刘原源;黄书晨;高珺珊;王晓岑;宫鹏涛;张壮志;李建华;杨举;李赫;张西臣;

• 1:吉林大学动物医学学院
• 2:新疆畜牧科学院兽医研究所

摘要(Abstract)：

目的建立简便、特异的检测犬粪便中细粒棘球绦虫抗原的双抗体夹心ELISA方法,并进行验证及初步应用。方法以细粒棘球绦虫EdiagA864蛋白为抗原,免疫日本大耳白兔,制备多克隆抗体;利用HRP标记兔抗细粒棘球绦虫EdiagA864多克隆抗体,通过溶解、超声方法处理犬粪便样品;以抗细粒棘球绦虫单克隆抗体2D12作为捕获抗体,HRP标记的兔抗细粒棘球绦虫EdiagA864多克隆抗体为检测抗体,通过棋盘法确定抗体最佳包被浓度、最佳封闭剂、待检粪样最佳稀释比例及酶标抗体最佳浓度。应用建立的双抗体夹心ELISA法对来自长春地区的64份犬粪便样品及来自新疆的8份犬粪便阳性样品进行检测。结果兔抗EdiagA864多克隆抗体的效价为105。建立的双抗体夹心ELISA法的最佳检测条件为:抗体包被浓度为1∶50,封闭剂为1%BSA,粪液稀释度为1∶5,酶标二抗稀释度为1∶800。建立的双抗体夹心ELISA法与犬贾第虫和犬蛔虫阳性样品均无交叉反应;检测不同稀释度的粪便液,当稀释至1∶20时,P/N仍大于2;检测6份阳性样品与4份阴性样品的批间和批内变异系数均小于8。用建立的方法检测8份阳性样品的结果均为阳性,64份待检样品的结果均为阴性。结论建立的双抗夹心ELISA方法特异性较强,敏感性较高,重复性较好,为细粒棘球绦虫流行病学调查及诊断提供了一种更简便、快速、特异的免疫学检测方法。

关键词(KeyWords)： 细粒棘球绦虫;双抗体夹心ELISA;EdiagA864;多克隆抗体

Abstract:

Keywords:

基金项目(Foundation): 国家公益性行业(农业)科研专项(201303042)

作者(Author): 刘原源;黄书晨;高珺珊;王晓岑;宫鹏涛;张壮志;李建华;杨举;李赫;张西臣;

Email:

DOI: 10.13200/j.cnki.cjb.001619

参考文献(References)：

• [1]陈洁,吾拉木·马木提.犬感染细粒棘球绦虫的诊断研究进展及展望[J].新疆医科大学学报,2011,34(3):251-253.
• [2]ZIADINOV I,MATHIS A,TRACHSEL D,et al.Canine echinococcosis in Kyrgyzstan:epidemiology and transmission analysis incorportaing diagnostic uncertainty[J].Int J Parasitol,2008,38:1179-1190.
• [3]CRAIG P S.Diagnosis of canine echinococcosis:Comparison of coproantigen and serum antibody tests with arecoline purgation in uraguay[J].Vet Parasitol,1995,56:293.
• [4]CRAIG P S.Immunodiagnosis of Echinococcus granulosus//Andersen FI,Chai JJ,Liu FJ,et al.Compendium on cystic echino-coccosis with special reference[M].Provo:Brigham Young University,1993:105.
• [5]ALLAN J,CRAIG P,GARCIA NOVAL J,et al.Coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans[J].Parasitology,1992,104(2):347-355.
• [6]ALLAN J,CRAIG P.Coproantigens in taeniasis and echinococcosis[J].Parasitol Int,2006,55(8):75-80.
• [7]ZHANG X,GUNUR T,MI X Y,et al.Establishment of an antibody sandwich ELISA for detection of Echinococcus granulosus antigen in canine feces[J].Prog in Vet Med,2012,33(3):19-23.(in Chinese)张旭,古努尔·吐尔逊,米晓云,等.犬细粒棘球绦虫粪抗原夹心ELISA检测方法的建立[J].动物医学进展,2012,33(3):19-23.
• [8]JIAO W,CHAI J J,ISRAYIL O,et al.Diagnosis of Echinococcus granulosus infection in dogs by using coproantigen detectionⅠ.Establishment of methods by using E.granulosus adult somatic antigen-antibody system for coproantigen detection in dogs[J].Endem Dis Bull,1998,13(2):16-21.(in Chinese)焦伟,柴君杰,伊斯拉音·乌斯曼,等.粪抗原检测法诊断犬细粒棘球绦虫感染的研究Ⅰ.用细粒棘球绦虫成虫虫体抗原-抗体系统检测粪抗原方法的建立[J].地方病通报,1998,13(2):16-21.
• [9]JIAO W,CHAI J J,ISRAYIL O,et al.Coproantigen detection on dogs infected with Echinococcus granulosusⅢ.A field evaluation of coproantigen detection in diagosis of E.granulosus infection in dogs with double antibody sandwich ELISA[J].Chin J Zoon,1999,15(6):28-29.(in Chinese)焦伟,柴君杰,伊斯拉音·乌斯曼,等.粪抗原检测法诊断犬细粒棘球绦虫感染的研究III.双抗体夹心ELISA法检测粪抗原诊断家犬细粒棘球绦虫感染的现场评价[J].中国人兽共患病杂志,1999,15(6):28-29.
• [10]JIAO W,CHAI J J,ISRAYIL O,et al.Comparative assessment of Echinococcus granulosus protoscolex somatic and excretory/secretory antigen-antibody systems for coproantigen detection in dogs[J].Endem Dis Bull,2001,16(1):1-4.(in Chinese)焦伟,柴君杰,伊斯拉音·乌斯曼,等.细粒棘球蚴原头节虫体和排泄分泌抗原抗体系统检测犬粪抗原的比较研究[J].地方病通报,2001,16(1):1-4.
• [11]CHEN J,MA H M,GULIPALL M,et al.Development of sandwich-ELISA detection system using recombinant protein of Eg AB8/3 for diagnosis of dogs infected with Echinococcus spp[J].J Xingjiang Med Univ,2011,34(3):240-245.(in Chinese)陈洁,马海梅,古力帕丽·麦曼提依明,等.用Eg Ag B8/3重组蛋自ELISA-双抗夹心法建立棘球绦虫感染犬粪抗原检测系统的研究[J].新疆医科大学学报,2011,34(3):240-245.
• [12]HUANG Y,DAVID H D,CHRISTINE A J,et al.An immunological coproantigen test used to detect infection of Echinococcus in dogs[J].J Prev Med,Inf,2008,24(6):407-411.(in Chinese)黄燕,DAVID H D,CHRISTINE A J,等.犬粪便棘球绦虫抗原的双抗体夹心酶联免疫吸附试验检测法建立与应用[J].预防医学情报杂志,2008,24(6):407-411.