猪白细胞介素-4特异性抗体的鉴定及夹心ELISA和ELISPOT检测系统的建立。
Characterization of specific antibodies and the establishment of sandwich ELISA and ELISPOT systems for swine IL-4.
作者信息
Nuntaprasert A, Mori Y, Fujita K, Yoneda M, Miura R, Tsukiyama-Kohara K, Kai C
机构信息
Laboratory of Animal Research Center, Institution of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan.
出版信息
Comp Immunol Microbiol Infect Dis. 2004 Nov;27(6):457-70. doi: 10.1016/j.cimid.2004.03.011.
We produced four monoclonal antibodies (mAb) and two polyclonal antibodies using the purified cytokine expressed in bacteria and characterized them. Specific binding of each of the mAb and polyclonal antibodies to recombinant swine IL-4 (rSwIL-4) purified from Escherichia coli and baculovirus was demonstrated in an indirect ELISA and/or in western blotting. We established a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring concentration of SwIL-4 in biological samples and established an enzyme-linked immunospot (ELISPOT) assay for detecting IL-4-secreting cells using a mAb and a polyclonal IgG from goat. The detection limit of the sandwich ELISA for SwIL-4 was 78 pg/ml. Using sandwich ELISA, SwIL-4 was detected in the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Mycoplasma hyopneumoniae and could quantitate in supernatants of mitogen-stimulated PBMC culture. The ELISPOT system is useful for the detection of IL-4 producing cells in swine PBMC culture.
我们使用在细菌中表达的纯化细胞因子制备了四种单克隆抗体（mAb）和两种多克隆抗体，并对它们进行了表征。在间接ELISA和/或蛋白质印迹中证明了每种单克隆抗体和多克隆抗体与从大肠杆菌和杆状病毒中纯化的重组猪IL-4（rSwIL-4）的特异性结合。我们建立了一种夹心酶联免疫吸附测定（ELISA）来测量生物样品中SwIL-4的浓度，并建立了一种酶联免疫斑点（ELISPOT）测定法，使用一种单克隆抗体和山羊的多克隆IgG来检测分泌IL-4的细胞。SwIL-4夹心ELISA的检测限为78 pg/ml。使用夹心ELISA，在实验感染猪肺炎支原体的猪的支气管肺泡灌洗液（BALF）中检测到了SwIL-4，并且可以在丝裂原刺激的PBMC培养上清液中进行定量。ELISPOT系统可用于检测猪PBMC培养物中产生IL-4的细胞。
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