Production and Application of New Monoclonal Antibodies Specific for a FecalHelicobacter pyloriAntigen

Abstract

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.

Characterization and usefulness of stool antigen tests using a monoclonal antibody to Helicobacter pylori catalase

“…8 Peroxidase-labeled MAb 21G2 was conjugated with peroxidase-N-succinimidyl ester according to the manufacturer's instructions (LK11-10 Peroxidase Labeling Kit-NH2 Unit: Dojindo Molecular Technologies, Inc., Tokyo, Japan). We used phosphate buffered saline (PBS; Dulbecco's PBS[-]) containing 0.05% Tween 20 as a washing buffer, PBS containing 0.05% Tween 20 plus 5% BSA, Fr V (Serologicals Proteins Inc., Kankakee, IL, USA) as a diluent buffer, 3,3′,5,5′-tetramethylbenzidine (TMB: BioFX, Owings Mills, MD, USA) as the substrate solution, and 1N H2SO4 as the stop reagent for the reaction.…”

“…Culturing and whole-cell disruption of all of the bacterial strains was carried out as described previously. 8 The standard H. pylori cellular antigen was prepared from H. pylori ATCC 43504 and cultured on Difco Brain Heart Infusion Agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) plates containing 5% horse blood in a microaerophilic environment (Anaero Pack Helico System; Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan). The culture plate was incubated for 7 days in an anaerobic environment (Anaero Pack Anaero System; Mitsubishi Gas Chemical Co. Inc.).…”

“…We have established three MAbs with high specificity for the native H. pylori catalase antigen. 8,9 Using one of these MAbs (21G2), we developed a single-step direct sandwich EIA: Testmate Pylori Antigen EIA (TPAg EIA), and an immunochromatographic test: Testmate Rapid Pylori Antigen (Rapid TPAg). Several studies in the USA and Japan have verified the accuracy and usefulness of TPAg EIA and Rapid TPAg in confirming the results of eradication therapy.…”

Applicability of a rapid stool antigen test, using monoclonal antibody to catalase, for the management of Helicobacter pylori infection

“…Stool antigen tests based on monoclonal antibody have been shown to be more accurate than those based on polyclonal antibodies [4,9]. The new Japanese guidelines recommend monoclonal antibody-based stool antigen tests for confirmation of H. pylori eradication [1].…”

“…Testmate pylori antigen (TPAg; Wakamoto Pharmaceutical Co., Ltd, Tokyo, Japan) utilizes monoclonal antibody to native H. pylori catalase [3,4] and is reported to be as accurate as UBT for the evaluation of the outcome of eradication therapy in Japanese patients [5,6]. However, the results of testing with TPAg cannot be obtained quickly because stool samples are usually transferred to central laboratories and subjected to enzyme immunoassay (EIA).…”