斑点免疫结合法测定卵黄抗体的活性

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pH值为7(中性),不加酸或碱。1～7号管均放在37℃孵箱中3h,每隔1h各管分别取10μg样品,采用斑点免疫结合法检测卵黄抗体不同pH值条件下的稳定性。⑤卵黄抗体分别置于6个EP管,10μL/管,编号1～6。按顺序编号各管分别于30,40,50,60,70,80℃水浴15min,然后4℃冰箱冷却,每管取样10μg,以未经过加热处理的样品作为标准对照,采用斑点免疫结合法检测卵黄抗体热稳定性。主要观察指标:①卵黄抗体SDS-PAGE电泳检测。②卵黄抗体室温稳定性检测。③卵黄抗体不同pH值稳定性检测结果。④卵黄抗体热稳定性检测。结果:①纯化的卵黄抗体经SDS-PAGE电泳后有两条主带,重链相对分子质量约66000,轻链相对分子质量约25000。②1,0.1,0.01g/L的卵黄抗体,室温下放置4个月后仍然保留部分活性。③pH5～9时37℃孵育3h后,卵黄抗体仍有部分活性;在pH低于5或高于9的条件下37℃孵育3h后,卵黄抗体失去全部活性。④纯化的卵黄抗体分别置于6个EP管中,1～4号管的卵黄抗体仍有活性,第5管和第6管出现白色沉淀,可能是较高温度下蛋白变性引起的。结论:卵黄抗体稳定性较好,斑点免疫结合法能够快速简单的证明和描述卵黄抗体的功能活性,且只需要微量抗原和抗体,斑点特异,能同时处理大量样品。

Dot-immunobinding assay for the activity of egg yolk anibody

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 byusing dot-immunobinding assay.DESIGN: Open trail.SETTING: Department of Transfusion, Kunming General Hospital of Chinese PLA.MATERTALS: 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: ①HLA-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. ②1 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 positive reaction was represented by adeep brown spot,Irdlcating that IgY had better activity; if the spot became lighter IgY lost part activity, and when the spot disappeared, the IgY lost a the activty.According to intensity (gray degree)of the dot compared tothe standard, the remained percent of activity of the IgY was calculated. ③IgY was adjusted to three different protein concentrations with PBS: 1, 0.1, 0.01 g/L and stayed at room temperature for four months. 10 μg lgY was taken out from each concentration sample every month to detect the activity by dot-immunobinding assay. ④IgY was put into seven EP tubes with 100 μL per tube and numbered 1-7. Number 1 to 3 was adjusted pH to 5, 3 and 2, respectively with 1 mol/L HCI; Number 4 to 6 was to 9, 11 and 12, respectively with 1 mol/L NaOH. The pH of number 7 was neutral without adding acid or base. The samples were stayed in incubator at 37 ℃ for 3 hours. 10 μg IgY from each tube was taken every hour to detect the stability at different pH by dot-immunobinding assay. ⑤IgY was added to six EP tubes (10 μL per tube) and numbered 1-6. Number 1-6 was put into waterbath at 30, 40, 50, 60, 70 and 80 ℃ for 15 minutes. After cooled in refrigerator at 4 ℃, 10 mg samples from each tube and standard sample (untreated sample) taken to check the thermal stability by dot-immunobinding assay.MAIN OUTCOME MEASURES: ①SDS-PAGE of IgY. ②IgY stability at room temperature. ③IgY stability at different pH. ④ Detection of IgY thermal stability.RESULTS: ①Purified IgY after SDS-PAGE had two major binds, the molecular mass of the heavy chain was 66.000,and the light chain was 25 000. ②1, 0.1, 0.01 g/L IgY still had partial activity after staying at room temperature for four months. ③When pH ranged from 5 to 9, IgY still had partial activity after staying in 37 ℃ for 3 hours. If pH was lower than 5 or higher than 9, it lost the whole activity in above condition. ④Purified IgY was added to six EP tubes, the number 1-4 still had partial activity, but number 5 and 6 showed some white precipitate, which was caused by protein denaturation at higher temperature.CONCLUSION: IgY stability is higher than others. The dot-immunobinding assay described a rapid and simple method for the demonstration and characterization of functional activity of egg yolk antibody. With only small volume antigen and antibody, and specific dot, the dot-immunobinding assay method could process many samples at the same time.