CN110133290B - 一种诊断犬恶丝虫病的elisa试剂盒 - Google Patents
一种诊断犬恶丝虫病的elisa试剂盒 Download PDFInfo
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Abstract
本发明涉及生物技术领域,公开了一种诊断犬恶丝虫病的ELISA试剂盒。本发明所述试剂盒包括包被有犬恶丝虫小热休克蛋白的固相载体。本发明提供了犬恶丝虫小热休克蛋白作为犬恶丝虫病诊断抗原的相关应用,相关实验结果显示,犬恶丝虫小热休克蛋白能被自然感染犬恶丝虫病的犬血清识别,犬恶丝虫小热休克蛋白制备的兔抗IgG能识别犬恶丝虫小热休克蛋白,具有良好的免疫原性和反应原性;同时在间接ELISA方法中表现出极高敏感性和特异性,种种结果证明犬恶丝虫小热休克蛋白可以作为犬恶丝虫病的诊断抗原,以及应用到相关检测试剂盒中。
Description
技术领域
本发明涉及生物技术领域,特别涉及一种诊断犬恶丝虫病的ELISA试剂盒。
背景技术
犬恶丝虫病是一种通过蚊媒传播的人畜共患寄生虫病。犬恶丝虫(Dirofilariaimmitis)主要寄生于犬、猫的肺动脉和右心室,寄生在肺动脉的犬恶丝虫成虫可引发宿主的血管病变以及肺动脉高血压等反应。由于全球变暖增加了蚊媒的活动性,近年来有关人感染犬恶丝虫病的病例报道呈上升趋势。
犬恶丝虫病的常规诊断方法主要有病原检测,血清学检测和分子生物学检测。目前,血清学检测是最常用的检测方法,分为抗原检测和抗体检测。抗原检测的假阴性结果常见于轻微感染、雌虫尚未成熟、仅感染雄虫等情况;抗体检测较抗原检测的优势在于,不论雌虫还是雄虫都能检测出来。宿主感染犬恶丝虫2个月后,幼虫刺激机体所产生的免疫反应就能被检测出来,抗体检测比抗原检测能更早地检测出犬恶丝虫,适用于犬恶丝虫病的早期检测。探索发现有效的犬恶丝虫病新诊断方法对于犬恶丝虫病的高效诊断具有重要的现实意义。
发明内容
有鉴于此,本发明的目的在于提供一种诊断犬恶丝虫病的ELISA试剂盒,使得所述试剂盒在检测时具备较高的特异性和敏感性。
为实现上述发明目的,本发明提供如下技术方案:
一种诊断犬恶丝虫病的ELISA试剂盒,包括包被有犬恶丝虫小热休克蛋白(Di-sHSP12.6)的固相载体。在本发明具体实施方式中,所述固相载体可选择为96孔培养板或与其相似的固相载体,所述犬恶丝虫小热休克蛋白包被浓度为100μl/孔,可采用包被液包被于载体上,包被液由0.39g Na2CO3,35mM NaHCO3,0.2M NaCl,调PH至9.6获得。
在确定了试剂盒核心组分后,所述ELISA试剂盒还包括酶标二抗、洗涤液、显色液、封闭液、稀释液、终止液中的一种或两种以上。
其中,所述酶标二抗优选为HRP标记的兔抗犬IgG,在本发明具体实施方式中,所述酶标二抗采用购自于武汉博士德生物工程有限公司的产品,酶标二抗稀释比例为1:2000;
所述洗涤液优选为PBS-T洗涤液,在本发明具体实施方式中,所述PBS-T洗涤液组成为:8gNaCl,0.2gKCl,1.42gNa2HPO4,0.27gKH2PO4,溶于800mL去离子水中,加入0.5mLTween20,定溶至1L;所述显色液优选为TMB显色液;
所述封闭液优选为脱脂牛奶,在本发明具体实施方式中,所述脱脂牛奶浓度为5%。
所述终止液优选为硫酸溶液,浓度优选为2mol/L;配制方法为178mL去离子水中缓慢滴加21.7mL的98%的浓硫酸,冷却至室温,4℃保存;
所述稀释液优选为PBS;配制方法为8gNaCl,0.2gKCl,1.42gNa2HPO4,0.27gKH2PO4,溶于800mL去离子水中,定溶至1L,灭菌后,室温保存。
在本文中,犬恶丝虫小热休克蛋白可以是非天然的,例如是合成的或者由人工载体表达的(业内常称为重组蛋白rDi-sHSP12.6)。术语“非天然的”是指目标物质不是自然界天然存在的,这并不排除所述非天然物质与天然存在的物质具有相同的结构和/或组成。
在线虫中,现在已经发现了14种小分子热休克蛋白(small heat shockprotein,sHSP),根据分子质量的不同可以分成6组:sHSP12s(包括HSP12.1、HSP12.2、HSP12.3和HSP12.6)、sHSP16s(HSP16.1、HSP16.2、HSP16.41、HSP16.48以及新发现的F08H9.3、F08H9.4)、HSP25、HSP43、HSP17.5和胁迫诱导蛋白-1(stress-induced protein-1,SIP-1),研究证实它不仅能够在应激条件下维持细胞必需的蛋白质空间构象,保护细胞生命活动以维持细胞生存,而且在蛋白质折叠、跨膜运输、转运、机体免疫、细胞凋亡、细胞骨架及核骨架稳定等基本功能上发挥着重要的作用。目前,犬恶丝虫小热休克蛋白(Di-sHSP12.6)在犬恶丝虫病中的相关研究尚未见报道。
本发明通过原核表达得到重组犬恶丝虫小热休克蛋白(rDi-sHSP12.6,其和Di-sHSP12.6具备完全相同的氨基酸序列,如SEQ ID NO:1所示),对其进行免疫印迹,ELISA以及早期诊断方法的建立。免疫印迹结果显示重组蛋白rDi-sHSP12.6制备的兔抗IgG能识别重组蛋白rDi-sHSP12.6,重组蛋白rDi-sHSP12.6能被自然感染犬恶丝虫病的犬血清识别,不能被健康犬血清识别,说明犬恶丝虫小热休克蛋白具有良好的免疫原性和反应原性。
间接ELISA结果显示,用rDi-sHSP12.6蛋白检测24份犬恶丝虫阳性血清和24份犬恶丝虫阴性血清,结果显示,22份阳性血清OD450>临界值,2份阳性血清和24份阴性血清OD450<临界值(0.699),则评估该方法的敏感性为91.6%(22/24);同时,不与犬细粒棘球绦虫阳性血清、犬细颈囊尾蚴阳性血清和犬钩虫阳性血清(共18份)发生交叉反应,检测的18份犬弓首蛔虫阳性血清中仅有2份发生轻微的交叉反应,则评估该方法的特异性为91.6%(34/36);综上所述,rDi-sHSP12.6具有较高的敏感性和特异性,且交叉反应率低,具有较好的诊断效果,适合作为犬恶丝虫病的诊断抗原以及作为诊断抗原制备相关的检测试剂盒。
由以上技术方案可知,本发明提供了犬恶丝虫小热休克蛋白作为犬恶丝虫病诊断抗原的相关应用,相关实验结果显示,犬恶丝虫小热休克蛋白能被自然感染犬恶丝虫病的犬血清识别,犬恶丝虫小热休克蛋白制备的兔抗IgG能识别犬恶丝虫小热休克蛋白,具有良好的免疫原性和反应原性;同时在间接ELISA方法中表现出极高敏感性和特异性,种种结果证明犬恶丝虫小热休克蛋白可以作为犬恶丝虫病的诊断抗原,以及应用到相关检测试剂盒中。
附图说明
图1所示为Di-sHSP12.6基因的PCR扩增;其中,M为DNA分子质量标准(DL2000);1-4:Di-sHSP12.6PCR产物;
图2所示为重组质粒pET32a(+)-sHSP12.6的双酶切鉴定;其中,M:DNA分子质量标准(DL2000);1-4:pET32a(+)-sHSP12.6的双酶切产物;
图3所示为重组Di-sHSP12.6蛋白的表达;其中,M:蛋白质分子质量标准;1:IPTG诱导的未含pET32a(+)-sHSP12.6的大肠杆菌;2-4:IPTG诱导的含pET32a(+)-sHSP12.6的大肠杆菌;
图4所示为rDi-sHSP12.6的可溶性分析;其中,M:蛋白质分子质量标准;1:8M尿素溶解;2:4M尿素溶解;3:2M尿素溶解;4:上清;
图5所示为rDi-sHSP12.6的表达、纯化与免疫印迹分析;其中,1:IPTG诱导的未含有重组pET32a(+)-sHSP12.6的大肠杆菌;2:IPTG诱导的含有重组pET32a(+)-sHSP12.6的大肠杆菌;3:经镍柱纯化后的兔抗rDi-sHSP12.6的IgG;4:经镍柱纯化后的rDi-sHSP12.6;5:自然感染犬恶丝虫的犬阳性血清特异性识别rDi-sHSP12.6;6:经镍柱纯化后的兔抗rDi-sHSP12.6的IgG识别rDi-sHSP12.6;7:健康的阴性犬血清识别rDi-sHSP12.6;
图6所示为雌性和雄性犬恶丝虫横切面Di-sHSP12.6蛋白的间接免疫荧光定位;其中,I:肠;UT:子宫;TE:睾丸;MU:肌肉;HY:侧索;PS:假体腔;比例尺:200μm;A,B:雌虫;C,D:雄虫;注:绿色荧光区域为蛋白分布大概位置;
图7所示为重组蛋白rDi-sHSP12.6的间接ELISA(特异性);
图8所示为重组蛋白rDi-sHSP12.6的间接ELISA(敏感性)。
具体实施方式
本发明公开了一种诊断犬恶丝虫病的ELISA试剂盒,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明所述试剂盒已经通过实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述试剂盒进行改动或适当变更与组合,来实现和应用本发明技术。
本发明通过提取犬恶丝虫成虫虫体总RNA,并逆转录为cDNA,从cDNA中扩增犬恶丝虫小热休克蛋白(Di-sHSP12.6)编码序列。经T克隆后,将扩增产物以酶切连接的方式导入表达载体中,以大肠杆菌进行原核表达获得重组的rDi-sHSP12.6。
在具体实施方的实验中,所有实验动物严格按照“中华人民共和国动物保护法”(2009年9月18日发布的草案)进行处理。所有程序严格按照“四川农业大学动物伦理委员会实验动物护理指南”(中国,雅安;批准号:2013-028)实施。所有方法均按照相关准则和规定进行,包括任何相关细节。
犬恶丝虫虫体来自四川省自然感染并剖检的犬,虫体自犬心脏分离后使用PBS清洗,参照有关文献资料,经形态学鉴定为犬恶丝虫。犬恶丝虫病阳性血清采自四川省自然感染犬恶丝虫的阳性犬。用于交叉反应的血清采自经过粪检或者自然死亡后剖检的犬(死亡前已采集血液);阴性血清采自春季经体内外驱虫处理后的3月龄比格犬(实验专用犬)。
实验动物为2只健康新西兰兔,雌性,3月龄左右,1.5~2.0kg,购自成都达硕生物有限公司。
菌株和质粒:大肠杆菌DH5α、大肠杆菌BL21(DE3),购自天根公司;TaKaRa pMD19-TVector,购自大连宝生物工程有限公司;原核表达载体pET32a(+),购自Invitrogen。
以下就本发明所提供的一种诊断犬恶丝虫病的ELISA试剂盒做进一步说明。
实施例1:基因扩增
1、犬恶丝虫总RNA的提取
预先准备好-80℃预冷的研钵,从液氮中取出犬恶丝虫成虫虫体(约20mg),剪碎后,加入少许液氮并充分研磨,接着按照天根动物组织RNA提取试剂盒指示,提取犬恶丝虫总RNA,-80℃保存。
2、合成cDNA第一条链
以提取的犬恶丝虫总RNA为模版,OligodT(18)为引物,按照Thermo公司反转录试剂盒指示,合成cDNA的第一条链。
3、引物的设计与合成
参照NCBI中马来丝虫sHSP12.6核苷酸序列(gb:XM_001900555.1),利用Primerpremier5.0设计引物,下划线部分为酶切位点(BamHⅠ和XhoⅠ):
Di-sHSP12.6上游引物(F):CGGATCCATGGAAGAAAAGGTAGTGGA
Di-sHSP12.6下游引物(R):CGAGCTCTCATGCTTTCTTTTTGGC
4、扩增程序与体系
以犬恶丝虫cDNA为底物模板进行PCR扩增。扩增程序见表1,扩增体系见表2:
表1
表2
5、产物回收
将上述产物进行核酸凝胶电泳后,在凝胶成像***下比对条带大小是否正确。然后在紫外灯下切取目的条带,并按照天根生物科技公司胶回收试剂盒对目的条带进行DNA回收,-20℃保存。
6、Di-sHSP12.6基因的扩增和基本理化属性
以犬恶丝虫cDNA为模板扩增出一条340bp左右的条带(图1,1-4泳道),生工生物有限公司测序结果显示该序列与NCBI中的序列同源性达到100%。
本发明扩增的Di-sHSP12.6基因长度为342bp,经氨基酸序列分析,编码113个氨基酸(最后3个碱基为终止密码子,不编码氨基酸),分子质量为13052.71,推测其分子式为C575H910N162O179S3,ProtParam预测Di-sHSP12.6蛋白主要含有Val、Thr、Lys和Asp。信号肽分析结果显示Di-sHSP12.6无信号肽,跨膜区预测显示Di-sHSP12.6位于胞外区。
实施例2:克隆与鉴定
1、连接
将回收的目的条带DNA片段与pMD19-T载体相连接,16℃过夜(不超过16h),连接体系见表3:
表3
2、转化
取出连接好的产物8μL置于1mL的EP管中,加入30μLE.coliDH5α感受态细胞,用微量加样器轻轻混匀,冰浴30min,42℃水浴90s,再冰浴5min。向EP管中加入600μLLB液体培养基,于37℃,180r/min环境中培养1.5h后,取100μL菌液均匀接种于含氨苄(AMP)抗性的固体培养基上,37℃恒温培养箱中培养12h。
3、测序鉴定
挑选形态正常,分散的单个菌落接种于1mLLB液体培养基(含1μLAMP)中,37℃180r/min环境中培养6h,菌液PCR鉴定是否能扩增出目的片段大小的条带,将能扩增出目的片段的菌液取400μL送生工生物有限公司测序。菌液PCR反应体系见表4:
表4
实施例3:构建表达载体
1、提取质粒
将实施例2测序正确的菌液进行扩大培养,按照天根质粒小提试剂盒说明书抽提Di-sHSP12.6的质粒,并最后吸取5μL混合5μLloadingbuffer进行核算凝胶电泳,检测质粒的纯度。
2、目的基因质粒和表达载体质粒的酶切回收
将目的基因质粒及按照2.3.1的方法获得表达载体pET32a(+)的质粒,用相同的Takara快切酶进行酶切,获得相同的黏性末端。用(BamHⅠ和SacⅠ)快切酶对Di-sHSP12.6和pET32a(+)表达载体质粒进行酶切。按照实施例1中的方法对酶切后目的基因质粒和表达载体质粒的DNA片段进行胶回收。
3、目的基因连接表达载体
分别取2中回收的DNA片段,用T4连接酶进行连接,构建pET32a(+)-重组蛋白表达载体质粒。瞬时离心后,于PCR仪中22℃连接2h。按照实施例2中方法进行转化。
4、重组质粒的双酶切鉴定
按照1中方法获取重组质粒,分别用相应的快切酶对重组质粒双酶切,并进行核酸凝胶电泳,确定目的基因已经连接到表达载体上。将确定的菌液取400μL送生工生物有限公司测序。
经核酸凝胶电泳鉴定,重组质粒能酶切出与目的基因大小的片段,与预期结果相同(图2)。将重组Di-sHSP12.6质粒转入BL21大肠杆菌中,经由AMP抗性的的平板筛选后,挑取形态规则的单个菌落进行菌落PCR鉴定,阳性菌送生工生物有限公司测序,测序序列与目的基因序列相同,表明构建表达载体成功。
实施例4:蛋白的原核表达
(1)将实施例2中测序正确的菌液进行扩大培养,并按照实施例3中方法获取重组质粒。
(2)取10μL质粒置于1mL的EP管中,加入50μLE.coliBL21感受态细胞,用微量加样器轻轻混匀,冰浴30min,42℃水浴90s,再冰浴5min。向EP管中加入600μLLB液体培养液,于37℃,180r/min环境中培养1.5h后,取150μL菌液均匀接种于含氨苄(AMP)抗性的固体培养基上,在37℃恒温培养箱中培养12h。
(3)按照实施例2中方法进行PCR鉴定,将PCR鉴定为阳性的菌液进行5mL扩大培养,37℃,180r/min培养至OD值=0.6,加入诱导剂IPTG(1mmol/L)诱导6h,另一瓶不加IPTG做空白对照。
(4)取1mL诱导后的菌液,4℃,12000r/min,离心1min,收集菌体沉淀,加入40μLddH2O和10μL5×SDS上样Buffer混匀。
(5)沸水中加热10min后,4℃,12000r/min,离心1min,取适量上清进行SDS-PAGE。
(6)取下PAGE胶,放入装有考马斯亮蓝染色剂的容器中,染色30min左右,于沸水中脱色10min,于凝胶成像***下观察结果。
SDS-PAGE显示得到约30KDa(Di-sHSP12.6大小约为12KDa,His标签大小约为18KDa)(图3)。
实施例5:重组蛋白的可溶性分析
将能够成功表达的菌接种于1LLB液体培养基(含AMP100μg/mL)中,37℃,180r/min培养3h,待OD=0.6,加入10mLIPTG诱导6h。将所有菌液4℃,12000r/min离心10min,弃上清保留余下菌体,用10mL(50mmol/L)的Tris-HCl裂解细胞壁,然后将混悬液放入冰水混合物中,超声破碎细胞。4℃,12000r/min,离心10min,取上清按照实施例4中方法制样。沉淀物分别用梯度尿素溶解,再分别离心收集上清按照实施例4中方法制样。将所得样品进行SDS-PAGE后,于凝胶成像***成像。结果显示rDi-sHSP12.6蛋白均表达在上清(图4)。
实施例6:重组蛋白的纯化
将能够成功表达的菌接种于1LLB液体培养基(含AMP100μg/mL)中,37℃,180r/min培养3h,待OD=0.6,加入10mLIPTG诱导6h,获得重组蛋白菌液。
(1)将所有菌液4℃,12000r/min,离心10min,弃上清,保留菌体。
(2)用10mL(50mmol/L)的Tris-HCl裂解细胞壁,然后将混悬液放入冰水混合物中,超声破碎细胞。
(3)4℃,12000r/min,离心10min,收集上清。
(4)沉淀分别用2mol/L,4mol/L,6mol/L,8mol/L的尿素溶液洗涤,再4℃,12000r/min,离心10min,收集上清。
(5)用0.45μm滤膜过滤上述所得上清液,置于冰水混合物中备用。
(6)用0.22μm滤膜过滤BandingBuffer和ElutionBuffer,同20%酒精一起置于冰水混合物中备用,使用前超声1-2min去除气泡。
(7)取出镍离子亲和层析柱,连接到蛋白纯化仪上,用BandingBuffer平衡5-10个床体积。
(8)注入蛋白,待BandingBuffer平衡5-10个床体积,开始分别用13%,25%,50%,75%和100%的ElutionBuffer(含400mM咪唑)进行洗脱,待出现洗脱峰,开始收集蛋白样品。
(9)按照超滤管使用方法进行重组蛋白的超滤,期间加入0.22μm滤膜过滤后并灭菌的PBS进行咪唑和其他物质的置换。
(10)超滤后的蛋白,使用超微量紫外分光光度计测定蛋白浓度,加入1-2滴甘油后-80℃保存,以保证蛋白的稳定。
结果显示rDi-sHSP12.6(图5,泳道4)蛋白的纯化效果良好。
实施例7:重组蛋白免疫原性和反应原性的分析
2.7.1兔抗重组蛋白IgG的制备
将纯化后的蛋白中加入等量的弗氏不完全佐剂或者弗氏完全佐剂,使得蛋白终浓度为0.2mg/mL,于超声破碎仪中超声至油包水状态乳制剂(滴入水中1min不扩散)。分4次对新西兰兔进行皮下注射免疫,具体免疫程序见表5:
表5
第四次免疫后7天,从兔耳缘静脉采血,并分离血清-20℃保存。
2、IgG的粗提
(1)取10mL血清加生理盐水10mL,再逐滴加入(NH4)2SO4饱和溶液5mL(直至产生絮状沉淀为止),使之成为20%(NH4)2SO4溶液,边加边搅拌,静置30min
(2)4℃,3000r/min,离心20min,弃沉淀(除去纤维蛋白)。
(3)向上清中加入(NH4)2SO4饱和溶液15mL左右,使之成为50%(NH4)2SO4溶液,静置30min。
(4)4℃,3000r/min,离心20min,弃上清。
(5)沉淀用A2BangdingBuffer充分溶解后可上柱纯化。
3、IgG的纯化
(1)按正确步骤将纯化柱安装于仪器上,用前20%酒精冲洗仪器管子,直至平衡。
(2)用A2BangdingBuffer平衡柱子,直至离子线平衡。
(3)向仪器中注入粗提好的IgG,继续A2BangdingBuffer平衡柱子至离子线平衡。
(4)接着用BBuffer洗脱,出现洗脱峰后,控制为低流速,收集纯化后的IgG样品,立即用Tris中和,PH=8.0左右。
(5)重复加样纯化,重复(2)-(4)的步骤。
(6)纯化完后,用20%的酒精充满仪器管,直至离子线平衡。
(7)取少量纯化后的IgG按照实施例4中方法制样验证。
按照上述方法制备兔抗r-Di-sHSP12.6(图5,泳道3)蛋白IgG,并通过镍柱亲和层析对兔抗重组蛋白IgG进行纯化,效果良好,分别出现一条50KDa左右的重链和25KDa左右的轻链。
4、Di-sHSP12.6蛋白的免疫印迹
(1)将重组蛋白进行SDS-PAGE。
(2)切去凝胶多余部分,分别裁剪24层滤纸(2张)和硝酸纤维素膜(NC膜),共同置于转膜缓冲液中平衡3次,每次5min。
(3)将平衡好后的凝胶,滤纸和NC膜,按照阴性电板,滤纸,凝胶,NC膜,滤纸的顺序叠放,并用玻棒去除中间多余气泡。
(4)盖上阳极电板,电流按照1mA/cm2进行设置,共转移30min。
(5)转膜结束后,取出凝胶用考马斯亮蓝染色,于凝胶成像***中观察转膜的效率。
(6)将NC膜放入容器中,用TBST进行洗涤3次,每次5min。
(7)向容器中加入5%的脱脂奶粉封闭2h,加入一抗(犬恶丝虫阳性血清或者制备的兔抗IgG),按照1:100稀释后,4℃,过夜。
(8)弃一抗,用TBST洗涤3次,每次5min,加入二抗(HRP-标记兔抗犬IgG),按照PBS1:2000稀释后,室温下孵育2h。
(9)弃二抗,用TBST洗涤3次,每次5min,取出NC膜置于干净容器中,向NC膜上滴加新鲜配置的二氨基联苯胺(DAB)显色液。
(10)出现条带后,立即用双蒸水终止反应,于凝胶成像***中拍照。
按照上述中的方法对rDi-sHSP12.6蛋白的免疫原性和反应原性进行分析。免疫印迹结果显示r-Di-sHSP12.6蛋白具有良好的免疫原性和反应原性。重组蛋白制备的兔抗IgG能够识别重组蛋白(图5,泳道6,白色箭头所示),重组蛋白能被自然感染犬恶丝虫病的犬血清识别,不能被健康犬血清识别,说明该蛋白具有良好的反应原性和免疫原性(图5,泳道5,白色箭头所示)。
5、间接免疫荧光定位
(1)切片:将4%多聚甲醛固定的犬恶丝虫雌性成虫用石蜡包埋并切片(厚度4mm)。
(2)烤片:把切片置于60℃恒温箱中2h。
(3)将烤好的切片按以下顺序进行脱蜡和水化:二甲苯Ⅰ(7min)、二甲苯Ⅱ(7min)、100%酒精Ⅰ(3min)、100%酒精Ⅱ(3min)、95%酒精(3min)、85%酒精(3min)、75%酒精(3min)、蒸馏水(8min)。
(4)将脱腊和水化完成后的切片放入柠檬酸钠缓冲液中抗原热修复,95℃以上加热15min。冷却后用PBS冲洗切片3次,5min/次。
(5)用3%H2O2滴在组织上,37℃,25min,用PBS洗涤3次,5min/次。
(6)向组织上滴加5%BSA封闭液,室温,45min。
(7)弃多余液体,加兔抗重组蛋白IgG(1:100稀释),在湿盒中4℃孵育过夜;PBS洗涤3次,4min/次。
(8)加入用0.1%的伊文氏蓝稀释的FITC标记的羊抗兔IgG(1:100稀释),37℃避光孵育1h。
(9)PBS洗涤3次,4min/次。
(10)用适量甘油缓冲液进行封片,将切片置于荧光显微镜下观察并成像。
间接免疫荧光染色检测Di-sHSP12.6在雌性和雄性犬恶丝虫成虫横切面的分布,结果显示sHSP12.6主要分布于雌性犬恶丝虫的表皮和肠中,肌肉中有少量分布;在雄性犬恶丝虫体内主要分布于表皮,并且能分泌到肠道中,肌肉中也只有少量分布(图6,A,B,C,D,其中A、C为阳性,B、D为阴性)。
实施例8:rDi-sHSP12.6蛋白间接ELISA方法的建立
1、操作方法
(1)取96孔酶标板,用包被液稀释重组蛋白(抗原),加入稀释好的蛋白100μL/孔,4℃,包被过夜。
(2)弃蛋白液,用PBST洗涤3次,5min/次,于微量震荡仪上震荡清洗,(尽量加满,但不能串孔)。
(3)加入5%脱脂奶粉,250μL/孔,37℃,孵育1.5h。
(4)PBST洗涤3次,5min/次。
(5)加入用PBS稀释好的血清,100μL/孔,37℃,孵育1h。
(6)PBST洗涤3次,5min/次。
(7)加入用PBS按比例稀释HRP标记的兔抗犬IgG,100μL/孔,37℃,孵育1h。
(8)PBST洗涤4次,5min/次。
(9)向96孔酶标板中加入可溶性单组分底物TMB显色液,100μL/孔,室温,避光孵育20min。
(10)加入2mol/L的H2SO4,100μL/孔,终止反应。
(11)将96孔板置于酶标仪上,测定其OD值(λ=450nm)。
可根据本实施例的间接ELISA试剂组成一种试剂盒。
2、条件优化
(1)确定最佳的抗原包被浓度和血清浓度:参照棋盘滴定法,设置6个抗原包被浓度如:(20.20μg、10.10μg、5.05μg、2.53μg、1.26μg和0.63μg/每孔)和4个血清稀释度(1:20、1:40、1:80和1:160),按照2.8.1方法完成试验后,于酶标仪上测定读数,计算出P/N值,确定最佳的抗原包被浓度和血清浓度。以阳性血清OD450接近1,P/N最大的条件作为最佳。
(2)确定最佳的封闭液:按照最佳的抗原包被浓度和血清浓度,分别使用5%脱脂奶粉和5%的BSA作为封闭液,完成试验后于酶标仪上测定读数,计算出P/N值,确定最佳的封闭液。以阳性血清OD450接近1,P/N最大的条件作为最佳。
(3)确定最佳的一抗孵育时间:按照最佳的抗原包被浓度、血清浓度和封闭液,设立4个不用的孵育时间(0.5h,1h,1.5h和2h),完成试验后于酶标仪上测定读数,计算出P/N值,确定最佳的一抗孵育时间。以阳性血清OD450接近1,P/N最大的条件作为最佳。
(4)确定最佳的二抗孵育浓度:按照最佳的抗原包被浓度、血清浓度和一抗孵育时间,设立5个不同的二抗稀释度(1:1000,1:2000,1:3000,1:4000和1:5000),完成试验后于酶标仪上测定读数,计算出P/N值,确定最佳的二抗孵育浓度。以阳性血清OD450接近1,P/N最大的条件作为最佳。
(5)确定最佳的显色时间:按照以上的最佳条件,设立5个不同的显色时间(10min,15min,20min,25min,30min),完成试验后于酶标仪上测定读数,计算出P/N值,确定最佳的显色时间。以阳性血清OD450接近1,P/N最大的条件作为最佳。
3、临界值的确定
按照2.8.2中优化的试验条件进行试验,采用cutoff值法确定临界值。测定24份犬恶丝虫病阴性血清OD450,计算出所有血清OD450的平均值和标准差。cutoff值=平均值+3倍标准差,设置3个重复。
4、特异性和敏感性的确定
按照2中优化的试验条件进行试验,分别对本实验室采集到的犬细粒棘球绦虫阳性血清(8份),犬细颈囊尾蚴阳性血清(2份),犬钩虫阳性血清(8份)和犬弓首蛔虫血清(18份)分别进行交叉反应性的测定。按照3中确定的临界值,计算出相应的特异性和敏感性,计算公式如下:特异性=真阴性血清数/(真阴性血清数+假阳性血清数)×100%,敏感性=真阳性血清数/(真阳性血清数+假阴性血清数)×100%,每组设置3个重复。
按照优化的间接ELISA方法,用rDi-sHSP12.6蛋白检测犬细粒棘球绦虫阳性血清(8份),犬细颈囊尾蚴阳性血清(2份),犬钩虫阳性血清(8份)和犬弓首蛔虫血清(18份),结果显示,不与犬细粒棘球绦虫阳性血清、犬细颈囊尾蚴阳性血清和犬钩虫阳性血清发生交叉反应,检测的18份犬弓首蛔虫阳性血清中仅有2份发生轻微的交叉反应(图7),特异性为94.4%(34/36)。
5、检测试验的重复性
(1)批内重复性检验:在同一张96孔酶标板中包被相同浓度的重组蛋白,检测6份确定的犬恶丝虫病阳性血清,每份血清重复3个孔,按照优化好的ELISA方法,进行批内重复试验,计算变异系数,检验批内重复性。
(2)批间重复性检验:分别在3张96孔酶标板中包被相同浓度的重组蛋白,检测6份确定的犬恶丝虫病阳性血清,每份血清重复3个孔,按照优化好的ELISA方法,进行批间重复试验,计算变异系数,检验批间重复性。
批间变异系数为(5.63%-8.15%),批内变异系数为(0.96%-3.89%),批间变异系数小于10%,批内变异系数小于5%,表明该试验具有良好的重复性。
6、临床检测
用建立好的间接ELISA方法分别对24份犬恶丝虫病阳性血清和24犬恶丝虫病阴性血清进行检测,根据临界值,特异性和敏感性判断ELISA方法的可靠性。
按照优化的间接ELISA方法,用rDi-sHSP12.6蛋白检测24份犬恶丝虫阳性血清和24份犬恶丝虫阴性血清,结果显示,22份阳性血清OD450>临界值,2份阳性血清和24份阴性血清OD450<临界值(0.699),则评估该方法的敏感性为91.6%(22/24)(图8)。
以上所述仅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,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 四川农业大学
<120> 一种诊断犬恶丝虫病的ELISA试剂盒
<130> MP1908394
<160> 1
<170> SIPOSequenceListing 1.0
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Lys Glu Ile Glu Val Lys Val Cys Gly Asp Asn Leu Val Ile His Cys
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Claims (8)
1.一种诊断犬恶丝虫病的ELISA试剂盒,其特征在于,包括包被有犬恶丝虫小热休克蛋白的固相载体,所述犬恶丝虫小热休克蛋白的序列如SEQ ID NO:1所示。
2.根据权利要求1所述ELISA试剂盒,其特征在于,还包括酶标二抗、洗涤液、显色液、封闭液、稀释液、终止液中的一种或两种以上。
3.根据权利要求2所述ELISA试剂盒,其特征在于,所述酶标二抗为HRP标记的兔抗犬IgG。
4.根据权利要求2所述ELISA试剂盒,其特征在于,所述洗涤液为PBS-T洗涤液。
5.根据权利要求2所述ELISA试剂盒,其特征在于,所述显色液为TMB显色液。
6.根据权利要求2所述ELISA试剂盒,其特征在于,所述封闭液为脱脂牛奶。
7.根据权利要求2所述ELISA试剂盒,其特征在于,所述稀释液为PBS。
8.根据权利要求2所述ELISA试剂盒,其特征在于,所述终止液为硫酸溶液。
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Evaluation of a Multivalent Vaccine against Lymphatic Filariasis in Rhesus macaque Model;Gajalakshmi Dakshinamoorthy等;《PLOS ONE》;20141117;第9卷(第11期);第1页右栏第1段第6页右栏第2段、第7页左栏第1段 * |
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