori cellular antigen. The fecal antigens had catalase activity. The amino-terminal amino acid sequences (five residues) of the human fecal antigen and H. pylori catalase were the same. The monoclonal antibodies reacted with the native cellular antigen, but did not react with the denatured antigen, human catalase, and bovine catalase. The results show that the target antigen of the monoclonal antibodies is native H. pylori catalase and that the monoclonal antibodies are able to specifically detect the antigen, which exists in an intact form, retaining the catalase activity in human feces.Helicobacter pylori infection can be diagnosed by tests requiring endoscopic biopsy of the gastric mucosa (culture, histology, and a rapid urease test) and by noninvasive tests (serology and urea breath test) (20). Recently, two enzyme immunoassays (EIAs) for the direct detection of the fecal H. pylori antigens have been developed. One uses polyclonal rabbit antibody (Premier Platinum HpSA; Meridian Diagnostics, Inc., Cincinnati, Ohio), and the other uses plural kinds of monoclonal antibodies (MAbs) (FemtoLab H. pylori; Connex GmbH, Martinsried, Germany). They have been shown to be reliable tools for noninvasive diagnosis of H. pylori infection (3,11,12,19). However, the lower specificity and considerable lot-to-lot variation of Premier Platinum HpSA have been reported in several papers (4-6, 18), and the H. pylori antigen profile in human feces recognized by the polyclonal antibody or the plural kinds of MAbs remains uncertain.To develop a diagnostic test for H. pylori infection with higher specificity and reproductivity, we previously reported three new MAbs (21G2, 41A5, and 82B9) that recognize the same fecal H. pylori antigen, partial characterization of the cellular and fecal antigen, and development of a new singlestep EIA that uses one kind of MAb, 21G2, for the detection of fecal H. pylori antigen (17). The developed EIA was able to detect 41 H. pylori isolates and fecal samples from seven H. pylori-positive h...