CN114573677A - 细粒棘球蚴AgB抗原的提取方法及该抗原在包虫感染抗体检测上的应用 - Google Patents
细粒棘球蚴AgB抗原的提取方法及该抗原在包虫感染抗体检测上的应用 Download PDFInfo
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Abstract
本发明提供一种细粒棘球蚴AgB抗原的提取方法及该抗原在包虫感染抗体检测上的应用，属于生物检测技术领域，一种细粒棘球蚴AgB抗原的提取方法，包括以下步骤：步骤1、动物脏器上包囊的收集：在疫区屠宰场查找带包囊脏器，取回实验室后，冻至冰箱；步骤2、将步骤1收集的包囊进行安全性验证；步骤3、将包囊中AgB抗原进行提取并纯化；步骤4、将步骤3提取的AgB抗原进行验证。本发明通过分离纯化囊液中特异的AgB抗原，利用荧光免疫层析技术，使得包虫感染抗体检测更准确。
Description
技术领域
本发明涉及生物检测技术领域，具体涉及一种细粒棘球蚴AgB抗原的提取方法及该抗原在包虫感染抗体检测上的应用。
背景技术
棘球蚴病是由棘球绦虫（Echinococcus granulo-sus，E.g）的中绦期幼虫寄生在哺乳动物脏器内引起的疾病，是一种严重的人畜共患寄生虫病。抗原B（AgB）是由细粒棘球蚴病病原性幼虫产生的高丰度蛋白，是由原头节和包囊合成和分泌的，现有针对动物细粒棘球蚴病的检测中，对免疫抗体大多采用酶免试剂盒，针对感染抗体仅见到采用胶体金方法。
因此，需要提供一种细粒棘球蚴AgB抗原的提取方法及该抗原在包虫感染抗体检测上的应用，以解决上述现有存在的问题。
发明内容
有鉴于此，本发明提供一种细粒棘球蚴AgB抗原的提取方法及该抗原在包虫感染抗体检测上的应用。
为解决上述技术问题，本发明提供一种细粒棘球蚴AgB抗原的提取方法及该抗原在包虫感染抗体检测上的应用。
一种细粒棘球蚴AgB抗原的提取方法，包括以下步骤：
步骤1、动物脏器上包囊的收集：在疫区屠宰场查找带包囊脏器，取回实验室后，冻至冰箱；
步骤2、将步骤1收集的包囊进行安全性验证；
步骤3、将包囊中AgB抗原进行提取并纯化：取不具有感染力的包囊，融化后在通风厨中剪开，收集囊液，将囊液离心后取上清，通过预包埋有多抗的免疫亲和柱进行层析纯化，收集过滤后的囊液，弃掉，用0.1M咪唑对亲和柱进行洗脱，收集目标物，即为AgB抗原，取500ul目标物放置于3万孔径的超滤膜中，加入2ml 0.01M pH7.2磷酸盐缓冲液，5000转/min离心10min，取超滤膜上层，即为AgB抗原；
步骤4、将步骤3提取的AgB抗原进行验证。
进一步的，所述步骤2的具体操作如下：
将步骤1取回包囊的脏器分别冻至-20±2度冰箱，-80±2度冰箱，1周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊，2周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊，3周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊，4周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊。将饲喂过包囊的SPF羊隔离喂养7个月后，进行剖检发现，-20±2度冻存4周的包囊仍具有感染力，-80±2度冻存1周的包囊有感染力，冻存2周及之后的包囊饲喂SPF羊，剖检均未发现包囊，证明取回的包囊在-80度冰箱冻存2周后失去感染能力。
进一步的，所述步骤3中免疫亲和柱制备方法如下：
步骤3.1、AgB多抗包的制备：购买市售重组AgB抗原5mg，取0.5mg抗原进行乳化后，在新西兰大白兔背部进行多点皮下注射，以手触摸到鼓包为准，对兔子进行初次免疫；两周后取0.5mg抗原乳化后，进行二次免疫，再过两周后进行三次免疫，三免后一周采集兔子耳静脉血，用AgB抗原包被进行三免兔子血液的效价检测，效价不低于1：10000后，采集兔子耳静脉血20ml，37±2度放置2h，2--8度过夜，将处理后的血液均分至4个离心管中，5000转/min离心10min，取血清，硫酸铵沉淀法获得AgB多抗包；
步骤3.2、免疫亲和柱的制备：将AgB多抗包埋于琼脂糖凝胶上后，填入柱体中，制成特异免疫亲和柱，用于囊液中AgB抗原的纯化。
进一步的，将所述步骤3中细粒棘球蚴AgB抗原制备成试纸，包括以下步骤：
步骤a 、检测垫的制备；
步骤b、结合垫的制备；
步骤c. 将步骤a、步骤b的关键组分组装成试纸进行检测，与市售抗原组成的产品进行比对。
进一步的，所述步骤a的具体操作如下：
制备浓度为1.5mg/mL的AgB多抗包被液，浓度为1.6mg/mL的AgB抗原包被液，将两种包被液各自单独吸至划膜机管线中，均按照0.8μL/cm在硝酸纤维素膜上依次划出两条线，制得检测垫。
进一步的，所述步骤b的具体操作如下：
将荧光微球与步骤3中的AgB抗原进行连接后，经过封闭离心复溶制得结合垫。
一种上述的细粒棘球蚴AgB抗原的提取方法提取得到的细粒棘球蚴AgB抗原在包虫感染抗体检测上的应用。
本发明的上述技术方案至少包括以下有益效果：
1、本发明通过利用成虫期特征蛋白，与荧光免疫层析技术相结合，可检测羊细粒棘球蚴感染情况，检测时间短，对操作人员实验操作水平要求不高，无需洗板等繁琐步骤，只需一步加样后，读数即可；
2、本发明通过分离纯化囊液中特异的AgB抗原，利用荧光免疫层析技术，使得包虫感染成虫期检测更准确；
3、本发明可实现包虫感染抗体检测，且检测时间短，针对感染包虫的动物，检出阳性后可进行相应的治疗，避免直接杀害造成过大的经济损失，给养殖企业带来了裨益。
附图说明
图1为本发明实施例中跑电泳胶试验中的示意图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
一种细粒棘球蚴AgB抗原的提取方法，包括以下步骤：
步骤1、动物脏器上包囊的收集：在疫区屠宰场查找带包囊脏器，取回实验室后，冻至冰箱；
步骤2、将步骤1收集的包囊进行安全性验证；所述步骤2的具体操作如下：将步骤1取回包囊的脏器分别冻至-20±2度冰箱，-80±2度冰箱，1周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊，2周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊，3周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊，4周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊。将饲喂过包囊的SPF羊隔离喂养7个月后，进行剖检发现，-20±2度冻存4周的包囊仍具有感染力，-80±2度冻存1周的包囊有感染力，冻存2周及之后的包囊饲喂SPF羊，剖检均未发现包囊，证明取回的包囊在-80度冰箱冻存2周后失去感染能力。
步骤3、将包囊中AgB抗原进行提取并纯化：取在-80±2度冰箱中冻存2周后不具有感染力的包囊，融化后在通风厨中剪开，收集囊液，将囊液离心后取上清，通过预包埋有多抗的免疫亲和柱（提前用磷酸盐缓冲液进行平衡）进行层析纯化，收集过滤后的囊液，弃掉，用0.1M咪唑对亲和柱进行洗脱，收集目标物，即为AgB抗原，取500ul目标物放置于3万孔径的超滤膜中，加入2ml 0.01M pH7.2磷酸盐缓冲液，5000转/min离心10min，取超滤膜上层，即为AgB抗原。
所述步骤3中免疫亲和柱制备方法如下：步骤3.1.AgB多抗包的制备：购买市售重组AgB抗原5mg，取0.5mg抗原进行乳化后，在新西兰大白兔背部进行多点皮下注射，可选5-6个点，以手触摸到鼓包为准，对兔子进行初次免疫；两周后取0.5mg抗原乳化后，进行二次免疫，再过两周后进行三次免疫，三免后一周采集兔子耳静脉血，用AgB抗原包被进行三免兔子血液的效价检测，效价不低于1：10000后，采集兔子耳静脉血20ml，37±2度放置2h，2--8度过夜（15--18h），将处理后的血液均分至4个离心管中，5000转/min离心10min，取血清，硫酸铵沉淀法获得AgB多抗包；步骤3.2：免疫亲和柱的制备：将AgB多抗包埋于琼脂糖凝胶上后，填入柱体中，制成特异免疫亲和柱，用于囊液中AgB抗原的纯化。
步骤4、将步骤3提取的AgB抗原进行验证，将步骤3提取的AgB抗原进行跑电泳胶验证，与市售重组合成AgB抗原大小基本一致，见图1，泳道1为市售抗原，泳道2、3为自提抗原，证明自提抗原为目标物。
实施例2
一种细粒棘球蚴AgB抗原在包虫感染抗体检测上的应用，在本发明中，将所述步骤3中细粒棘球蚴AgB抗原制备成试纸用于在包虫感染抗体检测中。
将所述步骤3中细粒棘球蚴AgB抗原制备成试纸，包括以下步骤：
步骤a 、检测垫的制备；
所述步骤a的具体操作如下：制备浓度为1.5mg/mL的AgB多抗包被液，AgB多抗包被液用于质控线，浓度为1.6mg/mL的AgB抗原包被液，AgB抗原包被液用于检测线，将两种包被液各自单独吸至划膜机管线中，均按照0.8μL/cm在硝酸纤维素膜上依次划出两条线，制得检测垫。
步骤b、结合垫的制备；
所述步骤b的具体操作如下：将荧光微球与步骤3中的AgB抗原进行连接后，经过封闭离心复溶等步骤制得结合垫。
步骤c、将步骤a和步骤b的关键组分组装成试纸进行检测，与市售抗原组成的产品进行比对。
分别检测未免疫未感染羊血清10份，经过三次免疫Eg95疫苗羊血清10份，经过剖检有包囊的羊血清（即感染细粒棘求蚴）10份，检测结果如下：
表一
表二
表三
通过表一可看出，两个抗原特异性均合格，可满足市场检测需求；
通过表二可看出，自提抗原做出产品与免疫抗体无交叉，特异性好，市售抗原做出产品检出1份阳性，特异性较自提抗原略差；
通过表三可看出，自提抗原做出产品有1份漏检，市售抗原产品有2份漏检，敏感性不如自提抗原。
通过上述试验证明，自提抗原特异性敏感性均优于市售抗原。
产生此结果的原因是，提纯天然抗原较重组抗原更能维持其天然构象，便于其与机体产生抗体相结合。
以上是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种细粒棘球蚴AgB抗原的提取方法，其特征在于，包括以下步骤：
步骤1、动物脏器上包囊的收集：在疫区屠宰场查找带包囊脏器，取回实验室后，冻至冰箱；
步骤2、将步骤1收集的包囊进行安全性验证；
步骤3、将包囊中AgB抗原进行提取并纯化：取不具有感染力的包囊，融化后在通风厨中剪开，收集囊液，将囊液离心后取上清，通过预包埋有多抗的免疫亲和柱进行层析纯化，收集过滤后的囊液，弃掉，用0.1M咪唑对亲和柱进行洗脱，收集目标物，即为AgB抗原，取500ul目标物放置于3万孔径的超滤膜中，加入2ml 0.01M pH7.2磷酸盐缓冲液，5000转/min离心10min，取超滤膜上层，即为AgB抗原；
步骤4、将步骤3提取的AgB抗原进行验证。
2.根据权利要求1所述的细粒棘球蚴AgB抗原的提取方法，其特征在于，所述步骤2的具体操作如下：
将步骤1取回包囊的脏器分别冻至-20±2度冰箱，-80±2度冰箱，1周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊，2周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊，3周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊，4周后各取出含有2个10mm直径包囊的脏器，饲喂SPF羊；将饲喂过包囊的SPF羊隔离喂养7个月后，进行剖检发现，-20±2度冻存4周的包囊仍具有感染力，-80±2度冻存1周的包囊有感染力，冻存2周及之后的包囊饲喂SPF羊，剖检均未发现包囊，证明取回的包囊在-80度冰箱冻存2周后失去感染能力。
3.根据权利要求1所述的细粒棘球蚴AgB抗原的提取方法，其特征在于，所述步骤3中免疫亲和柱制备方法如下：
步骤3.1、AgB多抗包的制备：购买市售重组AgB抗原5mg，取0.5mg抗原进行乳化后，在新西兰大白兔背部进行多点皮下注射，以手触摸到鼓包为准，对兔子进行初次免疫；两周后取0.5mg抗原乳化后，进行二次免疫，再过两周后进行三次免疫，三免后一周采集兔子耳静脉血，用AgB抗原包被进行三免兔子血液的效价检测，效价不低于1：10000后，采集兔子耳静脉血20ml，37±2度放置2h，2--8度过夜，将处理后的血液均分至4个离心管中，5000转/min离心10min，取血清，硫酸铵沉淀法获得AgB多抗；
步骤3.2、免疫亲和柱的制备：将AgB多抗包埋于琼脂糖凝胶上后，填入柱体中，制成特异免疫亲和柱，用于囊液中AgB抗原的纯化。
4.根据权利要求1所述的细粒棘球蚴AgB抗原的提取方法，其特征在于，将所述步骤3中细粒棘球蚴AgB抗原制备成试纸，包括以下步骤：
步骤a、检测垫的制备；
步骤b、结合垫的制备；
步骤c. 将步骤a、步骤b的关键组分组装成试纸进行检测，与市售抗原组成的产品进行比对。
5.根据权利要求4所述的细粒棘球蚴AgB抗原的提取方法，其特征在于，所述步骤a的具体操作如下：
制备浓度为1.5mg/mL的AgB多抗包被液，浓度为1.6mg/mL的AgB抗原包被液，将两种包被液各自单独吸至划膜机管线中，均按照0.8μL/cm在硝酸纤维素膜上依次划出两条线，制得检测垫。
6.根据权利要求4所述的细粒棘球蚴AgB抗原的提取方法，其特征在于，所述步骤b的具体操作如下：
将荧光微球与步骤3中的AgB抗原进行连接后，经过封闭离心复溶制得结合垫。
7.一种如权利要求1-6任一项所述的细粒棘球蚴AgB抗原在包虫感染抗体检测上的应用。
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