CN117368483A - 一种基于均相光激化学发光免疫分析技术的肾损伤分子-1检测方法及试剂盒 - Google Patents
一种基于均相光激化学发光免疫分析技术的肾损伤分子-1检测方法及试剂盒 Download PDFInfo
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Abstract
本发明公开了一种基于均相光激化学发光免疫分析技术的肾损伤分子‑1检测方法及试剂盒,属于免疫检测分析技术领域。本发明提供的均相光激化学发光免疫分析技术所述方法,包括偶联Kim‑1捕获抗体的供体微球的制备,偶联Kim‑1检测抗体的受体微球的制备,将待检测样品加入聚丙烯孔板中,加入偶联好的供体微球和受体微球,混匀孵育,无需清洗,最后用Tecan M200检测,获得被测样本的浓度等信息。本发明应用双抗体夹心免疫法和光激化学发光的特性,可以快速检测待测样本中Kim‑1的含量,具有检测范围宽、灵敏度高、准确度高、检测快速简便等特点,可用于临床对急性肾损伤的早期辅助诊断,具有较好的应用前景。
Description
技术领域
本发明属于生物技术检测领域,具体涉及一种基于均相光激化学发光免疫分析技术的Kim-1的检测方法和涉及此方法的试剂盒,能够在待测样品中检测Kim-1浓度。
背景技术
均相光激化学发光免疫分析技术(amplified luminescent proximityhomogeneous assay linked immunosorbentassay,AlphaLISA)是以生物分子间相互作用为原理,荧光共振能量转移发光为基础,以硅胶微球为载体,时间分辨荧光为检测模式的免疫学研究技术。分子间相互作用(抗原抗体特异性相互作用)会将供体和受体微球拉近至单线态氧扩散范围以内,即200nm以内,从而激发级联放大的化学发光反应。其发光原理为光激化学发光,供体微球表面涂布光敏剂苯二甲蓝,受体微球表面涂布发光剂二甲噻吩衍生物并螯合有稀土原子铕。因而,当用680nm的激光照射时,供体微球表面光敏剂分解环境中的氧,形成单体氧分子(氧自由基),单体氧分子扩散到受体微球,将能量最终传递给稀土原子铕,激发波长为615nm、半衰期为0.3s的激发光,可以采用荧光扫描仪检测。
肾损伤分子-1(Kim-1)又称T细胞免疫球蛋白粘蛋白-1(Tim-1),是一种I型跨膜蛋白,被认为在肾小管间质损伤中发挥作用。该蛋白在近端肾小管细胞的顶端膜上表达,其外显子被裂解并释放到肾小管的管腔中。Kim-1是一种磷脂酰丝氨酸受体,可识别凋亡细胞并将其引导至溶酶体。它还是氧化脂蛋白的受体,因此善于接收凋亡细胞发出的"吃我"信号。此外,Kim-1还是第一个将肾脏近端上皮细胞转化为半特化吞噬细胞的已知分子。Kim-1可调节肾损伤时的免疫反应,因为它能促进存活的肾小管细胞清除死亡细胞。吞噬凋亡细胞可降低促炎免疫反应。此外,Kim-1与某些类型肾脏疾病的肾间质纤维化和炎症有关。这表明,Kim-1在肾脏疾病中发挥着重要作用。
急性肾损伤(Acute kidney injury,AKI)是一系列急性肾脏疾病中的一部分,在临床上被定义为肾功能的迅速丧失。目前,AKI是临床上急性病中最常见的严重疾病之一,如果没有及时治疗,可能是致命性的。早期诊断和干预不仅可以为AKI患者提供更好的治疗选择,而且可以改善预后,降低死亡率。
对动物和细胞培养的研究表明,在AKI中,Kim-1直接参与近端肾单位隔室上皮的结构、功能完整性的保存和恢复过程。据推测,Kim-1可以诱导对死亡细胞残余物的吞噬作用,从而清除近端小管腔中的细胞碎片,并降低滤液流动障碍的可能性。目前多数研究发现尿液Kim-1(uKim-1)有助于诊断AKI,且具有较高的敏感性和特异性,研究的汇总分析显示,uKim-1诊断AKI的估计敏感性为74%,而特异性为84%,这表明uKim-1对AKI具有良好的诊断效力。AKI可以由许多不同的机制触发,无论刺激原因是什么,AKI的发病特征均为血清肌酐水平迅速升高,同时肾小球滤过率(Estimated glomerular filtration rate,eGFR)迅速降低,而uKim-1的升高可能发生在血清肌酐升高之前,在肾小管损伤后24小时内就可以对uKim-1进行检测,来早期发现AKI。此外,不同肾病阶段的Kim-1值对急诊和危重患者有意义,因为许多研究表明,Kim-1可以区分不同类型急性肾小管坏死患者和无AKI患者。综上所述,Kim-1是一种有前途的识别早期AKI的血清标志物。因此,准确快速地检测人血清或尿中的Kim-1浓度具有重要的意义。
均相光激化学发光免疫分析技术,以时间分辨作为检测平台,背景影响低,可以在较短时间内对样品进行大规模的快速检测,检测灵敏度高,稳定性强,可实现对早期AKI的快速评估,具有较为广阔的应用空间。
发明内容
本发明的目的在于提供具有可以检测待测样品中Kim-1含量,效率高、特异性强、灵敏度高、稳定性好等技术特点的一种基于均相光激化学发光免疫分析技术的Kim-1检测方法以及涉及此方法的试剂盒。
本发明的技术解决方案如下:
本发明一方面提供了一种基于均相光激化学发光免疫分析技术的肾损伤分子-1检测方法,所述方法包括以下步骤:
1)供体微球与Kim-1捕获抗体偶联,其中Kim-1捕获抗体的重链可变区和轻链可变区的序列如SEQ ID NO.1和SEQ ID NO.2所示;
2)受体微球与Kim-1检测抗体偶联,其中Kim-1检测抗体的重链可变区和轻链可变区的序列如SEQ ID NO.3和SEQ ID NO.4所示;
3)利用分析缓冲液分别将偶联于供体微球的Kim-1捕获抗体和偶联于受体微球的Kim-1检测抗体均稀释成75μg/mL;
4)在聚丙烯孔板中依次加入30μL待检测样品、30μL偶联于供体微球的Kim-1捕获抗体和30μL偶联于受体微球的Kim-1检测抗体,避光震荡孵育20分钟,得到偶联于供体微球的Kim-1包被抗体-Kim-1抗原-偶联受体微球的Kim-1检测抗体荧光免疫复合物,其中荧光免疫复合物经激发光照射供体微球上的光敏剂产生单线态氧,受体微球上的发光剂接受单线态氧能量传递产生荧光,最后上机检测荧光值,根据Kim-1抗原标准品的浓度和荧光值建立标准曲线,将待检样品的荧光值代入标准曲线即可计算待检测样品中Kim-1的含量。
优选地,本发明所述步骤3)中的分析缓冲液为pH8.0 50mM Tris缓冲液。
优选地,本发明所述步骤4)中的Kim-1抗原标准品的浓度分别为50pg/mL、500pg/mL、1250pg/mL、2500pg/mL、5000pg/mL。
再一方面,本发明还提供了一种基于均相光激化学发光免疫分析技术的肾损伤分子-1检测试剂盒,所述试剂盒包括试剂盒本体以及放在试剂盒本体内的多个药剂盒;其中,具有放置偶联Kim-1捕获抗体的供体微球的药剂盒,具有放置偶联Kim-1检测抗体的受体体微球的药剂盒,具有放置Kim-1标准品溶液的药剂盒,具有放置缓冲液的药剂盒。
优选地,本发明所述试剂盒本体里设有可发性聚苯乙烯泡沫层,试剂盒本体包括盒体与盒盖,盒体与盒盖由酚醛塑料材质的连接轴连接,试剂盒本体内的底部预制有存放碎冰的冰槽。
本发明是一种基于均相光激化学发光免疫分析技术的Kim-1的检测方法,该检测方法包括:将Kim-1捕获抗体和检测抗体在MES标记缓冲液环境下分别共价偶联于供体微球和受体微球表面,1mg微球标记0.1mg抗体,抗体与微球偶联条件为室温避光震荡孵育2小时,利用分析缓冲液(50mM Tris,PH8.0)进行洗涤和分离,去除未结合的抗体,之后对偶联了抗体的微球的使用量进行优化,确定适合的微球使用剂量,用分析缓冲液进行稀释,避免检测微球过量。将优化剂量的偶联捕获抗体的供体微球,偶联检测抗体的受体微球和待测样品一起加入聚丙烯孔板中,孵育条件为37℃避光震荡20min,最终形成Kim-1捕获抗体-Kim-1抗原-Kim-1检测抗体荧光免疫复合物,经激发光照射供体微球上的光敏剂产生单线态氧,受体微球上的发光剂接受单线态氧能量传递产生荧光,最后经时间分辨仪进行荧光检测分析。其检测原理图如图1所示。
所述荧光检测分析是指利用镧系稀土螯合物具有长寿命荧光这一特性,在样品被脉冲光激发后、荧光信号采集之前,根据样品中所包含的不同的荧光物质其荧光衰变时间的各不相同通过延迟一定时间,在此时间段内待测样本中短寿命的背景荧光完全淬灭后,再对长寿命的稀土螯合物特异性荧光信号进行收集检测。通过时间分辨延迟可以有效消除来自样品、试剂、仪器等其它非特异性荧光的干扰,从而极大地提高检测的灵敏度。
优选的,将Kim-1的捕获抗体和检测抗体分别共价偶联于供体微球和受体微球表面,形成微球-抗体复合物后,利用分析缓冲液(50mM Tris,PH8.0)进行洗涤和分离,去除未结合的抗体。
优选的,供体(受体)微球与Kim-1捕获(检测)抗体形成偶联物过程的包被比例和孵育条件为:1mg/ml,抗体和微球的包被孵育条件为常温避光震荡2h。
优选的,Kim-1的捕获抗体和检测抗体分别共价偶联于供体微球和受体微球表面,利用偶联于供体微球的Kim-1捕获抗体、待测样品中的Kim-1抗原、偶联于受体微球的Kim-1检测抗体,通过双抗体夹心法进行检测。
优选的,荧光免疫复合体经激发光照射供体微球上的光敏剂产生单线态氧,受体微球上的发光剂接受单线态氧能量传递产生荧光,根据其荧光强度计算待测物含量。
优选的,偶联于微球的Kim-1抗体含量为0.1mg,每0.1mg抗体偶联1mg微球。
优选的,根据荧光强度进行检测分析,即识别利用时间分辨荧光测量法测定最后产物中的荧光强度,从而有效地提高了检测的灵敏度。
有益效果:本发明提供的方法以及在此基础上制备的检测试剂盒可以快速检测待测样本中Kim-1的含量,具有特异性强、灵敏度高、稳定性好等特点,可用于临床对早期AKI的辅助诊断,具有较好的应用前景;试剂盒使用可发性聚苯乙烯泡沫材质,盒体与盒盖由酚醛塑料材质的连接轴连接,试剂盒底部预制有存放碎冰的冰槽,结构简单,实用性强。
另外,本发明提供的Kim-1捕获抗体和Kim-1检测抗体均具有良好的特异性和敏感性,且配合使用,可以实现对Kim-1的特异性检测,因此,这2株抗体不仅可以应用于本发明的方法和试剂盒,同时也可以应用于其他Kim-1的检测方法和试剂盒(如化学发光检测方法和试剂盒、ELISA检测方法和试剂盒等)。
附图说明
图1Kim-1检测方法原理图。
图2Kim-1检测标准曲线。
图3Kim-1-AlphaLISA与Kim-1-TRFIA的相关性分析。
具体实施方式
为使本发明的目的、方法及优点更加清楚明确,下面结合具体实施例对本发明做出详细说明,所述的实施例只用于说明本发明,而不是用于对本发明进行限定,任何在本发明基础上所做的修改、等同替换等均在本发明的保护范围内。
实施例1供体(受体)微球-Kim-1捕获(检测)抗体偶联
1、供体微球与Kim-1捕获抗体偶联
供体微球(粒径200nm)购自苏州为度生物技术有限公司,Kim-1捕获抗体为使用常规的杂交瘤细胞技术制备的鼠源单克隆抗体,其重链可变区和轻链可变区的序列如SEQ IDNO.1和SEQ ID NO.2所示。一般1mg微球标记0.1mg抗体。其步骤简述如下所示:
首先对微球进行稀释,取100μL供体微球和900μL标记缓冲液(50Mm MES,PH6.0),17000rpm离心20分钟,弃上清,加入1000μL标记缓冲液超声15分钟,震散混匀。微球活化,称取0.01g NHS和0.01g EDC分别混入500μL标记缓冲液,配制成20mg/mL的溶液,现配现用,取10μL NHS溶液加入到清洗后的微球中,快速混匀,然后再取5μL EDC溶液加入到微球中,快速混匀,室温避光震荡孵育20分钟。清洗残留EDC,将活化后的供体微球17000rpm离心20分钟,第一遍,去掉上清,用1000μL标记缓冲液重悬微球,超声混散;17000rpm离心20分钟,第二遍,去掉上清,用1000μL标记缓冲液重悬微球,超声混散,备用。供体微球与捕获抗体偶联,取0.1mg捕获抗体于2mL离心管中,加入活化且清洗残留EDC后的微球,快速混匀,室温避光震荡孵育2小时。微球偶联抗体后,加入100μL BSA封闭液,室温避光混匀孵育1小时。封闭后,高速离心去除游离未结合微球的抗体,12000rpm离心10分钟,第一遍,弃上清,加入1000μL分析缓冲液(50mM Tris,PH8.0)超声15分钟,震散混匀;12000rpm离心10分钟,第二遍,弃上清,加入1000μL分析缓冲液超声15分钟,震散混匀。最后配制成1mg/mL抗体-微球标记复合物,4℃放置备用。
2、受体微球与Kim-1检测抗体偶联
受体微球(粒径200nm)购自苏州为度生物技术有限公司,Kim-1检测抗体为使用常规的杂交瘤细胞技术制备的鼠源单克隆抗体,其重链可变区和轻链可变区的序列如SEQ IDNO.3和SEQ ID NO.4所示。步骤与供体微球和Kim-1捕获抗体的偶联相同。
实施例2偶联于供体微球的Kim-1包被抗体-Kim-1抗原-偶联受体微球的Kim-1检测抗体荧光免疫复合物形成
首先,利用分析缓冲液(50mM Tris,PH8.0)将偶联于供体微球的Kim-1捕获抗体稀释成75μg/mL,相同的,将偶联于受体微球的Kim-1检测抗体稀释成75μg/mL。
其次,在聚丙烯孔板中依次加入30μL待检测样品、30μL偶联于供体微球的Kim-1捕获抗体和30μL偶联于受体微球的Kim-1检测抗体,避光震荡孵育20分钟,得到偶联于供体微球的Kim-1包被抗体-Kim-1抗原-偶联受体微球的Kim-1检测抗体荧光免疫复合物,其中荧光免疫复合物经激发光照射供体微球上的光敏剂产生单线态氧,受体微球上的发光剂接受单线态氧能量传递产生荧光,最后上机检测荧光值,根据Kim-1抗原标准品的浓度和荧光值建立标准曲线,将待检样品的荧光值代入标准曲线即可计算待检测样品中Kim-1的含量。
实施例3Kim-1检测的线性范围、灵敏度、特异性、精密度及回收率。
配制Kim-1抗原标准品(购自上海优宁生物科技有限公司),利用分析缓冲液(50mMTris,PH8.0)将Kim-1抗原标准品进行稀释(50,500,1250,2500,and5000pg/mL)。利用本试剂盒进行检测,绘制标准曲线,利用荧光值和浓度值绘制标准曲线,Kim-1抗原检测的对数线性方程为:Y=1.04+1.08X,R2=0.9969,线性范围为3.83pg/mL~5000pg/mL,结果图2所示。
平行测定10次0pg/mL参考标准品的荧光值,并计算其均值(mean)及标准差(SD),采用mean+2SD代入标准曲线方程,计算得到本试剂盒对Kim-1的检测灵敏度为3.83pg/mL。
测定3次L-FABP,NGAL,MMP-3的抗原,并通过标准曲线计算其浓度。交叉反应率(%)=检测浓度/实际浓度。结果如表1,计算得到本试剂盒对L-FABP,NGAL和MMP-3几乎没有交叉反应率,特异性较好。
表1Kim-1检测特异性
分别检测Kim-1低值样本、中值样本、高值样本各10次,结果如表2,得到Kim-1检测的批内变异系数为3.36%-4.71%(<10%),批间变异系数为5.61%-11.84%(<15%)。
表2Kim-1分析精密度
将三种Kim-1抗原标准品(500、2500和5000pg/mL)与已知浓度为2047.65pg/mL的血清样本按1:9的比例混合。使用标准曲线计算混合溶液的AlphaLISA信号,以确定混合溶液的实际浓度。将血清和抗原标准溶液按比例混合,制备出理论浓度为1892.89、2092.89和2342.89pg/mL的混合溶液。回收率(%)的计算方法如下:回收率(%)=实际浓度/理论浓度。结果如表3,回收率为92.31%-99.58%。
表3Kim-1检测回收率
综上所述,本发明可以检测待测样品中Kim-1抗原含量,效率高、特异性强、灵敏度高、稳定性好,可用于早期AKI诊断和筛查,具有较好的应用前景。
实施例4与现有试剂盒的比较试验
先前开发的一种基于时间分辨荧光免疫分析法(TRFIA)的Kim-1抗原检测的试剂盒,其检测步骤为于包被后的Kim-1聚丙烯孔板同时加入铕离子标Kim-1检测抗体和待测样品孵育,孵育条件为37℃震荡60min,得到包被抗体-Kim-1抗原-铕离子标记检测抗体荧光免疫复合物,2洗涤后加入增强液震荡5min后上机检测荧光强度确定检测指标含量。与之相比,本发明均相光激化学发光免疫分析技术的Kim-1抗原检测的试剂盒检测步骤简单,耗时明显减少。
此外,由于本发明的均相光激化学发光免疫分析技术的Kim-1抗原检测的试剂盒免清洗,不需要很长的反应时间,消除了TRFIA中多步洗涤过程对精度的干扰,避免了外部环境的脆弱性,灵敏度也有明显提高(3.83pg/mL VS11pg/mL)。
采用AlphaLISA-Kim-1试剂盒和TRFIA-Kim-1试剂盒分别检测13例肾病患者和13例健康体检者血清中kim-1浓度。两种方法的相关系数R2为0.9086(图3)。说明AlphaLISA-Kim-1试剂盒检测性能良好。
实施例5试剂盒的组装
根据以上的制备和检测方法,本发明是一种基于均相光激化学发光免疫分析技术的Kim-1抗原检测的试剂盒,包括试剂盒本体以及放在试剂盒本体内的多个药剂盒;其中,具有放置偶联Kim-1捕获抗体的供体微球的药剂盒;具有放置偶联Kim-1检测抗体的受体体微球的药剂盒,具有放置Kim-1标准品溶液的药剂盒,具有放置缓冲液的药剂盒,所述缓冲液为分析缓冲液(50mM Tris,PH8.0);试剂盒本体里设有可发性聚苯乙烯泡沫层,试剂盒本体包括盒体与盒盖,盒体与盒盖由酚醛塑料材质的连接轴连接,试剂盒本体内的底部预制有存放碎冰的冰槽。
有益效果:本发明可以检测待测样品中Kim-1抗原含量,效率高、特异性强、灵敏度高、稳定性好,可用于早期AKI诊断和筛查,具有较好的应用前景;试剂盒使用可发性聚苯乙烯泡沫材质,盒体与盒盖由酚醛塑料材质的连接轴连接,试剂盒底部预制有存放碎冰的冰槽,结构简单,实用性强。
最后,需要注意的是,本发明不限于以上实施例,还可以有很多变形。本领域的普通技术人员能从本发明公开的内容中直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (5)
1.一种基于均相光激化学发光免疫分析技术的肾损伤分子-1检测方法,其特征在于,所述方法包括以下步骤:
1)供体微球与Kim-1捕获抗体偶联,其中Kim-1捕获抗体的重链可变区和轻链可变区的序列如SEQ ID NO.1和SEQ ID NO.2所示;
2)受体微球与Kim-1检测抗体偶联,其中Kim-1检测抗体的重链可变区和轻链可变区的序列如SEQ ID NO.3和SEQ ID NO.4所示;
3)利用分析缓冲液分别将偶联于供体微球的Kim-1捕获抗体和偶联于受体微球的Kim-1检测抗体均稀释成75μg/mL;
4)在聚丙烯孔板中依次加入30μL待检测样品、30μL偶联于供体微球的Kim-1捕获抗体和30μL偶联于受体微球的Kim-1检测抗体,避光震荡孵育20分钟,得到偶联于供体微球的Kim-1包被抗体-Kim-1抗原-偶联受体微球的Kim-1检测抗体荧光免疫复合物,其中荧光免疫复合物经激发光照射供体微球上的光敏剂产生单线态氧,受体微球上的发光剂接受单线态氧能量传递产生荧光,最后上机检测荧光值,根据Kim-1抗原标准品的浓度和荧光值建立标准曲线,将待检样品的荧光值代入标准曲线即可计算待检测样品中Kim-1的含量。
2.根据权利要求1所述的方法,其特征在于,所述步骤3)中的分析缓冲液为pH8.0 50mMTris缓冲液。
3.根据权利要求1所述的方法,其特征在于,所述步骤4)中的Kim-1抗原标准品的浓度分别为50pg/mL、500pg/mL、1250pg/mL、2500pg/mL、5000pg/mL。
4.一种基于均相光激化学发光免疫分析技术的肾损伤分子-1检测试剂盒,其特征在于,所述试剂盒包括试剂盒本体以及放在试剂盒本体内的多个药剂盒;其中,具有放置偶联Kim-1捕获抗体的供体微球的药剂盒,具有放置偶联Kim-1检测抗体的受体体微球的药剂盒,具有放置Kim-1标准品溶液的药剂盒,具有放置缓冲液的药剂盒。
5.根据权利要求4所述的试剂盒,其特征在于,所述试剂盒本体里设有可发性聚苯乙烯泡沫层,试剂盒本体包括盒体与盒盖,盒体与盒盖由酚醛塑料材质的连接轴连接,试剂盒本体内的底部预制有存放碎冰的冰槽。
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