Production and Application of New Monoclonal Antibodies Specific for a Fecal Helicobacter pylori Antigen
Production and Application of New Monoclonal Antibodies Specific for a Fecal Helicobacter pylori Antigen
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粪便幽门螺杆菌抗原特异性单克隆抗体的制备及应用
DOI：
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发表时间：
2002
影响因子：
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通讯作者：
T. Shimoyama
中科院分区：
文献类型：
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作者：
Nobuyuki Suzuki;M. Wakasugi;S. Nakaya;K. Okada;R. Mochida;Masami Sato;H. Kajiyama;R. Takahashi;H. Hirata;Y. Ezure;Y. Koga;Y. Fukuda;T. Shimoyama
摘要本研究的目的是建立单克隆抗体，可用于生产一种单克隆抗体识别粪便幽门螺杆菌抗原组成的诊断试验。开发这种测试的需要源于使用多克隆抗体或多种单克隆抗体的诊断测试的缺点，例如对H. pylori抗原和难以以一致的质量繁殖。用超声处理的球形H. pylori和来自H.通过直接夹心酶免疫测定法（EIA）从32个杂交瘤克隆中筛选幽门螺杆菌阳性受试者的抗体产生。三个稳定的克隆产生与相同可溶性抗原反应的抗体（21G2、41A5和82B9）。凝胶过滤层析显示细胞抗原和粪便抗原的分子量相同，为260 kDa。该抗原对十二烷基硫酸钠和热处理反应不稳定。建立了一种单克隆抗体21G2直接夹心酶免疫分析法。酶免疫法可检测到41个H. pylori临床分离株和来自7个H.幽门阳性的受试者。除H. pylori，粪便中的主要细菌（空肠弯曲杆菌，普通拟杆菌，短双歧杆菌，双歧杆菌和大肠杆菌）和来自6个H.幽门阴性受试者显示阴性结果。这些结果表明，新的单克隆抗体和新的特异性EIA将作为一种非侵入性诊断H.幽门感染
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.