本文選題：花生 + 過敏原��； 參考：《南昌大學》2007年碩士論文

【摘要】： 花生是世界糧農(nóng)組織(FAO，1995)認定的八大類食物過敏原之一。相對其它食物過敏而言，花生過敏發(fā)生率較高、臨床癥狀更為嚴重，且不容易隨年齡的增長而產(chǎn)生免疫耐受，在公共衛(wèi)生和食品安全領域倍受人們的關注。因此，建立準確、靈敏、快速地檢測花生過敏原的方法具有重要意義。 本研究主要內(nèi)容包括花生過敏原Ara h2的分離純化、兔抗Ara h2多克隆抗體的制備及間接競爭ELISA檢測Ara h2方法的建立。 研究中以四粒紅花生(Arachis hypogaea L．)為實驗對象，經(jīng)粉碎、脫脂、蛋白抽提、陰離子交換層析、SDS-PAGE電泳回收，得到了電泳純(純度＞95％)的Ara h2純品，建立了DEAE-Sepharose Fast Flow結合SDS-PAGE電泳回收分離Ara h2的方法。 在制備抗Ara h2抗體過程中，以純化的Ara h2為抗原，初次免疫采用弗式完全佐劑，加強免疫采用弗式不完全佐劑，皮下多點免疫，每隔兩周加強免疫一次，共計免疫6次，每次免疫劑量為200μg／只。首次免疫12周后，經(jīng)ELISA檢測和雙向瓊脂擴散法檢測，抗體效價分別為1∶200000及1∶16。雙向瓊脂擴散的交叉試驗表明，Ara h2特異性好，與大豆蛋白、雞蛋清蛋白、BSA均無交叉反應。此實驗建立了間接ELISA檢測Ara h2抗體效價的檢測方法及制備抗Ara h2多克隆抗體的操作程序。 在用板外競爭反應模式建立Ara h2的間接競爭ELISA檢測方法過程中，用160ng／mL抗原包被酶標板(37℃包被2小時)，以5％脫脂奶粉37℃封閉1小時，，選擇抗血清稀釋度為1∶16000。該方法的檢測靈敏度為51ng／mL，最低檢出限為0.26ng／mL，樣品加標回收率為90％-100％，檢測Ara h2的線性范圍在12.5ng／mL-400ng／mL之間。同時，用此方法對6份樣品進行了檢測，測得花生牛奶飲料中Ara h2含量為812mg／kg，其它樣品中未檢出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

本文編號：1875158