CN116256512A - 一种复合免疫荧光检测外泌体的试剂盒及方法 - Google Patents
一种复合免疫荧光检测外泌体的试剂盒及方法 Download PDFInfo
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Abstract
本发明涉及一种复合免疫荧光检测外泌体的试剂盒及方法，属于免疫检测领域，本发明采用一种能够结合多种外泌体特异性Cargoes抗体磁珠，被均匀标记后，来捕获生物液中的外泌体，再采用流式技术进行检测。此方法最大程度上降低了由于外泌体的异质性导致捕获不完全，从而提高了外泌体的捕获率；另外采用膜特异性结合荧光染料和直标流式抗体的检测方式使成本更低廉，结果更准确。
Description
技术领域
本发明属于免疫检测领域，涉及一种外泌体的检测方法，具体涉及一种复合免疫荧光检测外泌体的试剂盒及方法。
背景技术
细胞外囊泡（extracellular vesicles, EVs）是一种由细胞释放到细胞外基质的膜性小囊泡，其粒径分布范围为30 nm～1000 nm，它们通过膜上的蛋白质、脂类等信号分子，以及膜内包裹的内容物（神经递质、酶、激素和核酸等）在细胞之间的通讯和机体调控中发挥着至关重要的作用。根据分泌途径的不同，EVs主要分为外泌体（exosomes, 30～160nm）和微囊泡（microvesicles, 100～1,000 nm）两大类。外泌体经由细胞内多泡体（multivesicularbody, MVB）与细胞膜融合而释放，而微囊泡则是通过细胞膜包裹信号分子直接释放到细胞外。
外泌体是健康细胞和癌细胞均可释放的小膜泡，广泛存在于人体的血液、乳汁、尿液以及唾液等多种体液中，其种类和数量与机体的生理状态息息相关。细胞来源的物质，如基因组DNA（脱氧核糖核酸）、各种RNA（核糖核酸）片段、蛋白质和脂质被封装入外泌体并释放到细胞外环境。重要的是，外泌体其可近距离或远距离将来源细胞的细胞质成分转移到其它细胞。一旦到达受体细胞，来源细胞的胞浆成分可以改变靶细胞的生物学功能。因此，外泌体有望成为新型的疾病诊断标志物、纳米药物载体、治疗制剂和药物作用靶标，在疾病的诊断和治疗领域具有非常广阔的应用前景。外泌体可以通过超速离心、过滤、以及其他方法从体液中分离，这提供了一个非常有吸引力的替代侵入活检用于诊断和监测癌症的非侵入方法。
由于外泌体粒径微小（30-160nm）、异质性高，折射率低，生物标志物含量低，对其进行表征极具挑战性。尽管纳米流式检测技术可以检测低至30nm 的外泌体的散射光信号，荧光通道可以实现单个藻红蛋白分子的检测，对于多数拥有以细胞为检测对象流式的单位，对NanoFCM的购置无疑将会增加成本。因此开发使用传统检测范围在200nm以上的适合常用流式检测的试剂和方法是非常有必要的。
案例1：《CN201910961754.6一种流式细胞仪检测外泌体的方法》，本案例采用了CytoFLEX系列的流式细胞仪，能够直接进行囊泡的标记检测，但需要购置高昂的流式设备，成本较高。
案例2：《CN202110077720.8一种用于纳米流式细胞仪检测的外泌体的制备方法及应用》，本案例除了需要能够检测纳米级别的流式细胞仪外泌，还需要先对样品进行纯化的超速离心机，无疑增加了检测成本。
案例3：《CN202110360754.8 基于免疫磁珠和滚环扩增的外泌体膜蛋白的流式检测方法及应用》，本发明提供了一种基于免疫磁珠和滚环扩增的外泌体膜蛋白的流式检测方法及应用。其包括：(1)将免疫磁珠捕获的外泌体和核酸适配体、封闭剂混合反应，获得第一产物，核酸适配体包括膜蛋白特异性识别区、第一连接区和环模板杂交区；(2)将第一产物和环模板序列混合反应，获得的第二产物和连接酶、聚合酶混合，进行滚环扩增，获得扩增产物；环模板序列包括核酸适配体杂交区、第二连接区和荧光探针杂交区，环模板杂交区和核酸适配体杂交区至少部分互补；(3)将扩增产物和荧光探针混合杂交，获得荧光标记产物，并利用流式细胞术进行检测。本方法部分与作者的方法相似，但部发采用了对外泌体核酸的一系列复杂的操作，增加了操作的复杂性和出错几率。
发明内容
为了解决现有技术中的不足，本发明的目的一在于一种复合免疫荧光检测外泌体的试剂盒，目的二在提供上述试剂盒在外泌体检测方面的应用，目的三在于提供一种外泌体检测的方法。
本发明采用的具体方案为：
第一方面，一种复合免疫荧光检测外泌体的试剂盒，包括试剂A、试剂B和试剂C；所述试剂A包括捕获珠和抗体，所述捕获珠为偶联有Protein A/G的磁珠或琼脂糖凝胶珠，所述抗体为针对外泌体特异性Cargoes的抗体；所述试剂B为与质膜直接结合的荧光染料或/和与外泌体质膜上特异性Cargoes结合的指标荧光抗体；所述试剂C为缓存洗涤液。
作为对上述方案的进一步优化，试剂A中，所述磁珠或琼脂糖凝胶珠的直径在5-10μm。
作为对上述方案的进一步优化，试剂A中，所述针对外泌体特异性Cargoes的抗体为anti-CD63、anti-CD81和anti-CD9抗体。
作为对上述方案的进一步优化，试剂B中，所述与质膜直接结合的荧光染料为Dil、DiO、DiD。
作为对上述方案的进一步优化，试剂B中，所述指标荧光抗体为与荧光光染料结合的anti-CD63、anti-CD81和anti-CD9。进一步地， 所述荧光光染料为FITC、PE或APC。
作为对上述方案的进一步优化，所述试剂C为含有2.5%BSA的PBS缓冲液。
第二方面，上述试剂盒在外泌体检测中的应用。
第三方面，一种复合免疫荧光检测外泌体的方法，采用偶联有Protein A/G的磁珠或琼脂糖凝胶珠为捕获珠，在其表面连接针对外泌体特异性Cargoes的抗体，然后在外加磁场作用下，通过抗体与磁珠相连的外泌体被吸附而滞留在磁场中从而使外泌体得以分离；将分离的外泌体直接与Dil、DiO或DiD荧光染料结合，或/和利用经过荧光标记的抗体与外泌体质膜上的特异Cargoes进行结合，通过流式对荧光进行检测的方式来进行检测。
作为对上述方案的进一步优化，所述针对外泌体特异性Cargoes的抗体为anti-CD63、anti-CD81和anti-CD9抗体。
作为对上述方案的进一步优化，所述经过荧光标记的抗体为与荧光光染料结合的anti-CD63、anti-CD81和anti-CD9。进一步地， 所述荧光光染料为FITC、PE或APC。
有益效果：本发明是关于一种对生物液包括人体或动物体血液、尿液、乳液、组织液和细胞培养上清液中分离纯化和鉴定外泌体的技术。本发明采用一种能够结合多种外泌体特异性Cargoes抗体磁珠，被均匀标记后，来捕获生物液中的外泌体，再采用流式技术进行检测。此方法最大程度上降低了由于外泌体的异质性导致捕获不完全，从而提高了外泌体的捕获率；另外采用膜特异性结合荧光染料和直标流式抗体的检测方式使成本更低廉，结果更早准确。
附图说明
图1是本发明外泌体分选检测试剂盒的组成原理图。
图2是试剂盒的外泌体分选检测结果图。
具体实施方式
本发明的技术原理为：免疫磁珠法分离外泌体是基于外泌体表面抗原能与连接有磁珠的特异性单抗相结合，在外加磁场中，通过抗体与磁珠相连的外泌体被吸附而滞留在磁场中，无该种表面抗原的外泌体由于不能与连接着磁珠的特异性单抗结合而没有磁性，不在磁场中停留，从而使外泌体得以分离。分离的外泌体含有质膜相同的成分能够与Dil（与细胞膜能够直接结合）或者利用经过荧光标记的抗体与外泌体质膜上的特异Cargoes进行结构，而通过流式对荧光进行检测的方式来进行检测。通过在免疫磁珠的吸附能够使检测事件的粒径达到μm级别，归入传统流式检测范围内。模式图见图1。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述。
本发明了一种从生物液包括人体或动物体血液、尿液、乳液、组织液和细胞培养上清液中分离纯化和鉴定外泌体的技术。
本产品主要包括以下几部分组成：
（1）一种偶联有Protein A/G 直径在5-10μm磁珠（Magnetic Beads）/琼脂糖凝胶株。
（2）几种针对外泌体特异性Cargoes的抗体：anti-CD63、anti-CD81、anti-CD9抗体。（1）+（2）构成试剂A。
（3）几种能与质膜结合的荧光染料：Dil、DiO、DiD。
（4）几种能够与外泌体质膜上特异性Cargoes结合的指标荧光抗体：anti-CD63、anti-CD81、anti-CD9（注：它们被荧光光染料：FITC、PE、APC）。.(3)/(4)为试剂B
（5）一种缓存洗涤液：含有2.5%+BSA的PBS。试剂C
技术方案：
（1）用旋涡重悬捕获珠(试剂A)约15-30秒。
（2）在每个12×75 mm的聚苯乙烯圆底管(流式细胞仪管)中加入50μL的试剂 A。
（3）将差异超离心法分离的10-100µl直接外泌体加入相应的试管中。用移液管上下移液几次，并旋转几秒钟，轻柔地混合反应。
（4）室温黑暗过夜孵育。不要搅拌。
（5）孵育过夜后，加入1ml检测试剂C清洗样品(珠状外泌体)。
（6）收集磁珠，将样品置于磁架上孵育5分钟或2500×g离心5分钟。，弃去试管中的上清液。
（7）将检测试剂B建议的体积(5μL/所提供抗体的检验)加到珠状外泌体管中。通过移液和/或轻击轻轻混合。建议准备一个额外的管与适当的同型对照或没有外泌体，以作背景测定。
（8）2-8ºC避光孵育60分钟，不搅拌。
（9）加入1ml检测试剂C清洗样品(珠状外泌体)。
（10）收集磁珠，将试管放在磁架上，孵育5分钟，或以2,500 x g离心5分钟。使用磁架或抽吸时，弃去上清液。
（11）将样品在350 μL试剂C中重悬，在流式细胞仪进行分析。
检测结果如图2所示。我们采用了一两种细胞MSC和melanocyte;检测细胞和生成的外泌体的分选效果。以MSC的外泌体为检测对象。Sam-msccell-c是MSC细胞的阴性对照，Sam-msccell是细胞检测组；BEADS、BEAD-PE、BEAD-EXO是外泌体检测的阴性对照组；Sam.8.23.001是未超速离心的MSC外泌体组；Sam-MSC1是超速离心MSC外泌体组；Sam-mealn是melancyte细胞对照组。从结果中可以看出我们的试剂盒能够得到较好的分选检测效果。
需要说明的是，以上所述的实施方案应理解为说明性的，而非限制本发明的保护范围，本发明的保护范围以权利要求书为准。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对本发明作出的一些非本质的改进和调整仍属于本发明的保护范围。
Claims (10)
1.一种复合免疫荧光检测外泌体的试剂盒，其特征在于：包括试剂A、试剂B和试剂C；所述试剂A包括捕获珠和抗体，所述捕获珠为偶联有Protein A/G的磁珠或琼脂糖凝胶珠，所述抗体为针对外泌体特异性Cargoes的抗体；所述试剂B为与质膜直接结合的荧光染料或/和与外泌体质膜上特异性Cargoes结合的指标荧光抗体；所述试剂C为缓存洗涤液。
2.根据权利要求1所述的一种复合免疫荧光检测外泌体的试剂盒，其特征在于：试剂A中，所述磁珠或琼脂糖凝胶珠的直径在5-10μm。
3.根据权利要求1所述的一种复合免疫荧光检测外泌体的试剂盒，其特征在于：试剂A中，所述针对外泌体特异性Cargoes的抗体为anti-CD63、anti-CD81和anti-CD9抗体。
4.根据权利要求1所述的一种复合免疫荧光检测外泌体的试剂盒，其特征在于：试剂B中，所述与质膜直接结合的荧光染料为Dil、DiO、DiD。
5.根据权利要求1所述的一种复合免疫荧光检测外泌体的试剂盒，其特征在于：试剂B中，所述指标荧光抗体为与荧光光染料结合的anti-CD63、anti-CD81和anti-CD9。
6.根据权利要求5所述的一种复合免疫荧光检测外泌体的试剂盒，其特征在于：所述荧光光染料为FITC、PE或APC。
7.根据权利要求1所述的一种复合免疫荧光检测外泌体的试剂盒，其特征在于：所述试剂C为含有2.5%BSA的PBS缓冲液。
8.根据权利要求1-7任意一种所述的试剂盒在外泌体检测中的应用。
9.一种复合免疫荧光检测外泌体的方法，其特征在于：采用偶联有Protein A/G的磁珠或琼脂糖凝胶珠为捕获珠，在其表面连接针对外泌体特异性Cargoes的抗体，然后在外加磁场作用下，通过抗体与磁珠相连的外泌体被吸附而滞留在磁场中从而使外泌体得以分离；将分离的外泌体直接与Dil、DiO或DiD荧光染料结合，或/和利用经过荧光标记的抗体与外泌体质膜上的特异Cargoes进行结合，通过流式对荧光进行检测的方式来进行检测。
10.根据权利要求9所述的方法，其特征在于：所述针对外泌体特异性Cargoes的抗体为anti-CD63、anti-CD81和anti-CD9抗体。
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