抗花生过敏原Ara h2多克隆抗体的制备及其应用

发布时间：2018-05-11 18:54

  本文选题：花生 + 过敏原　； 参考：《南昌大学》2007年硕士论文

【摘要】： 花生是世界粮农组织(FAO，1995)认定的八大类食物过敏原之一。相对其它食物过敏而言，花生过敏发生率较高、临床症状更为严重，且不容易随年龄的增长而产生免疫耐受，在公共卫生和食品安全领域倍受人们的关注。因此，建立准确、灵敏、快速地检测花生过敏原的方法具有重要意义。 本研究主要内容包括花生过敏原Ara h2的分离纯化、兔抗Ara h2多克隆抗体的制备及间接竞争ELISA检测Ara h2方法的建立。 研究中以四粒红花生(Arachis hypogaea L．)为实验对象，经粉碎、脱脂、蛋白抽提、阴离子交换层析、SDS-PAGE电泳回收，得到了电泳纯(纯度＞95％)的Ara h2纯品，建立了DEAE-Sepharose Fast Flow结合SDS-PAGE电泳回收分离Ara h2的方法。 在制备抗Ara h2抗体过程中，以纯化的Ara h2为抗原，初次免疫采用弗式完全佐剂，加强免疫采用弗式不完全佐剂，皮下多点免疫，每隔两周加强免疫一次，共计免疫6次，每次免疫剂量为200μg／只。首次免疫12周后，经ELISA检测和双向琼脂扩散法检测，抗体效价分别为1∶200000及1∶16。双向琼脂扩散的交叉试验表明，Ara h2特异性好，与大豆蛋白、鸡蛋清蛋白、BSA均无交叉反应。此实验建立了间接ELISA检测Ara h2抗体效价的检测方法及制备抗Ara h2多克隆抗体的操作程序。 在用板外竞争反应模式建立Ara h2的间接竞争ELISA检测方法过程中，用160ng／mL抗原包被酶标板(37℃包被2小时)，以5％脱脂奶粉37℃封闭1小时，，选择抗血清稀释度为1∶16000。该方法的检测灵敏度为51ng／mL，最低检出限为0.26ng／mL，样品加标回收率为90％-100％，检测Ara h2的线性范围在12.5ng／mL-400ng／mL之间。同时，用此方法对6份样品进行了检测，测得花生牛奶饮料中Ara h2含量为812mg／kg，其它样品中未检出Ara h2。
[Abstract]:Peanut is one of the eight food allergens identified by FAO. Compared with other food allergies, peanut allergies have a higher incidence, more severe clinical symptoms, and are not easy to produce immune tolerance with age, which has attracted much attention in the field of public health and food safety. Therefore, it is important to establish an accurate, sensitive and rapid method for the detection of peanut allergens. The main contents of this study include the isolation and purification of peanut allergen Ara H2, the preparation of rabbit anti Ara H2 polyclonal antibody and the establishment of indirect competitive ELISA method for detection of Ara H2. Arachis hypogaea L. The purified Ara H2 was obtained by SDS-PAGE electrophoresis after being crushed, defatted, extracted by protein and recovered by anion exchange chromatography. The method of recovering Ara H2 by DEAE-Sepharose Fast Flow combined with SDS-PAGE electrophoresis was established. In the process of preparation of anti Ara H2 antibody, the purified Ara H2 was used as antigen, the first immunization was carried out with complete adjuvant of Freund type, and the adjuvant was used as partial adjuvant for the first time, and the subcutaneous multi-point immunization was carried out. The immunization was intensified once every two weeks for a total of 6 times. The dose of each immunization was 200 渭 g / mouse. After 12 weeks of first immunization, the titers of the antibodies were 1: 200000 and 1: 16, respectively, after ELISA detection and bidirectional Agar diffusion assay. The cross test of bidirectional Agar diffusion showed that Ara H2 had good specificity and had no cross reaction with soybean protein and egg white protein BSA. In this experiment, a method for detecting the titer of Ara H2 antibody by indirect ELISA and a procedure for preparing anti Ara H2 polyclonal antibody were established. In the process of establishing an indirect competitive ELISA detection method for Ara H2 by using the off-board competitive reaction model, 160ng/mL antigen was coated on the enzyme labeled plate at 37 鈩

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