CN114544532A - 一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒及其检测方法 - Google Patents
一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒及其检测方法 Download PDFInfo
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Abstract
本发明公开了一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒,包括聚苯乙烯材质的96微孔板、破碎后含有P72蛋白的非洲猪瘟病毒、封闭剂、P72蛋白捕获抗体、钯金纳米枝晶颗粒模拟酶标记的P72蛋白单克隆抗体、双氧水溶液(H2O2)、邻苯二胺溶液、洗涤液、稀释液、以及终止液。还提供了检测方法,在96微孔板上依次构建P72蛋白捕获抗体,非洲猪瘟病毒(灭活),以及钯金纳米枝晶标记的P72蛋白单克隆抗体,然后加入显色底物OPD和H2O2,利用钯金纳米枝晶颗粒模拟酶材料高效的类过氧化物酶性能,生成具有吸收和荧光发射的氧化态邻苯二胺(oxidized‑OPD),以实现对非洲猪瘟病毒(灭活)的定量检测。
Description
技术领域
本发明属于免疫分析、动物传染诊断技术领域,具体为一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒及其检测方法。
背景技术
传统ELISA广泛应用于生物小分子和肿瘤标记物的检测领域。ELISA技术以抗原抗体为识别元件,使用酶引发显色反应引起溶液的颜色变化,将被测物质的浓度信号转换成光学信号,通过肉眼或者测量溶液吸光度的变化对待测物质进行定性定量分析,由于酶具有专一性且有高效的催化效率,能实现快速灵敏的检测。但酶这类物质在使用过程中易失活,且储存运输条件苛刻,而且价格高昂,所以寻找一种高效的类酶材料来替代传统的酶变得尤为重要。
金、铂、银和钯等贵金属纳米颗粒因其生物相容性,优良的催化和电子传递性等优点成为免疫传感器中常用的纳米材料。钯金纳米枝晶,其制备过程绿色简便,具有催化性能好,生物相容性好、长期分散性稳定等特点;钯金纳米粒子表面可与氨基发生共价键结合,我们可将抗体直接固定在纳米材料表面形成钯金纳米枝晶颗粒-抗原复合物。该复合材料的应用具有提高免疫传感器的灵敏度和稳定性,降低传感器的检出限,增强信号等优势。
有研究表明非洲猪瘟病毒含有多种衣壳蛋白,分别是主要衣壳蛋白P72(Majorcapsid protein)、P54蛋白、P72蛋白、五邻体蛋白(Penton protein)以及三种不同的次要衣壳蛋白(Minor capsid protein)。P72是非洲猪瘟病毒衣壳上含量最高的蛋白,位于衣壳的外层,是一种比较典型的双“果冻卷”(Jelly-roll,JR)结构分子。本领域一直致力于研究一种能够快速检验出非洲猪瘟病毒的试剂盒以及检测方法。
发明内容
为了解决现有技术中的检测非洲猪瘟病毒试剂盒稀少的问题,本发明提供了一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒及其检测方法,实现的目的为合成一种具有高类过氧化物酶活性的钯金纳米枝晶颗粒材料,并将其应用于免疫夹心型传感器的构建,并基于此传感器,开发出一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒及检测方法。
为了实现上述目的,本发明提供以下技术方案:本发明提供的一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒,该试剂盒包括聚苯乙烯材质的96微孔板12孔×8条、破碎后含有P72或P54蛋白或P32蛋白的非洲猪瘟病毒0.5mL、封闭剂20mL、P72蛋白捕获抗体1mL、钯金纳米枝晶颗粒模拟酶标记的P72或P54或P32蛋白单克隆抗体0.5mL、双氧水溶液50mL、显色底物邻苯二胺溶液80mL、洗涤液150mL、稀释液200mL、以及终止液50mL。
所述钯金纳米枝晶颗粒模拟酶标记的P72或P54或P32蛋白单克隆抗体,制备步骤如下:先将50μL HAuCl4(0.1M)和70μL Na2PdCl4(0.06M)溶液加入到15mL超纯水中得到透明单一的黄色溶液,再加入20mg聚乙烯吡咯烷酮;然后,加入2mL对苯二酚溶液(0.1M)并开始加热至30-80℃,保温反应10-60分钟;反应结束后,对样品进行离心分离(10000r/min),取沉淀物水洗三次;再分散于5mL磷酸盐缓冲液中备用;将1mL的浓度为100μg/mL的P72或P54或P32蛋白单克隆抗体溶液加入上述含有钯金纳米枝晶颗粒的磷酸盐缓冲液中,并在4℃下震荡孵化12h后离心样品(12000r/min),PBS洗三次后冻干得到钯金纳米枝晶颗粒标记的P72或P54或P32蛋白单克隆抗体粉末。
进一步的,所述钯金纳米枝晶颗粒直径为20-50nm。
进一步的,所述的P72或P54或P32蛋白单克隆抗体或来源于鼠抗重组单克隆抗体。
进一步的,所述封闭剂是质量浓度为1-5%的牛血清蛋白溶液。
进一步的,所述的稀释液是pH=7.4含有质量浓度为0.05-0.2%Tween-20的磷酸盐缓冲液。
进一步的,所述的洗涤液是pH=7.4的浓度为0.01M的磷酸盐缓冲液。
进一步的,所述的终止剂为1M的硫酸。
进一步的,所述显色底物邻苯二胺溶液的浓度为1-10mM,双氧水溶液的浓度为40mM。
本发明还提供了采用上述试剂盒的检测方法,包括以下步骤:取200μL P72或P54或P32蛋白捕获抗体溶液加入96微孔板后在4℃下孵育12h后,吸取出孔中液体弃用,并用200μL洗涤液洗3次,然后在孔内加入200μL封闭液,室温下孵育1h后弃去液体,再用200μL洗涤液洗3次,之后加入用稀释液稀释过的不同浓度的非洲猪瘟病毒标准溶液并在室温下孵育1h,弃去孔中液体后用200μL洗涤液洗3次,加入钯金纳米枝晶标记的非洲猪瘟病毒抗体溶液,室温下孵育1h后弃去孔内液体并用300μL洗涤液洗3次;然后加入180μL的OPD溶液(5mM),20μL双氧水溶液(40mM),避光孵育10min后加入终止液;使用酶标仪读取孔中的吸光值和荧光强度(激发波长400nm)。
综上,本发明采用上述技术方案,所具有的有益效果包括:1、钯金纳米枝晶颗粒具有很高的类过氧化物酶活性和良好的分散性,从而提高传感器的灵敏度和稳定性。
2、标志物抗体直接孵化,利用贵金属优异的生物相容性,在抗体的标记中不必使用酶,避免了因酶的失活和泄漏造成的检测误差,显著提高了免疫传感器的重现性和稳定性。
3、本发明利用抗原、抗体的免疫反应,提高了检测方法的特异性。
4、本发明利用高活性的钯金纳米枝晶颗粒催化底物邻苯二胺反应生成具有紫外吸收和荧光的物质,其双模态的信号输出方式相比于传统的单模态的信号输出方式更加具有稳定性。
附图说明
图1为本发明实施例1中钯金纳米枝晶颗粒的透射电子显微镜图像。
图2为本发明实施例1中钯金纳米枝晶颗粒标记的P72蛋白单克隆抗体抗体合成示意图。
图3为本发明实施例1中一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒及检测方法的原理图。
图4为本发明实施例1中紫外吸收强度与非洲猪瘟病毒浓度的曲线关系。
图5为本发明实施例1中荧光强度与非洲猪瘟病毒浓度的曲线关系。
图6为本发明实施例2中紫外吸收强度与非洲猪瘟病毒浓度的曲线关系。
图7为本发明实施例2中荧光强度与非洲猪瘟病毒浓度的曲线关系。
图8为本发明实施例3中紫外吸收强度与非洲猪瘟病毒浓度的曲线关系。
图9为本发明实施例3中荧光强度与非洲猪瘟病毒浓度的曲线关系。
具体实施方式
下面通过具体的实施例对本发明做进一步的详细描述。实施例中涉及到的原料均为商购所得。
实施例1:本发明首先提供一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒,包括:
聚苯乙烯材质的96微孔板,非洲猪瘟病毒(破碎后含有P72蛋白),封闭剂,P72蛋白捕获抗体,钯金纳米枝晶颗粒模拟酶标记的P72蛋白单克隆抗体(钯金-抗体),双氧水溶液(H2O2),显色底物邻苯二胺溶液(OPD),洗涤液,稀释液,终止液。
本发明所述的聚苯乙烯材质的96微孔板,非洲猪瘟病毒(破碎后含有P72蛋白),P72蛋白捕获抗体,非洲猪瘟P72蛋白单克隆抗体的来源为商购。
本发明所述的钯金纳米枝晶标记的抗体的制备步骤如下:
先将50μL HAuCl4(0.1M)和70μL Na2PdCl4(0.06M)溶液加入到15mL超纯水中得到透明单一的黄色溶液,再加入20mg聚乙烯吡咯烷酮(PVP)。然后,加入2mL对苯二酚溶液(0.1M)并开始加热至70℃,保温反应60分钟。反应结束后,对样品进行离心分离(10000r/min),取沉淀物水洗三次。再分散于5mL磷酸盐缓冲液(PBS)中备用。将1mL的浓度为100μg/mL的P72非洲猪瘟病毒抗体溶液加入上述含有钯金纳米枝晶的磷酸盐缓冲液中,并在4℃下震荡孵化12h后离心样品(12000r/min),PBS洗三次后冻干并重新分散于磷酸盐缓冲液中得到钯金纳米枝晶颗粒标记的P72蛋白单克隆抗体溶液。其中,所述的钯金纳米枝晶颗粒是通过对苯二酚还原氯金酸和氯钯酸钠混合溶液得到的;所述钯金纳米枝晶颗粒粒径为20-50nm;所述的钯金纳米枝晶颗粒的合成温度优选为30-80℃,保温反应时间优选为10-60min。钯金纳米枝晶颗粒的透射电镜图如图1所示,钯金纳米枝晶标记的P72蛋白单克隆抗体制备如图2所示。
本发明所述的一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒,所述的封闭剂优选为浓度为1-5%的牛血清蛋白溶液。所述的稀释液优选为pH=7.4含有浓度为0.02-0.1%Tween-20的磷酸盐缓冲液。所述的洗涤液优选为pH=7.4的浓度0.01M的磷酸盐缓冲液;所述的终止剂优选为1M的硫酸。
本发明还提供一种非洲猪瘟病毒的检测方法,包括以下步骤:
取100μL P72蛋白捕获抗体PBS溶液加入96微孔板后在4℃下孵育12h后,吸取出孔中液体弃用并用200μL洗涤液洗3次,然后再孔内加入200μL封闭液,室温下孵育1h后弃去液体,再用200μL洗涤液洗3次,之后加入用稀释液稀释过的不同浓度的非洲猪瘟病毒标准溶液并在室温下孵育1h,弃去孔中液体后用200μL洗涤液洗3次,加入钯金纳米枝晶标记的P72蛋白单克隆抗体溶液,室温下孵育1h后弃去孔内液体并用300μL洗涤液洗3次。然后加入180μL的OPD溶液(5mM),20μL双氧水溶液(40mM),避光孵育10min后加入终止液。使用酶标仪读取孔中的吸光值和荧光强度(激发波长400nm)。在上述步骤中,P72蛋白捕获抗体的浓度为1-4μg/mL,钯金纳米枝晶标记的P72蛋白单克隆抗体浓度为4-10μg/mL,非洲猪瘟病毒的浓度为0.01-10ng/mL,OPD溶液的浓度为1-10mM,双氧水溶液的浓度为40mM,本发明免疫传感构建和信号检测示意图如图3所示,图4、图5为检测体系的荧光强度和紫外吸收强度随非洲猪瘟病毒浓度变化的曲线关系图,首先由图4A和图5A可知,随着非洲猪瘟病毒浓度的增加,吸光度和荧光强度均增大,紫外吸收吸光度波长是450nm,荧光强度对应的发射波长为570nm。图4B和5B是相对应的非洲猪瘟病毒浓度对吸光度和荧光强度的拟合工作曲线,由图可以看出在10~5000pg/mL范围内与吸光度和荧光强度呈现出良好的线性关系,拟合的线性方程分别为y=0.1092x+0.1235和y=0.94x+2628,比色吸光度的检出限是3.3pg/mL,荧光的检测限为3.5pg/mL,能够满足测试要求。
实施例2:本发明首先提供一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒,包括:
聚苯乙烯材质的96微孔板,非洲猪瘟病毒(破碎后含有P54蛋白),封闭剂,P54蛋白捕获抗体,钯金纳米枝晶颗粒模拟酶标记的P54蛋白单克隆抗体(钯金-抗体),双氧水溶液(H2O2),显色底物邻苯二胺溶液(OPD),洗涤液,稀释液,终止液。
本发明所述的聚苯乙烯材质的96微孔板,非洲猪瘟病毒(破碎后含有P54蛋白),P54蛋白捕获抗体,非洲猪瘟P54蛋白单克隆抗体的来源为商购。
本发明所述的钯金纳米枝晶标记的抗体的制备步骤如下:
先将50μL HAuCl4(0.1M)和70μL Na2PdCl4(0.06M)溶液加入到15mL超纯水中得到透明单一的黄色溶液,再加入20mg聚乙烯吡咯烷酮(PVP)。然后,加入2mL对苯二酚溶液(0.1M)并开始加热至70℃,保温反应60分钟。反应结束后,对样品进行离心分离(10000r/min),取沉淀物水洗三次。再分散于5mL磷酸盐缓冲液(PBS)中备用。将1mL的浓度为100μg/mL的P54非洲猪瘟病毒抗体溶液加入上述含有钯金纳米枝晶的磷酸盐缓冲液中,并在4℃下震荡孵化12h后离心样品(12000r/min),PBS洗三次后冻干并重新分散于磷酸盐缓冲液中得到钯金纳米枝晶颗粒标记的P54蛋白单克隆抗体溶液。其中,所述的钯金纳米枝晶颗粒是通过对苯二酚还原氯金酸和氯钯酸钠混合溶液得到的;所述钯金纳米枝晶颗粒粒径为20-50nm;所述的钯金纳米枝晶颗粒的合成温度优选为30-80℃,保温反应时间优选为10-60min。
本发明所述的一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒,所述的封闭剂优选为浓度为1-5%的牛血清蛋白溶液。所述的稀释液优选为pH=7.4含有浓度为0.02-0.1%Tween-20的磷酸盐缓冲液。所述的洗涤液优选为pH=7.4的浓度0.01M的磷酸盐缓冲液;所述的终止剂优选为1M的硫酸。
本发明还提供一种非洲猪瘟病毒的检测方法,包括以下步骤:
取100μL P54蛋白捕获抗体PBS溶液加入96微孔板后在4℃下孵育12h后,吸取出孔中液体弃用并用200μL洗涤液洗3次,然后再孔内加入200μL封闭液,室温下孵育1h后弃去液体,再用200μL洗涤液洗3次,之后加入用稀释液稀释过的不同浓度的非洲猪瘟病毒标准溶液并在室温下孵育1h,弃去孔中液体后用200μL洗涤液洗3次,加入钯金纳米枝晶标记的P54蛋白单克隆抗体溶液,室温下孵育1h后弃去孔内液体并用300μL洗涤液洗3次。然后加入180μL的OPD溶液(5mM),20μL双氧水溶液(40mM),避光孵育10min后加入终止液。使用酶标仪读取孔中的吸光值和荧光强度(激发波长400nm)。在上述步骤中,P54蛋白捕获抗体的浓度为1-4μg/mL,钯金纳米枝晶标记的P54蛋白单克隆抗体浓度为4-10μg/mL,非洲猪瘟病毒的浓度为0.01-10ng/mL,OPD溶液的浓度为1-10mM,双氧水溶液的浓度为40mM,图6、图7为检测体系的荧光强度和紫外吸收强度随非洲猪瘟病毒浓度变化的曲线关系图,首先由图6A和图7A可知,随着非洲猪瘟病毒浓度的增加,吸光度和荧光强度均增大,紫外吸收吸光度波长是450nm,荧光强度对应的发射波长为570nm。图6B和7B是相对应的非洲猪瘟病毒浓度对吸光度和荧光强度的拟合工作曲线,由图可以看出在10~5000pg/mL范围内与吸光度和荧光强度呈现出良好的线性关系,拟合的线性方程分别为y=0.1234x+0.1637和y=1.6x+2981,比色吸光度的检出限是2.8pg/mL,荧光的检测限为1.4pg/mL,能够满足测试要求。
实施例3:本发明首先提供一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒,包括:
聚苯乙烯材质的96微孔板,非洲猪瘟病毒(破碎后含有P32蛋白),封闭剂,P32蛋白捕获抗体,钯金纳米枝晶颗粒模拟酶标记的P32蛋白单克隆抗体(钯金-抗体),双氧水溶液(H2O2),显色底物邻苯二胺溶液(OPD),洗涤液,稀释液,终止液。
本发明所述的聚苯乙烯材质的96微孔板,非洲猪瘟病毒(破碎后含有P32蛋白),P32蛋白捕获抗体,非洲猪瘟P32蛋白单克隆抗体的来源为商购。
本发明所述的钯金纳米枝晶标记的抗体的制备步骤如下:
先将50μL HAuCl4(0.1M)和70μL Na2PdCl4(0.06M)溶液加入到15mL超纯水中得到透明单一的黄色溶液,再加入20mg聚乙烯吡咯烷酮(PVP)。然后,加入2mL对苯二酚溶液(0.1M)并开始加热至70℃,保温反应60分钟。反应结束后,对样品进行离心分离(10000r/min),取沉淀物水洗三次。再分散于5mL磷酸盐缓冲液(PBS)中备用。将1mL的浓度为100μg/mL的P32非洲猪瘟病毒抗体溶液加入上述含有钯金纳米枝晶的磷酸盐缓冲液中,并在4℃下震荡孵化12h后离心样品(12000r/min),PBS洗三次后冻干并重新分散于磷酸盐缓冲液中得到钯金纳米枝晶颗粒标记的P32蛋白单克隆抗体溶液。其中,所述的钯金纳米枝晶颗粒是通过对苯二酚还原氯金酸和氯钯酸钠混合溶液得到的;所述钯金纳米枝晶颗粒粒径为20-50nm;所述的钯金纳米枝晶颗粒的合成温度优选为30-80℃,保温反应时间优选为10-60min。
本发明所述的一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒,所述的封闭剂优选为浓度为1-5%的牛血清蛋白溶液。所述的稀释液优选为pH=7.4含有浓度为0.02-0.1%Tween-20的磷酸盐缓冲液。所述的洗涤液优选为pH=7.4的浓度0.01M的磷酸盐缓冲液;所述的终止剂优选为1M的硫酸。
本发明还提供一种非洲猪瘟病毒的检测方法,包括以下步骤:
取100μL P32蛋白捕获抗体PBS溶液加入96微孔板后在4℃下孵育12h后,吸取出孔中液体弃用并用200μL洗涤液洗3次,然后再孔内加入200μL封闭液,室温下孵育1h后弃去液体,再用200μL洗涤液洗3次,之后加入用稀释液稀释过的不同浓度的非洲猪瘟病毒标准溶液并在室温下孵育1h,弃去孔中液体后用200μL洗涤液洗3次,加入钯金纳米枝晶标记的P32蛋白单克隆抗体溶液,室温下孵育1h后弃去孔内液体并用300μL洗涤液洗3次。然后加入180μL的OPD溶液(5mM),20μL双氧水溶液(40mM),避光孵育10min后加入终止液。使用酶标仪读取孔中的吸光值和荧光强度(激发波长400nm)。在上述步骤中,P32蛋白捕获抗体的浓度为1-4μg/mL,钯金纳米枝晶标记的P32蛋白单克隆抗体浓度为4-10μg/mL,非洲猪瘟病毒的浓度为0.01-10ng/mL,OPD溶液的浓度为1-10mM,双氧水溶液的浓度为40mM,图8、图9为检测体系的荧光强度和紫外吸收强度随非洲猪瘟病毒浓度变化的曲线关系图,首先由图8A和图9A可知,随着非洲猪瘟病毒浓度的增加,吸光度和荧光强度均增大,紫外吸收吸光度波长是450nm,荧光强度对应的发射波长为570nm。图8B和9B是相对应的非洲猪瘟病毒浓度对吸光度和荧光强度的拟合工作曲线,由图可以看出在10~5000pg/mL范围内与吸光度和荧光强度呈现出良好的线性关系,拟合的线性方程分别为y=0.1611x+0.1476和y=0.86x+1786,比色吸光度的检出限是4.3pg/mL,荧光的检测限为3.1pg/mL,能够满足测试要求。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种非洲猪瘟病毒荧光与比色多模态精准检测的试剂盒,其特征在于,该试剂盒包括聚苯乙烯材质的96微孔板12孔×8条、破碎后含有P72或P54蛋白或P32蛋白的非洲猪瘟病毒0.5mL、封闭剂20mL、P72蛋白捕获抗体1mL、钯金纳米枝晶颗粒模拟酶标记的P72或P54或P32蛋白单克隆抗体0.5mL、双氧水溶液50mL、显色底物邻苯二胺溶液80mL、洗涤液150mL、稀释液200mL、以及终止液50mL。
2.根据权利要求1所述的试剂盒,其特征在于,所述钯金纳米枝晶颗粒模拟酶标记的P72或P54或P32蛋白单克隆抗体,制备步骤如下:
先将50μL HAuCl4(0.1M)和70μL Na2PdCl4(0.06M)溶液加入到15mL超纯水中得到透明单一的黄色溶液,再加入20mg聚乙烯吡咯烷酮;然后,加入2mL对苯二酚溶液(0.1M)并开始加热至30-80℃,保温反应10-60分钟;反应结束后,对样品进行离心分离(10000r/min),取沉淀物水洗三次;再分散于5mL磷酸盐缓冲液中备用;将1mL的浓度为100μg/mL的P72或P54或P32蛋白单克隆抗体溶液加入上述含有钯金纳米枝晶颗粒的磷酸盐缓冲液中,并在4℃下震荡孵化12h后离心样品(12000r/min),PBS洗三次后冻干得到钯金纳米枝晶颗粒标记的P72或P54或P32蛋白单克隆抗体粉末。
3.根据权利要求1或2所述的试剂盒,其特征在于,所述钯金纳米枝晶颗粒直径为20-50nm。
4.根据权利要求1所述的试剂盒,其特征在于,所述的P72或P54或P32蛋白单克隆抗体或来源于鼠抗重组单克隆抗体。
5.根据权利要求1所述的试剂盒,其特征在于,所述封闭剂是质量浓度为1-5%的牛血清蛋白溶液。
6.根据权利要求1所述的试剂盒,其特征在于,所述的稀释液是pH=7.4含有质量浓度为0.02-0.1%Tween-20的磷酸盐缓冲液。
7.根据权利要求1所述的试剂盒,其特征在于,所述的洗涤液是pH=7.4的浓度为0.01M的磷酸盐缓冲液。
8.根据权利要求1所述的试剂盒,其特征在于,所述的终止剂为1M的硫酸。
9.根据权利要求1所述的试剂盒,其特征在于,所述显色底物邻苯二胺溶液的浓度为1-10mM,双氧水溶液的浓度为40mM。
10.采用权利要求1、2、4-9任意一项权利要求所述试剂盒的检测方法,其特征在于,包括以下步骤:取200μL P72或P54或P32蛋白捕获抗体溶液加入96微孔板后在4℃下孵育12h后,吸取出孔中液体弃用,并用200μL洗涤液洗3次,然后在孔内加入100μL封闭液,室温下孵育1h后弃去液体,再用200μL洗涤液洗3次,之后加入用稀释液稀释过的不同浓度的非洲猪瘟病毒标准溶液并在室温下孵育1h,弃去孔中液体后用200μL洗涤液洗3次,加入钯金纳米枝晶标记的非洲猪瘟病毒抗体溶液,室温下孵育1h后弃去孔内液体并用300μL洗涤液洗3次;然后加入180μL的OPD溶液(5mM),20μL双氧水溶液(40mM),避光孵育10min后加入终止液;使用酶标仪读取孔中的吸光值和荧光强度(激发波长400nm)。
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