摘要
目的在常规ELISA检测中,以卵黄抗体取代哺乳动物来源的IgG类抗体,以探讨卵黄抗体应用于蛇毒检测的可行性.方法以甲醛减毒的眼镜王蛇毒抗原免疫莱航母鸡,制备抗眼镜王蛇毒抗体;采用过碘酸钠法对特异卵黄抗体进行酶标;将卵黄抗体及相应酶标抗体应用于常规ELISA中,对国内常见蛇毒样品抗原(包括眼镜王蛇毒、眼镜蛇毒、金环蛇毒、银环蛇毒、五步蛇毒及广东园斑蝰蛇毒)进行检测,并进行灵敏度、精确度、特异性及交叉反应等分析.结果检测的灵敏度可达32 μg·L-1,并且眼镜王蛇毒抗原浓度在32～750 μg·L-1的范围内,线性关系较好(r=0.963);初步应用表明卵黄抗体对眼镜王蛇毒具有较高的特异性,与蝰蛇科的五步蛇毒、园斑蝰蛇毒无交叉反应,与眼镜蛇科的金环及银环蛇毒仅在高剂量(剂量大于500 μg·L-1)时存在轻度交叉反应,不干扰实验检测,但与眼镜蛇毒之间交叉反应明显,干扰实验检测;应用本方法对低(100 μg·L-1)、中(300 μg·L-1)、高(1 000 μg·L-1)不同浓度的眼镜王蛇毒样品检测,结果平均板内变异系数均在1%～3%之内,板间变异系数在8%以内.结论 特异的卵黄抗体应用于微量蛇毒检测研究是可行的,具有灵敏度高、特异性较强等优点.该研究将为蛇伤诊断试剂盒的研制提供相应的理论依据.
Aim To discuss whether specific Egg yolk antibody （IgY） can be used for snake venom antigens detection. Methods Chickens （ white Leghorn） were immunized with detoxicated king cobra venom by form- aldehyde. Egg yolk antibody were isolated from egg yolk, and labeled with horse radish peroxidase （HRP）. Snake venom antigens samples, including king cobra venom, cobra venom, bungrarus fasciatus venom, bun- grams muhicinctus venom, agkistrodon actus venom and Guangdong viper venom, were detected using ELISA , and sensitivity,precision and specificity were tested, respectively. Results At about 32 μg · L^-1, the king cobra antigens were detected using the method ; Linear relation was better （ r = 0. 963 ） when the concentration of kingcobra venom was within 32-75μg · L^-1. The method had good specificity and no cross reactivity was observed among the reagents and agkistrodon acutus guenther venom and vipera russelli siamensis smith venom; little cross reactivity was shown with bungarus muhicinctus blyth venom and bungarus fasciatus chmeider venom;cross reactivity was obvious with cobra venom; the average intra-assay coefficient of variation （CV） was 1% - 3% ,and the inter-assay CV was within 8%. Conclusions The study indicates that IgY can be good reagents for snake venom antigens detecton,and the study provides foundation for the development of the diagnosis kits of snakebites.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2005年第11期1351-1354,共4页
Chinese Pharmacological Bulletin
基金
广州市科技局应用基础研究基金资助项目(No2002J1-C0171)
