CN117554612A - 非洲猪瘟病毒全自动化管式磁微粒化学发光免疫分析检测试剂盒及其应用 - Google Patents
非洲猪瘟病毒全自动化管式磁微粒化学发光免疫分析检测试剂盒及其应用 Download PDFInfo
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Abstract
本发明公开了一种非洲猪瘟病毒全自动化管式磁微粒化学发光免疫分析检测试剂盒及其应用,属于体外诊断产品技术领域。所述的试剂盒包括含有磁微粒偶联非洲猪瘟病毒p72蛋白的混悬液、非洲猪瘟病毒p72酶标单抗、酶标抗体稀释液、血清稀释液、浓缩洗涤液以及化学发光底物。本发明还提供该试剂盒的使用方法及应用。本发明所述方法操作简单、自动化、灵敏度高、重复性好,为我国防控、净化非洲猪瘟提供了物质保障和技术支撑,具有重要的意义。
Description
技术领域
本发明涉及一种基于非洲猪瘟病毒p72单抗的竞争全自动化管式化学发光免疫分析检测试剂盒及其应用。属于病毒检测技术领域。
背景技术
非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fevervirus,ASFV)引起的一种高传染和致死性病毒性疾病,各个品种猪均易感,给全球的生猪及其相关产业造成了巨大的经济损失。ASFV是非洲猪瘟病毒科非洲猪瘟病毒属的一种大型线性双链DNA病毒,编码至少68种结构蛋白和150-200种非结构蛋白。其中,p72作为ASFV的主要衣壳蛋白,约占整个病毒颗粒的31%~33%。由于其良好的免疫原性和反应性,p72被认为是抗体检测的最佳候选靶标之一。此外,有研究表明,基于p72的检测方法是目前最灵敏的检测技术,具有良好的发展前景。近年来由于弱毒株的出现,使得检测ASFV抗体能够用于猪场的排查以及“拔牙式”清除ASF疫情。因此,开发一种快速、准确、灵敏、简便的检测ASFV抗体方法,将为猪场大规模筛查以及快速控制疫情传播提供一种切实可行的方法。
目前研究中已经开发了多种用于检测ASFV特异性抗体的血清学诊断技术,如ELISA和横向流动试验(LFT)等。其中ELISA是当前最常用的血清学检测方法,而且也是OIE推荐使用的方法,但它存在操作复杂、耗时长等的缺点,使其无法满足大规模筛查及高通量分析。因此,目前迫切需要开发一种快速、成本低、操作简单以及适合高通量筛查ASFV抗体新方法。
近年来,化学发光免疫分析以其灵敏度高、特异性强、方法简便快速以及不受背景杂散光的干扰在兽医中被广泛应用。基于此,我们引进了功能化磁性颗粒(MP)作为化学发光免疫分析的新型固相载体材料,与传统的聚苯乙烯微孔板相比,其比表面积更大,使之与其偶联的抗原、抗体等生物活性物质结合的更加充分,同时由于MP的理化性质稳定、在基液中易均匀分散,使得免疫反应更加充分,另外配合全自动化化学发光检测仪进行全自动化检测,能实现多种病原的同时检测,大大提高了实验室诊断的效率和准确率,从而为有效防控和净化非洲猪瘟提供了一种新方法。
发明内容
针对目前检测非洲猪瘟病毒抗体所面临的问题,本发明的目的是提供一种非洲猪瘟病毒p72单抗竞争全自动化管式化学发光抗体检测试剂盒及其检测方法,能实现快速、自动化且高通量检测目的,可满足临床检测中使用方便操作简便的要求。
为了达到上述目的,本发明采用了以下技术手段:
本发明的一种非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,所述的试剂盒包括含有磁微粒偶联非洲猪瘟病毒p72蛋白的混悬液、非洲猪瘟病毒p72酶标单抗、酶标抗体稀释液、血清稀释液、浓缩洗涤液以及化学发光底物。
其中,优选的,所述的含有磁微粒偶联非洲猪瘟病毒p72蛋白的混悬液是通过以下方法制备得到:取1mg磁珠于1.5mL EP管中,用pH 5.0MES溶液洗涤3次后,定容到100μL,称取EDC、NHS并用MES定容到10mg/mL,向磁珠中加入EDC、NHS溶液各10μL,在混旋仪上混匀活化30min;将活化的磁珠用MES溶液洗涤3次,并定容至100μL,加入非洲猪瘟病毒p72三聚体蛋白4μg,室温混匀震荡3h;将偶联蛋白的磁珠用MES洗涤3次,定容至100μL,加入2%w/wBSA 10μL室温振荡封闭3h,洗涤3次,用0.1M pH5.0 MES溶液定容至4μg/mg,放于4℃保存备用。
其中,优选的,所述的非洲猪瘟病毒p72三聚体蛋白是通过以下方法制备得到:优化合成非洲猪瘟病毒p72基因和pB602L基因,分别如SEQ ID No.1以及SEQ ID No.2所示,p72基因N端还带有6×His-tag,并通过双酶切位点BamHⅠ和XholⅠ克隆到pcDNA3.1载体,得到重组载体pcDNA3.1-p72以及pcDNA3.1-pB602L;然后将重组质粒pcDNA3.1-p72以及pcDNA3.1-pB602L使用转染试剂盒转染至1LExpiCHO-S细胞,并将其置于含8%CO2的37℃培养箱中振荡培养,转染18–22小时后,添加ExpiFectamineTMCHO增强剂至培养瓶中,并立即放回培养箱中振荡培养8-10天;1,000rpm离心10min,收集细胞沉淀,置于冰上裂解20min,细胞超声仪超声10min,10,000rpm离心10min,收集上清,并使用Ni亲和层析进一步纯化含有目的蛋白质的上清液,得到非洲猪瘟病毒p72三聚体蛋白。
其中,优选的,所述的非洲猪瘟病毒p72酶标单抗是ALP标记的抗非洲猪瘟病毒p72蛋白单克隆抗体2B8D7-ALP。
其中,优选的,所述血清稀释液为含有1%w/w酪蛋白、0.05%v/wTween-20、0.1%v/wproclin-300、2%w/w蔗糖的PBS缓冲液。
其中,优选的,所述的酶标抗体稀释液为0.05M pH值为7.0的3-吗啉丙磺酸(MOPS)溶液。
其中,优选的,所述的浓缩洗涤液为0.1M pH值为7.0含有0.5%v/wTween-20的PBS缓冲液。
其中,优选的,血清稀释度为1:5,酶标抗体稀释度为1:500。
其中,优选的,,所述的试剂盒中还包括非洲猪瘟阳性对照血清、非洲猪瘟阴性对照血清。
进一步的,本发明还提出了所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒在制备检测非洲猪瘟病毒试剂中的应用。
本发明试剂盒的使用方法为:
1、将待测血清样本与含有磁微粒偶联非洲猪瘟病毒p72蛋白的混悬液及酶标抗体(2B8D7-ALP)混合孵育,磁分离,洗涤固相后与发光底物孵育,读取发光值。
检测结果的判定:标准阴性血清的化学发光值应≥1,000,000;标准阳性血清的阻断率(PI)≥95%,PI=(1-测试样本CLIA值/阴性对照平均CLIA值)×100%。
2、本发明利用哺乳动物表达并纯化p72蛋白偶联磁微粒,所述蛋白具有完整结构的p72三聚体,而具有天然结构的p72在抗体检测时有助于准确区分阳性、阴性血清及减少假阳性反应。并且以磁微粒偶联p72蛋白,与传统的聚苯乙烯微孔板相比,其比表面积更大,使之与其偶联的抗原、抗体等生物活性物质结合的更加充分,同时由于磁微粒的理化性质稳定、在基液中易均匀分散,使得免疫反应更加充分,另外配合全自动化化学发光检测仪进行全自动化检测,可实现精准、全自动化检测,大大提高了实验室诊断的效率和准确率。
3、本发明提供了一种基于非洲猪瘟病毒p72单克隆抗体的全自动化管式化学发光免疫分析检测试剂盒,本发明利用磁微粒偶联所述的p72三聚体蛋白,酶标抗体为所述单抗2B8D7-ALP,使得酶标抗体能够与待检血清中p72抗体竞争性结合偶联在磁微粒的p72蛋白,检测速度快,15min即可完成检测,检测结果稳定、可靠。本发明的检测方法敏感性好、特异性强、重复性好,检测时间短。
附图说明
图1是p72三聚体蛋白表达、纯化的WB图(A)、SDS-PAGE图(B)和非变性PAGE图(C);
其中,M表示蛋白分子量,1表示p72三聚体蛋白;
图2是非洲猪瘟病毒p72单抗竞争全自动化管式CLIA最佳检测条件的优化。
其中,A为血清稀释度优化结果;B为酶标抗体稀释度优化结果;C为使用最佳包被抗原浓度、血清稀释度以及单抗稀释度,进行反应时间优化的结果;D为最适封闭剂的筛选结果;
图3是非洲猪瘟病毒p72单抗竞争全自动化管式CLIA检测已知背景猪血清的ROC曲线图(A)和背景交互点图(B),其中0代表非洲猪瘟阴性血清,1代表非洲猪瘟阳性血清。
图4是非洲猪瘟病毒p72单抗竞争全自动化管式CLIA检测针对其它七种猪病毒血清的PI值。
具体实施方式
下面结合具体实施例和附图来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1非洲猪瘟病毒p72三聚体蛋白的表达
从GenBank数据库(MK333180.1)中检索非洲猪瘟病毒p72基因和pB602L基因,优化合成非洲猪瘟病毒p72基因和pB602L基因,分别如SEQ ID No.1以及SEQ ID No.2所示,p72基因N端还带有6×His-tag,并通过双酶切位点BamHⅠ和XholⅠ克隆到pcDNA3.1载体,得到重组载体pcDNA3.1-p72以及pcDNA3.1-pB602L。然后将这两种重组质粒pcDNA3.1-p72以及pcDNA3.1-pB602L使用转染试剂盒转染至1LExpiCHO-S细胞,并将其置于含8%CO2的37℃培养箱中振荡培养。转染18–22小时后,添加ExpiFectamineTMCHO增强剂至培养瓶中,并立即放回培养箱中振荡培养8-10天。1,000rpm离心10min,收集细胞沉淀,置于冰上裂解20min,细胞超声仪超声10min。10,000rpm离心10min,收集上清,使用抗His-tag抗体进行蛋白质印迹(WB)验证,结果显示p72重组蛋白表观分子量约为72kDa,与预期一致(图1A)。并使用Ni亲和层析进一步纯化含有目的蛋白质的上清液,并通过SDS-PAGE分析洗脱的蛋白质的纯度较好,可以用于后续实验(图1B)。纯化的p72蛋白通过非变性PAGE进一步分析(图1C),结果显示p72三聚体大小约为220kDa,与之前的报道一致。
实施例2非洲猪瘟病毒p72单抗全自动化管式竞争化学发光法的优化
标准阳性血清购于北京兽医药品监察所;标准阴性血清来自本实验室在非洲猪瘟疫情暴发前采集的猪血清,该血清使用p72-cELISA(PI=-5%)和商业化试剂盒(PI=1%)为阴性。
磁微粒偶联非洲猪瘟病毒p72蛋白混悬液的制备:取1mg磁珠于1.5mLEP管中,用MES溶液(pH 5.0)洗涤3次后,定容到100μL(1mg/100μL),称取EDC、NHS并用MES定容到10mg/mL,向磁珠中加入EDC、NHS溶液各10μL,在混旋仪上混匀活化30min;将活化的磁珠用MES溶液洗涤3次,并定容至100μL(1mg/100μL),分别加入非洲猪瘟病毒p72三聚体蛋白浓度为2μg、4μg以及8μg,室温混匀震荡3h;将偶联p72蛋白的磁珠用MES洗涤3次,定容至100μL,加入2%w/w BSA 10μL室温振荡封闭3h,洗涤3次,用磁珠稀释液(0.1M pH5.0 MES溶液)分别定容至8μg/mg、4μg/mg、2μg/mL,放于4℃保存备用。
p72-2B8D7-ALP的制备:首先使用NaIO4和乙二醇活化ALP,然后将活化后的ALP与1mg mAb-2B8D7(该单抗记载于以下文献中:Identification of p72 epitopes ofAfrican swine fever virus and preliminary application,Chun Miao et al.,Frontiers inMicrobiology,03February 2023,并由兰州兽医研究所保存、提供。)混合后置于500mL 0.05M CB溶液中过夜偶联,加入NaBH4进行还原,最后使用饱和硫酸铵沉淀法进行纯化,获得ALP标记的抗体(2B8D7-ALP),放于-20℃保存备用。
最佳检测条件的确定:取不同抗原浓度偶联的磁珠(8μg/mg、4μg/mg、2μg/mg)分别检测标准阳性和阴性血清,重复检测3次并取平均值,根据阴性血清与阳性血清的CL值比值(N/P)从而选择最适磁珠包被抗原浓度为4μg/mg。接着使用血清稀释液(含有1%w/w酪蛋白、0.05%v/wTween-20、0.1%v/wproclin-300、2%w/w蔗糖的0.1M PBS缓冲液)将标准阴性血清和阳性血清按照1:5、1:10、1:20、1:40稀释,酶标单抗2B8D7-ALP使用酶标抗体稀释液(0.05M pH值为7.0的3-吗啉丙磺酸(MOPS)溶液)按照1:500、1:1000、1:2000稀释,最终根据检测结果选出最佳的血清稀释度为1:5和酶标抗体稀释度为1:500(图2A、2B)。使用最佳包被抗原浓度、血清稀释度以及单抗稀释度,进行反应时间优化(10min、20min、30min、40min、50min、1h),最终确定最佳反应时间为10min(图2C)。
最适封闭剂的筛选:为了可以有效提高羧基磁珠偶联抗原效率,我们选用2%w/wBSA、1%w/w BSA、1%v/w牛血清、1%w/w海藻糖、1%w/w明胶、5%w/w脱脂奶粉作为封闭液,并按照上述实验流程进行单抗竞争全自动化管式CLIA反应,筛选最适封闭液为2%w/w BSA(图2D)。
实施例3通过已知背景血清确定该方法的Cut-off值、诊断敏感性和诊断特异性
一、该方法的操作步骤
设置全自动化学发光仪各项参数,按照实施例2优化后的反应条件,检测反应步骤如下(为仪器全自动):
将50μL稀释后的血清样本、25μL含有磁微粒偶联非洲猪瘟病毒p72蛋白的混悬液、50μL稀释后酶标单抗(2B8D7-ALP)加入反应杯中,37℃孵育10分钟;洗涤液清洗4次,加入100μL化学发光底物,37℃孵育5分钟,检测发光值。10×浓缩洗涤液为0.1M pH值为7.0含有0.5%v/wTween-20的PBS缓冲液。
检测结果用阻断率(PI)来评价,PI=(1-测试样本CLIA值/阴性对照平均CLIA值)×100%。
二、血清盘的建立
1、来自本实验室2018年从中国两个养猪场(甘肃天水秦安和庄浪)采集了432份猪血清,由于2018年之前中国没有发生过非洲猪瘟疫情暴发,这些血清被认为是非洲猪瘟阴性猪血清,并用于确定该方法的Cut-off值和诊断特异性。
2、68份阳性血清来自感染的猪,这些血清样本用于确定Cut-off值、诊断敏感性。
三、p72单抗竞争全自动化管式化学发光免疫分析法的Cut-off值、诊断敏感性和诊断特异性
用已知背景清楚的血清来确定该方法的Cut-off值,并对其诊断敏感性和诊断特异性进行评价。Cut-off值用ROC曲线(图3A)和背景交互点图(图3B)来分析。试验结果表明,在检测猪血清时,在PI为36%时,诊断敏感性为100%,诊断特异性为99.6%。
四、p72单抗竞争全自动化管式CLIA重复性、特异性实验
选取六份猪血清样本,使用相同和不同批次包被的抗原在不同的时间里检测三个重复。结果表明批内变异系数(CV)从2.75%到8.51%不等,批间CV从2.69%到9.78%不等。由于所有CV值均≤10%,该测定显示出良好的重复性。
表1 p72单抗竞争全自动化管式CLIA的批内重复性和批间重复性
使用该方法检测针对七种针对其它猪病毒(PCV2、PPV、FMDV、PRRSV、PDCoV、CSFV、PEDV)的猪血清。检测结果表明该方法与这些病毒感染血清无交叉反应,具有良好的特异性(图4)。
实施例4试剂盒的组装
所述的试剂盒含有磁微粒偶联非洲猪瘟病毒p72蛋白的混悬液、酶标单抗p72-2B8D7-ALP、酶标抗体稀释液、血清稀释液、浓缩洗涤液、非洲猪瘟阳性对照血清、非洲猪瘟阴性对照血清以及化学发光底物。
含有磁微粒偶联非洲猪瘟病毒p72蛋白的混悬液、p72-2B8D7-ALP酶标单抗按照实施例2制备。
血清稀释液为含有1%w/w酪蛋白、0.05%v/wTween-20、0.1%v/wproclin-300、2%w/w蔗糖的0.1M PBS缓冲液。
酶标抗体稀释液为0.05M pH值为7.0的3-吗啉丙磺酸(MOPS)溶液。
浓缩洗涤液(10×)为0.1M pH值为7.0含有0.5%v/w Tween-20的PBS缓冲液。
Claims (10)
1.一种非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,其特征在于,所述的试剂盒包括含有磁微粒偶联非洲猪瘟病毒p72蛋白的混悬液、非洲猪瘟病毒p72酶标单抗、酶标抗体稀释液、血清稀释液、浓缩洗涤液以及化学发光底物。
2.如权利要求1所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,其特征在于,所述的含有磁微粒偶联非洲猪瘟病毒p72蛋白的混悬液是通过以下方法制备得到:取1mg磁珠于1.5mL EP管中,用pH 5.0MES溶液洗涤3次后,定容到100μL,称取EDC、NHS并用MES定容到10mg/mL,向磁珠中加入EDC、NHS溶液各10μL,在混旋仪上混匀活化30min;将活化的磁珠用MES溶液洗涤3次,并定容至100μL,加入非洲猪瘟病毒p72三聚体蛋白4μg,室温混匀震荡3h;将偶联蛋白的磁珠用MES洗涤3次,定容至100μL,加入2%w/w BSA 10μL室温振荡封闭3h,洗涤3次,用0.1M pH5.0 MES溶液定容至4μg/mg,放于4℃保存备用。
3.如权利要求2所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,其特征在于,所述的非洲猪瘟病毒p72三聚体蛋白是通过以下方法制备得到:优化合成非洲猪瘟病毒p72基因和pB602L基因,分别如SEQ ID No.1以及SEQ ID No.2所示,p72基因N端还带有6×His-tag,并通过双酶切位点BamHⅠ和XholⅠ克隆到pcDNA3.1载体,得到重组载体pcDNA3.1-p72以及pcDNA3.1-pB602L;然后将重组质粒pcDNA3.1-p72以及pcDNA3.1-pB602L使用转染试剂盒转染至1LExpiCHO-S细胞,并将其置于含8%CO2的37℃培养箱中振荡培养,转染18–22小时后,添加ExpiFectamineTMCHO增强剂至培养瓶中,并立即放回培养箱中振荡培养8-10天;1,000rpm离心10min,收集细胞沉淀,置于冰上裂解20min,细胞超声仪超声10min,10,000rpm离心10min,收集上清,并使用Ni亲和层析进一步纯化含有目的蛋白质的上清液,得到非洲猪瘟病毒p72三聚体蛋白。
4.如权利要求1所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,其特征在于,所述的非洲猪瘟病毒p72酶标单抗是ALP标记的抗非洲猪瘟病毒p72蛋白单克隆抗体2B8D7-ALP。
5.如权利要求1所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,其特征在于,所述血清稀释液为含有1%w/w酪蛋白、0.05%v/wTween-20、0.1%v/wproclin-300、2%w/w蔗糖的PBS缓冲液。
6.如权利要求1所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,其特征在于,所述的酶标抗体稀释液为0.05M pH值为7.0的3-吗啉丙磺酸(MOPS)溶液。
7.如权利要求1所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,其特征在于,所述的浓缩洗涤液为0.1M pH值为7.0含有0.5%v/wTween-20的PBS缓冲液。
8.如权利要求1所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,其特征在于,血清稀释度为1:5,酶标抗体稀释度为1:500。
9.如权利要求1所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒,其特征在于,所述的试剂盒中还包括非洲猪瘟阳性对照血清、非洲猪瘟阴性对照血清。
10.权利要求1-9任一项所述的非洲猪瘟病毒全自动化管式化学发光抗体检测试剂盒在制备检测非洲猪瘟病毒试剂中的应用。
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