本文關(guān)鍵詞：花生主要過敏原Ara h1單克隆抗體的制備與應(yīng)用　出處：《免疫學(xué)雜志》2017年01期 　論文類型：期刊論文

  更多相關(guān)文章： 花生Ara h蛋白 單克隆抗體 特性鑒定 過敏原檢測(cè)

【摘要】：目的制備與鑒定抗花生主要過敏原Ara h1單克隆抗體,建立雙單抗ELISA法,檢測(cè)食品中花生過敏原。方法以花生主要過敏原Ara h1為抗原免疫Balb/c小鼠,融合免疫鼠脾細(xì)胞和小鼠骨髓瘤細(xì)胞NS-1。半固體培養(yǎng)基法結(jié)合有限稀釋法篩選穩(wěn)定分泌抗體的雜交瘤細(xì)胞株后誘生小鼠腹水,采用蛋白A親和層析法獲得純化抗體。利用Ig類與亞類鑒定試劑盒鑒定該單克隆抗體的Ig亞型。間接ELISA法和Western blot鑒定抗體效價(jià)和特異性以及與其他過敏源的交叉反應(yīng)性。建立雙單抗夾心ELISA法檢測(cè)食品中的花生過敏原。結(jié)果共獲得抗Ara h1細(xì)胞株4株,分別命名為2G9、5G4、5B5、2C7,效價(jià)均高于1∶106。經(jīng)抗體亞型鑒定,4株抗體均為Ig G1型。Western blot的結(jié)果表明4株抗體均能識(shí)別Ara h1,其中2G9與5G4結(jié)合能力較強(qiáng)。在特異性檢測(cè)實(shí)驗(yàn)中,2G9和5G4與其他種類過敏原無(wú)交叉反應(yīng)。通過建立雙單克隆抗體夾心ELISA法,發(fā)現(xiàn)花生Ara h1蛋白的檢出低限為:5 ng/ml,標(biāo)準(zhǔn)曲線在~80 ng/ml范圍內(nèi)線性良好。利用此法檢測(cè)了10種食品,結(jié)果顯示在5種含有花生成分的食品中均檢測(cè)到了花生過敏原。結(jié)論獲得高效價(jià)抗體4株,建立了高效、高特異性的食品中花生過敏原的檢測(cè)方法,為食品中花生過敏原的檢測(cè)提供了依據(jù)。
[Abstract]:Objective to prepare and identify monoclonal antibodies against Ara h1, a major peanut allergen, and to establish a double monoclonal antibody ELISA method. Methods the main allergen of peanut, Ara H1, was used as antigen to immunize Balb/c mice. Fusion immunized mouse spleen cells and mouse myeloma cells NS-1.The semi-solid medium combined with limited dilution method was used to screen hybridoma cell lines secreting antibody stably and then induce ascites in mice. Purified antibody was obtained by affinity chromatography of protein A. Ig subtype of monoclonal antibody was identified by Ig and subclass identification kit. Indirect ELISA and Western were used to identify the monoclonal antibody. Blot was used to identify antibody titers, specificity and cross-reactivity with other allergens. A double monoclonal antibody sandwich ELISA method was established for the detection of peanut allergens in food. H1 cell line 4. They were named as 2G9, 5G4, 5B5, 2C7, and their titers were higher than 1: 106. The antibody subtypes were identified. The results showed that all the four antibodies could recognize Ara H1. The binding ability of 2G9 to 5G4 was stronger. In the specific test, there was no cross reaction between 2G9 and 5G4 with other allergens. A sandwich ELISA method with double monoclonal antibodies was established. It was found that the detection limit of peanut Ara H1 protein was 1: 5 ng / ml, and the standard curve was linear in the range of 80 ng/ml. Ten foods were detected by this method. The results showed that peanut allergens were detected in 5 kinds of foods containing peanut ingredients. Conclusion High titer antibodies of 4 strains were obtained and a high efficiency and high specificity method was established for the detection of peanut allergens in food. It provides the basis for the detection of peanut allergen in food.
【作者單位】： 深圳大學(xué)過敏反應(yīng)與免疫學(xué)研究所;
【基金】：國(guó)家自然科學(xué)基金(81271950) 廣東省工程技術(shù)研究開發(fā)中心項(xiàng)目(2013158925) 廣東省對(duì)外科技合作項(xiàng)目(2013B051000088) 深圳市科技計(jì)劃國(guó)際科技合作項(xiàng)目(GJHZ20130408174112021) 深圳市科技計(jì)劃基礎(chǔ)研究項(xiàng)目(JCYJ20140418095735604)
【分類號(hào)】：R392
【正文快照】： concentration from 5 ng/ml to 80 ng/ml and the detection limit is 5 ng/ml of peanut allergen protein.Ten foodproducts were tested using this method,and 5 products were found contain the peanut allergen protein,which wereconsistent with the food allergen

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