CN106289926A - A kind of method using immuno magnetic cell separation serum China and foreign countries to secrete body - Google Patents
A kind of method using immuno magnetic cell separation serum China and foreign countries to secrete body Download PDFInfo
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- 210000002966 serum Anatomy 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000000926 separation method Methods 0.000 title claims description 38
- 239000011324 bead Substances 0.000 claims abstract description 57
- 239000000872 buffer Substances 0.000 claims description 20
- 239000000725 suspension Substances 0.000 claims description 18
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- 238000011534 incubation Methods 0.000 claims description 12
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- 239000002953 phosphate buffered saline Substances 0.000 claims description 6
- 108010090804 Streptavidin Proteins 0.000 claims description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
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- 210000001808 exosome Anatomy 0.000 abstract description 42
- 230000008878 coupling Effects 0.000 abstract description 3
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Abstract
本发明公开了一种使用免疫磁珠分离血清中外泌体的方法，方法包括血清预处理，单克隆抗体偶联磁珠，抗体‑磁珠复合物分离血清中外泌体步骤。本发明的优点是：操作简单，成本低；且可对血清中含有特定标志物的外泌体进行分离。
The invention discloses a method for separating exosomes in serum by using immune magnetic beads. The method includes the steps of serum pretreatment, monoclonal antibody coupling to magnetic beads, and antibody-magnetic bead complexes for separating exosomes in serum. The present invention has the advantages of simple operation and low cost; and the ability to separate exosomes containing specific markers in serum.
Description
技术领域technical field
本发明涉及生物医学领域，具体是关于一种使用免疫磁珠分离血清中外泌体的方法。The invention relates to the field of biomedicine, in particular to a method for separating exosomes in serum by using immunomagnetic beads.
背景技术Background technique
免疫磁珠分离技术是一项基于抗原-抗体结合作用发展起来的免疫学技术。它主要是靠在磁珠表面修饰氨基，羧基，链霉亲和素等基团，利用这些基团可以和抗体共价或非共价偶联，可用于结合相应的抗原，在外加磁场的作用下可达到相应抗原物质的分离，广泛应用于核酸提取，膜结构物质分离如细胞分离领域。Immunomagnetic bead separation technology is an immunological technology developed based on antigen-antibody binding. It mainly relies on modifying amino groups, carboxyl groups, streptavidin and other groups on the surface of magnetic beads. These groups can be used for covalent or non-covalent coupling with antibodies, which can be used to bind corresponding antigens. It can achieve the separation of corresponding antigenic substances, and is widely used in nucleic acid extraction, separation of membrane structure substances such as cell separation.
外泌体（exosome）是一类直径为30-150nm的膜结构囊泡，广泛存在于血液，尿液，唾液等体液中。外泌体膜表面含有CD9，CD63和CD81标志蛋白，膜内含有核酸如microRNA。近年来研究发现，肿瘤患者体内血清外泌体分泌异常，具体表现为：（1）肿瘤患者体内血清外泌体膜表面除含有CD9，CD63和CD81标志蛋白外，还含有特定标记蛋白，如胰腺癌患者体内血清外泌体中特定表达GPC1蛋白；（2）肿瘤患者体内血清外泌体中microRNA表达异常，如肝癌患者体内血清外泌体中microRNA-221表达量明显高于正常人表达量。因此，血清外泌体的表达分泌情况可作为肿瘤早期诊断的依据。Exosomes (exosomes) are a type of membrane-structured vesicles with a diameter of 30-150 nm, which are widely found in blood, urine, saliva and other body fluids. The surface of the exosome membrane contains CD9, CD63 and CD81 marker proteins, and the membrane contains nucleic acids such as microRNA. In recent years, studies have found that the secretion of serum exosomes in tumor patients is abnormal, and the specific manifestations are: (1) In addition to CD9, CD63 and CD81 marker proteins, the surface of serum exosome membranes in tumor patients also contains specific marker proteins, such as pancreatic GPC1 protein is specifically expressed in serum exosomes of cancer patients; (2) microRNA expression in serum exosomes of tumor patients is abnormal, for example, the expression of microRNA-221 in serum exosomes of liver cancer patients is significantly higher than that of normal people. Therefore, the expression and secretion of serum exosomes can be used as the basis for early diagnosis of tumors.
目前，血清外泌体分离有离心法，色谱法，大分子聚合物沉降法，免疫磁珠试剂盒法。离心法对外泌体得率较高，但其耗时耗力，不适合大量样本操作。色谱法提取的外泌体纯度较高，但其分离易受杂蛋白干扰，操作稳定性差。大分子聚合物沉淀外泌体耗时短，缺点是所得外泌体纯度低。以上所述技术均只能对血清中总的外泌体进行分离，缺乏对特定标志物外泌体(如CD9+/CD63+外泌体)的分离，免疫磁珠试剂盒法可对特定标志物的外泌体进行分离，但该项技术仅为少数国际巨头公司如美国SBI公司，日本MBL公司所掌握，使用成本高昂。Currently, serum exosomes are separated by centrifugation, chromatography, macromolecular polymer sedimentation, and immunomagnetic bead kit methods. The centrifugation method has a high yield of exosomes, but it is time-consuming and labor-intensive, and is not suitable for a large number of samples. The purity of exosomes extracted by chromatography is high, but its separation is easily interfered by foreign proteins, and the operation stability is poor. It takes a short time to precipitate exosomes with macromolecular polymers, but the disadvantage is that the purity of the obtained exosomes is low. The above-mentioned techniques can only isolate the total exosomes in serum, and lack the isolation of specific marker exosomes (such as CD9 + /CD63 + exosomes). The immunomagnetic bead kit method can isolate specific markers However, this technology is only mastered by a few international giants such as SBI in the United States and MBL in Japan, and the cost of use is high.
发明内容Contents of the invention
针对现有技术的缺陷，本发明的目的在于提供一种，操作简单，成本低，针对含有特定标志物外泌体分离方法。In view of the defects of the prior art, the purpose of the present invention is to provide a method for isolating exosomes containing specific markers, which is simple in operation and low in cost.
本发明的具体技术方案是：一种使用免疫磁珠分离血清中外泌体的方法，该方法包括以下各步骤：The specific technical solution of the present invention is: a method of using immunomagnetic beads to separate exosomes in serum, the method comprising the following steps:
（1）预处理，室温下以新鲜血清，经离心分离，去除细胞碎片，取上清；(1) For pretreatment, use fresh serum at room temperature, centrifuge to remove cell debris, and take the supernatant;
（2）取1倍体积链霉亲和素修饰磁珠悬液用100倍体积分离缓冲液清洗，磁力架分离磁珠，100倍体积分离缓冲液悬浮；将上述悬浮液加入1倍体积CD9或CD63单克隆抗体孵育；孵育完成后用100倍体积分离缓冲液清洗，磁力架分离磁珠，100倍体积分离缓冲液悬浮；(2) Take 1 volume of streptavidin-modified magnetic bead suspension and wash with 100 volumes of separation buffer, separate the magnetic beads on a magnetic stand, and suspend in 100 volumes of separation buffer; add the above suspension to 1 volume of CD9 or CD63 monoclonal antibody incubation; after the incubation is completed, wash with 100 times the volume of separation buffer, separate the magnetic beads with a magnetic frame, and suspend with 100 times the volume of separation buffer;
（3）将步骤（2）最终得到的磁珠悬浮液中加入2-10倍体积步骤（1）处理得到的血清上清，孵育；(3) Add 2-10 times the volume of the serum supernatant obtained in step (1) to the magnetic bead suspension finally obtained in step (2), and incubate;
（4）将步骤（3）孵育完成后用100倍体积磷酸缓冲盐溶液清洗，磁力架分离磁珠，即得分离有外泌体的复合物即磁珠-抗体-外泌体。(4) After step (3) is incubated, wash with 100 times the volume of phosphate-buffered saline, and separate the magnetic beads on a magnetic stand to obtain a complex with exosomes separated, that is, magnetic beads-antibody-exosomes.
进一步的，上述步骤（2）中悬浮液加入1倍体积CD9或CD63单克隆抗体孵育30分钟。Further, add 1 volume of CD9 or CD63 monoclonal antibody to the suspension in the above step (2) and incubate for 30 minutes.
进一步的，上述步骤（3）孵育2小时。Further, the above step (3) was incubated for 2 hours.
进一步的，上述步骤中磁力架分离磁珠，清洗三遍。Further, in the above steps, the magnetic beads are separated by the magnetic stand and washed three times.
进一步的，上述分离缓冲液为含0.1%牛血清白蛋白的磷酸缓冲盐溶液。Further, the above separation buffer is a phosphate buffered saline solution containing 0.1% bovine serum albumin.
进一步的，上述分离缓冲液以0.2微米滤膜过滤除菌。Further, the above-mentioned separation buffer is sterilized by filtration with a 0.2 micron filter membrane.
进一步的，上述的孵育条件为：30℃，孵育器参数为颠倒旋转90°，倾斜5秒，5°震动持续1秒。Further, the above-mentioned incubation conditions are: 30° C., the incubator parameters are upside-down rotation 90°, tilting for 5 seconds, and 5° vibration for 1 second.
附图说明Description of drawings
图1为免疫磁珠分离血清中CD9+外泌体的透射电镜效果图。其中A,B分别为偶联有/未偶联有生物素化CD9单克隆抗体的磁珠富集血清外泌体后的效果图。Figure 1 is a transmission electron microscope effect diagram of CD9 + exosomes in serum separated by immunomagnetic beads. Among them, A and B are the effect diagrams of serum exosomes enriched by magnetic beads coupled with/without biotinylated CD9 monoclonal antibody, respectively.
图2为免疫磁珠分离血清中CD63+外泌体的透射电镜效果图。其中A,B分别为偶联有/未偶联有生物素化CD63单克隆抗体的磁珠富集血清外泌体后的效果图。Figure 2 is a transmission electron microscope effect diagram of CD63 + exosomes in serum separated by immunomagnetic beads. Among them, A and B are the effects of enriching serum exosomes with magnetic beads coupled with/without biotinylated CD63 monoclonal antibody, respectively.
具体实施方式detailed description
下面结合附图和实施例对本发明作进一步说明。The present invention will be further described below in conjunction with drawings and embodiments.
本发明的基本思想是使用免疫磁珠分离血清中外泌体。 The basic idea of the present invention is to use immunomagnetic beads to separate exosomes in serum.
下述实施例中使用的主要仪器及试剂：链霉亲和素修饰Dynabeads® MyOne™Streptavidin T1磁珠（Invitrogen公司，美国）；生物素修饰的鼠抗人CD9单克隆抗体（Ancell公司，美国）；生物素修饰的鼠抗人CD63单克隆抗体（Ancell公司，美国）；HulaMixer® Sample Mixer孵育器（Invitrogen公司，美国）;透射电子显微镜（Hitachi，日本）Main instruments and reagents used in the following examples: Streptavidin-modified Dynabeads® MyOne™ Streptavidin T1 magnetic beads (Invitrogen, USA); biotin-modified mouse anti-human CD9 monoclonal antibody (Ancell Corporation, USA) ; Biotin-modified mouse anti-human CD63 monoclonal antibody (Ancell, USA); HulaMixer® Sample Mixer incubator (Invitrogen, USA); transmission electron microscope (Hitachi, Japan)
实施例1.免疫磁珠分离血清中CD9+外泌体Example 1. Separation of CD9 + exosomes in serum by immunomagnetic beads
一．方法one. method
（1）收集新鲜血清，置于离心机中，室温2000g离心30分钟，弃细胞碎片沉淀，取离心上清液（若血清处于冰冻状态，需先26℃水浴解冻后再离心）。(1) Collect fresh serum, put it in a centrifuge, and centrifuge at 2000g at room temperature for 30 minutes, discard the cell debris and precipitate, and take the centrifuged supernatant (if the serum is in a frozen state, it needs to be thawed in a water bath at 26°C before centrifuging).
（2）漩涡震荡链霉亲和素修饰Dynabeads® MyOne™ Streptavidin T1磁珠，待磁珠悬浮液均一后，取5微升磁珠悬液加入到500微升分离缓冲液中，悬浮清洗，磁力架分离磁珠，清洗三遍。清洗完成后，500微升分离缓冲液悬浮磁珠。(2) Vortex streptavidin-modified Dynabeads® MyOne™ Streptavidin T1 magnetic beads. After the magnetic bead suspension is uniform, take 5 microliters of the magnetic bead suspension and add it to 500 microliters of separation buffer, suspend and wash, and magnetically Magnetic beads were separated by rack and washed three times. After washing is complete, resuspend the beads in 500 µl of separation buffer.
（3）取5微升Ancell公司生产的鼠抗人生物素化CD9单克隆抗体加入到上述方法（2）所得的磁珠悬液中，颠倒混合均匀，置于HulaMixer® Sample Mixer孵育器上孵育30分钟（参数为颠倒旋转90°，倾斜5秒，5°震动持续1秒）；孵育完成，孵育液置于磁力架上5分钟，弃清液，磁珠用500微升分离缓冲液悬浮，悬浮清洗，磁力架分离磁珠，清洗三遍。清洗完成后，500微升分离缓冲液悬浮磁珠。(3) Take 5 microliters of the mouse anti-human biotinylated CD9 monoclonal antibody produced by Ancell Company and add it to the magnetic bead suspension obtained in the above method (2), mix evenly by inverting, and incubate on the HulaMixer® Sample Mixer incubator 30 minutes (the parameters are 90° upside-down rotation, 5 seconds of tilting, 5° shaking for 1 second); the incubation is completed, the incubation solution is placed on the magnetic stand for 5 minutes, the supernatant is discarded, and the magnetic beads are suspended with 500 microliters of separation buffer. Suspension wash, separate the magnetic beads with a magnetic stand, and wash three times. After washing is complete, resuspend the beads in 500 µl of separation buffer.
（4）取10微升方法（1）所得的血清加入到方法（3）所得的磁珠悬液中，颠倒混合均匀，置于HulaMixer® Sample Mixer孵育器上孵育2小时（参数为颠倒旋转90°，倾斜5秒，5°震动持续1秒）；孵育完成，孵育液置于磁力架上，弃清液，磁珠用500微升分离缓冲液悬浮，悬浮清洗，磁力架分离磁珠，清洗三遍；清洗完成后，即得分离有CD9+外泌体的复合物即磁珠-抗体-CD9+外泌体。(4) Add 10 microliters of the serum obtained in method (1) to the magnetic bead suspension obtained in method (3), mix evenly by inverting, and incubate on the HulaMixer® Sample Mixer incubator for 2 hours (the parameter is inverted rotation 90 °, tilt for 5 seconds, and shake at 5° for 1 second); after the incubation is completed, place the incubation solution on the magnetic stand, discard the supernatant, suspend the magnetic beads with 500 microliters of separation buffer, suspend and wash, separate the magnetic beads on the magnetic stand, and wash Three times; after the washing is completed, the complex with CD9 + exosomes is separated, that is, magnetic beads-antibody-CD9 + exosomes.
二．结果two. result
上述方法（4）所得到的磁珠-抗体-CD9+外泌体复合物经扫描电镜拍摄后得到附图1所示结果，显示磁珠表面The magnetic bead-antibody-CD9 + exosome complex obtained by the above method (4) was photographed by a scanning electron microscope and the results shown in Figure 1 were obtained, showing that the surface of the magnetic bead
实施例2.免疫磁珠分离血清中CD63+外泌体Example 2. Separation of CD63 + exosomes in serum by immunomagnetic beads
一．方法one. method
（1）收集新鲜血清，置于离心机中，室温2000g离心30分钟，弃细胞碎片沉淀，取离心上清液（若血清处于冰冻状态，需先26℃水浴解冻后再离心）。(1) Collect fresh serum, put it in a centrifuge, and centrifuge at 2000g at room temperature for 30 minutes, discard the cell debris and precipitate, and take the centrifuged supernatant (if the serum is in a frozen state, it needs to be thawed in a water bath at 26°C before centrifuging).
（2）漩涡震荡链霉亲和素修饰Dynabeads® MyOne™ Streptavidin T1磁珠，待磁珠悬浮液均一后，取5微升磁珠悬液加入到500微升分离缓冲液中，悬浮清洗，磁力架分离磁珠，清洗三遍。清洗完成后，500微升分离缓冲液悬浮磁珠。(2) Vortex streptavidin-modified Dynabeads® MyOne™ Streptavidin T1 magnetic beads. After the magnetic bead suspension is uniform, take 5 microliters of the magnetic bead suspension and add it to 500 microliters of separation buffer, suspend and wash, and magnetically Magnetic beads were separated by rack and washed three times. After washing is complete, resuspend the beads in 500 µl of separation buffer.
（3）取5微升Ancell公司生产的鼠抗人生物素化CD63单克隆抗体加入到上述方法（2）所得的磁珠悬液中，颠倒混合均匀，置于HulaMixer® Sample Mixer孵育器上孵育30分钟（参数为颠倒旋转90°，倾斜5秒，5°震动持续1秒）；孵育完成，孵育液置于磁力架上5分钟，弃清液，磁珠用500微升分离缓冲液悬浮，悬浮清洗，磁力架分离磁珠，清洗三遍。清洗完成后，500微升分离缓冲液悬浮磁珠。(3) Take 5 microliters of the mouse anti-human biotinylated CD63 monoclonal antibody produced by Ancell Company and add it to the magnetic bead suspension obtained in the above method (2), mix evenly by inverting, and incubate on the HulaMixer® Sample Mixer incubator 30 minutes (the parameters are 90° upside-down rotation, 5 seconds of tilting, 5° shaking for 1 second); the incubation is completed, the incubation solution is placed on the magnetic stand for 5 minutes, the supernatant is discarded, and the magnetic beads are suspended with 500 microliters of separation buffer. Suspension wash, separate the magnetic beads with a magnetic stand, and wash three times. After washing is complete, resuspend the beads in 500 µl of separation buffer.
（4）取10微升方法（1）所得的血清加入到方法（3）所得的磁珠悬液中，颠倒混合均匀，置于HulaMixer® Sample Mixer孵育器上孵育2小时（参数为颠倒旋转90°，倾斜5秒，5°震动持续1秒）；孵育完成，孵育液置于磁力架上，弃清液，磁珠用500微升分离缓冲液悬浮，悬浮清洗，磁力架分离磁珠，清洗三遍；清洗完成后，即得分离有CD63+外泌体的复合物即磁珠-抗体-CD63+外泌体。(4) Add 10 microliters of the serum obtained in method (1) to the magnetic bead suspension obtained in method (3), mix evenly by inverting, and incubate on the HulaMixer® Sample Mixer incubator for 2 hours (the parameter is inverted rotation 90 °, tilt for 5 seconds, and shake at 5° for 1 second); after the incubation is completed, place the incubation solution on the magnetic stand, discard the supernatant, suspend the magnetic beads with 500 microliters of separation buffer, suspend and wash, separate the magnetic beads on the magnetic stand, and wash Three times; after the washing is completed, the complex with CD63 + exosomes is separated, that is, magnetic beads-antibody-CD63 + exosomes.
二．结果two. result
上述方法（4）所得到的磁珠-抗体-CD63+外泌体复合物经扫描电镜拍摄后得到附图2所示结果，附图2中的A和B对比发现，A中未偶联有生物素化CD63单克隆抗体的磁珠富集血清外泌体后，磁珠表面未显示附着有外泌体，而B中偶联有生物素化CD63单克隆抗体的磁珠富集血清外泌体后，如图中箭头所示，磁珠表面显示有直径为30-150nm外泌体附着，表明本发明能够有效的富集血清样本中CD63+外泌体。The magnetic bead-antibody-CD63 + exosome complex obtained by the above method (4) was photographed by a scanning electron microscope and the results shown in Figure 2 were obtained. A comparison of A and B in Figure 2 revealed that there was no coupling in A After the magnetic beads of biotinylated CD63 monoclonal antibody enriched serum exosomes, the surface of the magnetic beads did not show exosomes attached, while the magnetic beads coupled with biotinylated CD63 monoclonal antibody in B enriched serum exosomes After ex vivo, as shown by the arrow in the figure, exosomes with a diameter of 30-150 nm were attached to the surface of the magnetic beads, indicating that the present invention can effectively enrich CD63 + exosomes in serum samples.
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