Production and application of new monoclonal antibodies specific for a fecal Helicobacter pylori antigen.
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.
Suzuki
Nobuyuki
N
Sagami Research Laboratories, Wakamoto Pharmaceutical Co., Ltd., Ohimachi, Ashigarakami-gun, Kanagawa 258-0018, Japan.
Wakasugi
Masahiko
M
Nakaya
Seigo
S
Okada
Keiko
K
Mochida
Ritsuko
R
Sato
Masami
M
Kajiyama
Hirofumi
H
Takahashi
Ryoki
R
Hirata
Haruhisa
H
Ezure
Yohji
Y
Koga
Yasuhiro
Y
Fukuda
Yoshihiro
Y
Shimoyama
Takashi
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000942:Antigens, Bacterial / Q000032:analysis* / Q000276:immunology*; D005243:Feces / Q000382:microbiology*; D016480:Helicobacter pylori / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D051379:Mice; D008875:Middle Aged; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial