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

  更多相關文章： 花生Ara h蛋白 單克隆抗體 特性鑒定 過敏原檢測

【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,融合免疫鼠脾細胞和小鼠骨髓瘤細胞NS-1。半固體培養(yǎng)基法結合有限稀釋法篩選穩(wěn)定分泌抗體的雜交瘤細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鑒定抗體效價和特異性以及與其他過敏源的交叉反應性。建立雙單抗夾心ELISA法檢測食品中的花生過敏原。結果共獲得抗Ara h1細胞株4株,分別命名為2G9、5G4、5B5、2C7,效價均高于1∶106。經抗體亞型鑒定,4株抗體均為Ig G1型。Western blot的結果表明4株抗體均能識別Ara h1,其中2G9與5G4結合能力較強。在特異性檢測實驗中,2G9和5G4與其他種類過敏原無交叉反應。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,標準曲線在~80 ng/ml范圍內線性良好。利用此法檢測了10種食品,結果顯示在5種含有花生成分的食品中均檢測到了花生過敏原。結論獲得高效價抗體4株,建立了高效、高特異性的食品中花生過敏原的檢測方法,為食品中花生過敏原的檢測提供了依據。
[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】： 深圳大學過敏反應與免疫學研究所;
【基金】：國家自然科學基金(81271950) 廣東省工程技術研究開發(fā)中心項目(2013158925) 廣東省對外科技合作項目(2013B051000088) 深圳市科技計劃國際科技合作項目(GJHZ20130408174112021) 深圳市科技計劃基礎研究項目(JCYJ20140418095735604)
【分類號】：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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