花生主要过敏原Ara h1单克隆抗体的制备与应用

Preparation and application of monoclonal antibodies against allergen Ara h1 from peanut

目的制备与鉴定抗花生主要过敏原Ara h1单克隆抗体,建立双单抗ELISA法,检测食品中花生过敏原。方法以花生主要过敏原Ara h1为抗原免疫Balb/c小鼠,融合免疫鼠脾细胞和小鼠骨髓瘤细胞NS-1。半固体培养基法结合有限稀释法筛选稳定分泌抗体的杂交瘤细胞株后诱生小鼠腹水,采用蛋白A亲和层析法获得纯化抗体。利用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型。间接ELISA法和Western blot鉴定抗体效价和特异性以及与其他过敏源的交叉反应性。建立双单抗夹心ELISA法检测食品中的花生过敏原。结果共获得抗Ara h1细胞株4株,分别命名为2G9、5G4、5B5、2C7,效价均高于1∶106。经抗体亚型鉴定,4株抗体均为Ig G1型。Western blot的结果表明4株抗体均能识别Ara h1,其中2G9与5G4结合能力较强。在特异性检测实验中,2G9和5G4与其他种类过敏原无交叉反应。通过建立双单克隆抗体夹心ELISA法,发现花生Ara h1蛋白的检出低限为:5 ng/ml,标准曲线在~80 ng/ml范围内线性良好。利用此法检测了10种食品,结果显示在5种含有花生成分的食品中均检测到了花生过敏原。结论获得高效价抗体4株,建立了高效、高特异性的食品中花生过敏原的检测方法,为食品中花生过敏原的检测提供了依据。

This study was performed to prepare and characterize monoclonal antibodies against allergen Ara hl from peanut, and to construct a method with sandwich-antibody enzyme linked immunosorbent assay （ELISA） to detect peanut allergen protein trace in food products. Balb/c mice were immunized with Ara hl protein, the main allergen from peanut; hybridoma and limit dilution technique were used to screen the fusion cell strain secreting antibodies stably. Then the fusion cells were used to induce ascites in mice and monoclonal antibodies were purified using affinity chromatography on immobilized protein A. The Ig subclass of the monoclonal antibody was identified with Ig and Ig subclass kits, while the specificity, titers and cross-reactivity of the antibodies were detected with indirect enzyme linked immunosorbent assay （ELISA） and Western blot. The these antibodies were used to construct a method of sandwich-ELISA for detecting Ara hl allergen protein trace in food products. Data showed that total of 4 hybridoma cell lines secreting McAbs against antigenic epitope of Ara hl protein from peanut were obtained, which were denominated as 2G9, 5G4, 5B5, 2C7. The titers of the four McAbs （2G9, 5G4, 5B5, 2C7） were higher than 1 ： 1 000 000 （P/N 〉 2.1）. The four McAbs belong to IgG1 subtype. Results of Western blot indicated that all antibodies could bind to Ara hl specifically. Moreover, 2G9 and 5G4 McAbs could specially recognize Ara hl protein and hadno cross-reaction with other familiar foods, but 5B5 and 2C7 had cross-reactivity with allergen from hazelnut. A sandwich-ELISA system was set up to detect the presence of peanut allergens. Our results indicated that the standard curve is linear with peanut allergen proteinconcentration from 5 ng/ml to 80 ng/ml and the detection limit is 5 ng/ml of peanut allergen protein. Ten food products were tested using this method, and 5 products were found contain the peanut allergen protein, which were consistent with the food allergen labels from their manufactures. Taken together, four monoclonal antibodies against Ara hl protein from peanut have prepared successfully, and a sandwich-ELISA system has been set up to detect the presence of peanut allergens, which would facilitate the establishment of detection method for peanut allergen.