抗旋毛虫肌幼虫ES抗原IgY的制备及鉴定

目的制备抗旋毛虫肌幼虫排泄-分泌(excretory-secretory,ES)抗原的鸡卵黄免疫球蛋白(IgY),测定其效价及用于检测抗原的敏感性。方法 4只24w龄罗曼母鸡用旋毛虫肌幼虫ES抗原经大腿外侧与胸部肌肉免疫4次(首次剂量为500μg/只,加强剂量为250μg/只),每次间隔10d。取免疫前和首次免疫后42d的鸡蛋卵黄,用水稀释法提取IgY,考马斯亮蓝法测定蛋白含量,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blot)及间接荧光抗体试验(IFAT)对IgY进行分析,ELISA检测纯化后IgY的效价及检测抗原的敏感性。结果罗曼鸡经ES抗原免疫后,每枚鸡蛋经提纯后均可得到约70mg抗体,SDS-PAGE表明纯化的IgY有2条主要蛋白带,分子量为67kDa、23kDa,Western blot与IFAT发现提纯的IgY可识别肌幼虫虫体与ES抗原。IgY的抗体效价为1∶107,IgY-夹心ELISA检测旋毛虫抗原的敏感性为1.17ng/mL。结论制备的抗旋毛虫ES抗原的IgY具有较高的效价与敏感性。

Production and identification of IgY against excretory-secretory antigens of Trichinella spiralis muscle larvae

To produce and purify egg yolk immunoglobulin（IgY）against excretory-secretory（ES）antigens of T.spiralis muscle larvae and assay its titer and sensitivity in antigens detection,twenty-four-week old hens were intramuscularly immunized with ES antigens of T.spiralis muscle larvae for 4 times.Four hens were firstly immunized with 500 μg of ES antigens and subsequent immunization was performed at 10-days intervals with 250 μg of ES antigens.IgY was extracted by water-dilution method from eggs of non-immunized hens and immunized hens 42 days after the first immunization.The protein concentration of IgY was measured by Bradford method,and IgY was analyzed by SDS-PAGE,Western blot and IFAT.ELISA was used to assay the titer of IgY and its sensitivity to detect antigens.The results showed that about 70 mg of IgY was extracted from each egg.SDS-PAGE showed IgY contained two major protein bands with molecular weight of 67 kDa and 23 kDa.The results of Western blot and IFAT demonstrated that somatic and ES antigens of T.spiralis muscle larvae could be recognized by IgY.The purified IgY antibody titer was 1∶107.The sensitivity of IgY-sandwich ELISA to detect antigens was 1.17ng/mL.It＇s demonstrated that the IgY to ES antigens of T.spiralis muscle larvae had high antibody titer and sensitivity.