抗甲型流感鸡卵黄抗体的制备及效价测定

Preparation and Titration of Influenza A Antibodies from Chicken Egg Yolk

目的:研究抗甲型流感卵黄抗体的制备与纯化,并探讨其效价随免疫时间的变化关系。方法:用灭活甲型流感病毒复合抗原免疫蛋鸡,用PEG6000对卵黄抗体进行分离提取,SDS-PAGE法对其进行分子量测定,考马斯亮蓝法对其含量和纯度进行测定,用微量凝集法检测蛋鸡血清抗体和卵黄抗体的效价。结果:提取得到的卵黄抗体重链分子量为66 kDa、轻链分子量分26 kDa,每毫升卵黄液可得到纯度为95.80%的卵黄抗体9.98mg,回收率93.01%;高效价持续时间90 d以上;免疫蛋鸡血清和卵黄中3种特异性抗体的消长规律基本相似,但抗体水平之间存在明显的差异。结论:采用灭活甲型流感病毒复合抗原免疫蛋鸡可制备高效价、高纯度抗甲型流感卵黄抗体,为卵黄抗体在甲型流感防治中的应用研究奠定了基础。

Objective：Preparation and purification of Egg-yolk antibodies（IgY） against influenza A was studied,and variations of IgY titer with time were investigated.Method：Chickens（white Leghorn） were immunized with inactivated influenza A virus（H732,H622 and H749） complex antigen.IgY was isolated with PEG6000 and identified by SDS-PAGE.Content of IgY was measured using Coomassie blue mtehod.Activities of serum antibody and IgY were evaluated using Micro-hemagglutination inhibition regularly.Result：Molecular weight of IgY heavy chain and light chain is 66 kDa and 26 kDa,respectively.Per milliliter of egg yolk could be gained IgY 9.98mg,purity 95.80%,its recovery 93.01%.The titer of IgY was lower than that of serum antibody in the early immunity,but with the immunity time,the titer of IgY exceeded that of serum antibody.The high titer IgY could maintain more than 90 days.Variation of 3 species-specific antibody with time in serum and yolk were similar,but it was significantly different among antibody levels.Conclusion：IgY with high titer and purity could be obtained immunizing chickens with inactivated influenza A virus complex antigen,which made a foundation for IgY using to prevent and treat influenza A.