斑点免疫结合法测定卵黄抗体的活性(英文)  

Dot-immunobinding assay for the activity of egg yolk anibody

作　　者：阮光萍[1] 安梅[1] 王桂华[1] 叶蕾[1] 邓淑芬[1] 

机构地区：[1]解放军成都军区昆明总医院输血科,云南省昆明市650032

出　　处：《中国组织工程研究与临床康复》2007年第12期2397-2400,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:哺乳动物产生的抗体和鸡产生的卵黄抗体在物理、生物活性方面存在差异。卵黄抗体能耐酸耐热,可口服用来防治动物和人类的肠内感染疾病,且对发展常规的诊断免疫实验有益。传统的ELISA法检测卵黄抗体比斑点免疫结合法费时。目的:应用斑点免疫结合法检测卵黄抗体的稳定性。设计:开放性实验。单位:解放军成都军区昆明总医院输血科。材料:实验于2006-01/06在解放军成都军区昆明总医院完成。选取30周龄的白色莱杭母鸡2只,以HLA-A*0201α链作为抗原,纯化抗原的总蛋白浓度0.04g/L,相对分子质量为32000(自行制备);硝酸纤维素膜(进口分装);脱脂奶粉(安怡产品,批号20051220);DAB(博士德公司);辛酸(上海星火化工厂生产);硫酸铵(汕头市光华化学厂生产)。方法:①抗原HLA-A*0201α链以总蛋白浓度0.04g/L纯化后,进行鸡卵黄抗体的纯化,采用SDS-PAGE电泳检测卵黄抗体的纯化结果。②1μL抗原被点样在硝酸纤维素膜的中心,于37℃孵箱中烤干,浸于1mL的PBST中,37℃孵箱90r/min封闭振荡15min,重新更换1mL的PBST,加入一抗卵黄抗体,终浓度为10mg/L。在37℃孵箱振荡30min后,硝酸纤维素膜用PBST洗3次。加入二抗标记HRP的鼠抗鸡卵黄抗体,孵育30min后,硝酸纤维素膜用PBST洗3次,通过用DAB试剂孵育来显色。阳性反应产生一个深棕色的斑点,表明卵黄抗体具有较好的活性;斑点变浅表明失去部分活性;斑点消失表明失去全部活性。根据斑点的灰度值变化对照标准样品,可计算卵黄抗体剩余的百分活性。③卵黄抗体用磷酸盐缓冲液调整为1,0.1,0.01g/L3种蛋白浓度,室温下放置4个月,每个月分别从各种浓度的样品中取10μg,采用斑点免疫结合法检测卵黄抗体室温稳定性。④卵黄抗体分别置于7个EP管,100μL/管,编号1～7。1～3号管用1mol/L的HCL分别调整pH值为5,3,2;4～6号管用1mol/L的NaOH分别调整pH值为9,11,12;7号管的BACKGROUND： There are differences in physical and biological activity between the antibody from mammals and egg yolk antibody （IgY） from chicken. IgY is acid- and heat-resistant, and can prevent and cure the infectious diseases in animals and human being, which is also benefit to develop routine diagnostic immunoassays. Conventional ELISA assay for IgY takes much more time than dot-immunobinding assay. OBJECTIVE： To detect the IgY stability by using dot-immunobinding assay DESIGN ： Open trail SETTING： Department of Transfusion, Kunming General Hospital of Chinese PLA. MATERIALS： The experiment was completed in the Kunming General Hospital of Chengdu Military Area Command of Chinese PLA from January to June 2006. Two White Leghorn hens （30 weeks old） were selected. HLA-A＊0201 α chain served as the antigen. The total protein concentration of the purified antigen was 0.04 g/L with the molecular mass of 32 000 （self-prepared）; nitrocellulose filter （NC, import and divided）; nonfat dry milk （Anyi Corp. No. 20051220）; DAB （Boshide Corp.）; caprylic acid （made by Shanghai Xinghuo Chemical Factory）; ammonium sulfate （Shantou Guanghua Chemical product）. METHODS： （1）HLA-A＊0201 α chain with the total protein concentration of 0.04 g/L was purified with egg yolk antibody, and identified by SDS-PAGE. （2）1 μL antigen was spotted into the center of NC membrane and dried in the incubator at 37 %. Then the NC membrane was blocked in 1 mL PBST and put in the incubator at 37 % with shaking in 90 r per minute for 15 minutes. Then the liquid was exchanged with 1 mL PBST and added the primary antibody at a final concentration of 10 mg/L. After 30 minutes shaking in the incubator at 37 %, the NC membrane was washed in PBST for three times. The second antibody, mouse anti-chicken IgY conjugated to horseradish peroxidase （HRP） was added and after 30 minutes incubation, the NC membrane was washed three times in PBST. Binding was revealed by incubation with a DAB reagent. A posit

关 键 词：卵蛋白质类 免疫球蛋白类 染色与标记/方法 

分 类 号：R446[医药卫生—诊断学]