抗眼镜王蛇毒鸡卵黄抗体的制备

目的　研制抗眼镜王蛇毒鸡卵黄抗体 ,并研究该抗体在鸡卵黄中表达过程。方法　以甲醛减毒处理的眼镜王蛇粗毒免疫莱航母鸡 ,母鸡免疫应答后 ,在卵黄中产生抗眼镜王蛇毒抗体 ,应用嗜硫色谱柱HiTrapIgYPurificationHP从鸡卵黄中提取抗眼镜王蛇毒抗体 ;以间接ELISA法及双向免疫抗散法测定抗体的效价、特异性及免疫活性 ;采用福林酚法测定蛋白含量 ;采用SDS PAGE法测定分子量及鉴定抗体纯度 ;抗体的特异性及交叉反应采用酶联免疫吸附法进行进行验证。结果　母鸡初次免疫第 9天即有抗体产生 ,初次免疫 6 0天后抗体效价超过 10 5;卵黄抗体经嗜硫色谱柱纯化后 ,经SDS PAGE检测为电泳纯 ,纯化后抗体效价较纯化前提高 4倍 ,每枚蛋中平均可获得较纯抗体 97 5mg ;所获取的抗体具有高度特异性 ,与蝰蛇科属的其它蛇毒无交叉反应 ,与眼镜蛇科蛇毒存在不同程度交叉反应 (蛇毒浓度大于5 0 0 μg·L-1时明显 )。结论　本研究首次研制并鉴定了抗眼镜王蛇毒鸡卵黄抗体 ,开拓了我国蛇伤治疗的新领域 ,并分析抗体在产蛋期卵黄液中表达的进度 ,为今后该抗体的持续、高质量诱导奠定基础 ,同时也促进其它抗蛇毒鸡卵黄抗体的研究开发及蛇毒单克隆抗体的研制。

King cobra egg yolk antibody from the egg yolks of immunized hens

Aim To develop King cobra antivenom from the egg yolks of immunized hens,and study dynamic expression of IgY in egg yolks.Methods chickens(white Leghorn) were immunized with detoxicated King cobra venom by formaldehyde ,Egg yolk antibody (IgY) was isotated by thiophilic interaction chromatography and identified by SDS-PAGE. Activity of IgY was evaluated by enzyme-linked immunoserbent assay(ELISA) and Double immuodiffusion. Protein was measured using folin-lowry method. Results Specific antivenom could be detected in the yolk laid by the hens 9 d after immunization, At the 60th day after primary immunization ,ELISA titers reached 1∶ 100 000,and 97.5 mg IgY·ml -1 yolk was obtained from thiophilic interaction chromatography. IgY was highly specific, No cross reactivity was observed among IgY and agkistrodon acutus venom and vipera venom ;Little cross reactivity was shown with cobra genus venoms.Conclusion King cobra IgY was obtained and purified from the egg yolks of immunized hens,The dynamic expression of IgY was manitered during the course of immunization,Further investigation is needed.