CN115469096A - Method for detecting autoimmune hemolytic anemia antibody based on flow microsphere technology - Google Patents
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Abstract
Description
技术领域technical field
本发明属于生物分子学检测及免疫学检测领域。具体的说，是一种基于流式微球技术检测自身免疫性溶血性贫血抗体的方法。The invention belongs to the fields of biomolecular detection and immunological detection. Specifically, it is a method for detecting autoimmune hemolytic anemia antibodies based on flow microsphere technology.
技术背景technical background
自身免疫性溶血性贫血（Autoimmune hemolytic anemia，AIHA）是指系体内免疫功能调节紊乱，自身免疫性溶血性贫血患者血清中含有针对自身红细胞的抗体和（或）补体吸附于红细胞表面，通过抗原抗体反应加速红细胞破坏而引起的一种溶血性贫血。Autoimmune hemolytic anemia (Autoimmune hemolytic anemia, AIHA) refers to the disorder of immune function regulation in the system. The serum of patients with autoimmune hemolytic anemia contains antibodies against their own red blood cells and (or) complement is adsorbed on the surface of red blood cells. A type of hemolytic anemia caused by the accelerated destruction of red blood cells.
流式细胞术是一种生物学技术，用于对悬浮于流体中的微小颗粒进行计数和分选。这种技术可以用来对流过光学或电子检测器的一个个细胞进行连续的多种参数分析。流式细胞术是对悬液中的单细胞或其他生物粒子,通过检测标记的荧光信号，实现高速、逐一的细胞定量分析和分选的技术。Flow cytometry is a biological technique used to count and sort tiny particles suspended in a fluid. This technique can be used for continuous multi-parameter analysis of individual cells flowing through optical or electronic detectors. Flow cytometry is a technique for high-speed, one-by-one quantitative analysis and sorting of single cells or other biological particles in suspension by detecting labeled fluorescent signals.
发明内容Contents of the invention
本发明的目的是提供一种基于流式微球技术检测自身免疫性溶血性贫血抗体的方法。The purpose of the present invention is to provide a method for detecting autoimmune hemolytic anemia antibody based on flow microsphere technology.
本发明的技术方案为：Technical scheme of the present invention is:
以功能性官能团修饰的聚合物微球作为载体，先将微球与检测抗原进行偶联，通过与样本中的自身免疫性溶血性贫血抗体和荧光标记的二抗进行反应，形成“聚合物微球-检测抗原-抗体-荧光标记的二抗”的复合物，通过流式细胞仪分析微球上二抗的荧光强度，进而分析待测样本中是否存在自身免疫性溶血性贫血的抗体以及抗体的浓度。Using polymer microspheres modified with functional functional groups as a carrier, the microspheres are first coupled to the detection antigen, and react with the autoimmune hemolytic anemia antibody in the sample and the fluorescently labeled secondary antibody to form a "polymer microsphere". Ball-Detection Antigen-Antibody-Fluorescently Labeled Secondary Antibody” complex, analyze the fluorescence intensity of the secondary antibody on the microsphere by flow cytometry, and then analyze whether there are autoimmune hemolytic anemia antibodies and antibodies in the sample to be tested concentration.
所采用的聚合物微球为羧基修饰的聚苯乙烯微球，粒径为微米级。The polymer microspheres adopted are polystyrene microspheres modified by carboxyl groups, and the particle diameter is micron order.
所采用的荧光标记的二抗为羊抗人免疫球蛋白IgG，二抗中所携带的荧光基团为异硫氰酸荧光素FITC。The fluorescently labeled secondary antibody used was goat anti-human immunoglobulin IgG, and the fluorescent group carried in the secondary antibody was fluorescein isothiocyanate FITC.
具体制备过程和检测方法为：The specific preparation process and detection method are as follows:
（1）聚合物微球与检测抗原偶联：取1.0×106个同一批次生产的羧基聚苯乙烯微球于离心管中，用洗涤液冲洗微球，再加入活化缓冲液将微球活化，室温避光条件下震荡0.5-1 h，向活化后的微球中加入检测抗原，混匀后室温避光震荡，震荡结束用洗涤液洗涤三次，最后加入封闭液震荡2h，将“聚合物微球-检测抗原”复合物置于保存液中4℃避光保存备用；(1) Coupling of polymer microspheres and detection antigen: take 1.0×10 6 carboxypolystyrene microspheres produced in the same batch in a centrifuge tube, wash the microspheres with washing solution, and then add activation buffer to dissolve the microspheres. After activation, shake for 0.5-1 h at room temperature and avoid light, add detection antigen to the activated microspheres, mix well, shake at room temperature and avoid light, wash three times with washing solution after shaking, finally add blocking solution and shake for 2 hours, and "polymerize The "microsphere-detection antigen" complex was stored in the preservation solution at 4°C and protected from light for later use;
（2）取一定量的“聚合物微球-检测抗原”复合物与离心管中，加入待测样本，混合均匀后室温避光震荡反应1h，反应结束后离心、洗涤去除未反应抗体，形成“聚合物微球-检测抗原-抗体”复合物；(2) Take a certain amount of "polymer microsphere-detection antigen" complex and a centrifuge tube, add the sample to be tested, mix well, and then shake it at room temperature for 1 hour in the dark. After the reaction, centrifuge and wash to remove unreacted antibodies to form "Polymer microsphere-detection antigen-antibody" complex;
（3）在“聚合物微球-检测抗原-抗体”复合物中加入FITC荧光标记的二抗，混合均匀后室温避光震荡反应1 h，反应结束后离心、洗涤去除未反应二抗，形成“聚合物微球-检测抗原-抗体-荧光标记的二抗”复合物；(3) Add FITC fluorescently-labeled secondary antibody to the "polymer microsphere-detection antigen-antibody" complex, mix well, and then shake and react at room temperature for 1 hour in the dark. After the reaction, centrifuge and wash to remove unreacted secondary antibody to form "Polymer microsphere-detection antigen-antibody-fluorescence-labeled secondary antibody" complex;
（4）将“聚合物微球-检测抗原-抗体-荧光标记的二抗”复合物加入稀释液稀释，用流式细胞仪对其进行荧光强度检测，检测得到抗体是否存在及其浓度。(4) Dilute the "polymer microsphere-detection antigen-antibody-fluorescence-labeled secondary antibody" complex by adding diluent, and detect the fluorescence intensity with a flow cytometer to detect the presence and concentration of the antibody.
优选的，所用的聚合物微球为羧基处理的聚苯乙烯荧光微球。Preferably, the polymer microspheres used are carboxy-treated polystyrene fluorescent microspheres.
优选的，荧光标记的二抗为羊抗人免疫球蛋白IgG。Preferably, the fluorescently labeled secondary antibody is goat anti-human immunoglobulin IgG.
优选的，荧光标记的二抗所用的荧光基团为异硫氰酸荧光素FITC。Preferably, the fluorescent group used in the fluorescently labeled secondary antibody is fluorescein isothiocyanate FITC.
优选的，活化缓冲液是NaH2PO4溶液。Preferably, the activation buffer is NaH 2 PO 4 solution.
优选的，封闭液是1%BSA的PBS溶液。Preferably, the blocking solution is 1% BSA in PBS.
本领域中，以及western、免疫组化、ELISA等都会有封闭这一步，并且其封闭液种类、浓度及时间都是对结果影响非常大的一步。但是蛋白并不是连续的，有很多空隙，而且比如说样品孔之间也有空隙，而抗体也是蛋白，也会被吸附在空的洞里，这样，就会有很多非特异性的信号，但如果用富含蛋白的封闭液与聚合物微球作用，那所有的空洞都会被BSA或者其他蛋白填满，但是，通过大量实验发明，只用1%BSA的PBS溶液，效果并不是最好的，聚合物微球仍然会有少量的空洞没有完全封闭，二抗会吸附到微球上，影响后期的信号准确度。In this field, as well as western, immunohistochemistry, ELISA, etc., there will be a blocking step, and the type, concentration and time of the blocking solution have a great impact on the result. But the protein is not continuous, there are many gaps, and for example, there are gaps between the sample wells, and the antibody is also a protein, and it will also be adsorbed in the empty holes, so there will be many non-specific signals, but if you use When the protein-rich blocking solution interacts with polymer microspheres, all the cavities will be filled with BSA or other proteins. However, through a large number of experiments, it is found that only 1% BSA in PBS solution is not the best. Polymerization There will still be a small amount of cavities in the microspheres that are not completely closed, and the secondary antibody will be adsorbed on the microspheres, which will affect the accuracy of the signal in the later stage.
本发明提供一种封闭液组合，优选的，封闭液还包括糖类物质、羟乙基纤维素、山梨醇。进一步的，封闭液具体包括如下质量百分比的成分：糖类物质为5％，羟乙基纤维素为0.1％，BSA为1％，山梨醇为0.5％，其余为PBS缓冲液。The present invention provides a blocking solution combination, preferably, the blocking solution further includes carbohydrates, hydroxyethyl cellulose, and sorbitol. Further, the blocking solution specifically includes the following components in mass percentage: 5% carbohydrate, 0.1% hydroxyethyl cellulose, 1% BSA, 0.5% sorbitol, and the rest is PBS buffer.
优选的，糖类物质是瓜尔胶、海藻糖、蔗糖、葡萄糖的任意一种。Preferably, the carbohydrate is any one of guar gum, trehalose, sucrose, and glucose.
本发明的有点在于：The advantages of the present invention are:
本发明以功能性官能团修饰的聚合物微球作为载体，先将微球与检测抗原进行偶联，再通过与样本中的自身免疫性溶血性贫血抗体进行偶联，对其进行离心、洗涤后再和荧光标记的二抗进行反应，形成“聚合物微球-检测抗原-抗体-荧光标记的二抗”的复合物，通过流式细胞仪分析微球上二抗的荧光强度，进而分析待测样本中的抗体的浓度。本发明的优点在于采用流式微球检测技术，可以提高检测效率，也可以制备成试剂盒，简易方便，为临床诊断及治疗提供依据。In the present invention, polymer microspheres modified with functional functional groups are used as carriers, and the microspheres are first coupled with the detection antigen, and then coupled with the autoimmune hemolytic anemia antibody in the sample, and then centrifuged and washed. Then react with the fluorescently labeled secondary antibody to form a complex of "polymer microspheres-detection antigen-antibody-fluorescently labeled secondary antibody". The fluorescence intensity of the secondary antibody on the microspheres is analyzed by flow cytometry, and then analyzed. Measure the concentration of antibody in the sample. The invention has the advantages of adopting the flow microsphere detection technology, which can improve the detection efficiency, and can also be prepared into a kit, which is simple and convenient, and provides a basis for clinical diagnosis and treatment.
本发明采用流式细胞仪进行检测，此项检测技术成熟，检测灵敏度高，检测时间短，可以提高检测效率，荧光基团具有多种选择性，二抗种类也具有多种选择性，可以制备成试剂盒，简易方便，为临床诊断及治疗提供依据。The invention adopts flow cytometer for detection, the detection technology is mature, the detection sensitivity is high, the detection time is short, and the detection efficiency can be improved. It is easy and convenient to form a kit, providing a basis for clinical diagnosis and treatment.
同时，本发明还提供一种封闭液组合物，可以降低抗体直接和聚合物微球结合的数量，提高检测准确度。At the same time, the invention also provides a blocking solution composition, which can reduce the number of antibodies directly bound to the polymer microspheres and improve detection accuracy.
具体实施方式detailed description
下面结合具体实施方式对本发明进行详细的说明。The present invention will be described in detail below in combination with specific embodiments.
实施例1：Example 1:
1、聚合物微球与检测抗原偶联：取1.0×106个同一批次生产的羧基聚苯乙烯微球于1.5 mL离心管中，加入定量的磷酸盐缓冲溶液(PBS溶液，Na2HPO4和KH2PO4组成，0.1mol/L,pH7.4)冲洗微球，再加入NaH2PO4(0.1 mol/L,pH6.2)将微球活化，室温避光条件下震荡0.5-1 h，向活化后的微球中加入100 μL检测抗原，混匀后室温避光震荡，震荡结束用PBS溶液洗涤三次，最后加入含有1%BSA的PBS溶液震荡2h，将“聚合物微球-检测抗原”复合物置于保存液中4℃避光保存备用。1. Coupling of polymer microspheres and detection antigen: Take 1.0×10 6 carboxypolystyrene microspheres produced in the same batch in a 1.5 mL centrifuge tube, add quantitative phosphate buffer solution (PBS solution, Na 2 HPO 4 and KH 2 PO 4 , 0.1mol/L, pH 7.4) to wash the microspheres, then add NaH 2 PO 4 (0.1 mol/L, pH 6.2) to activate the microspheres, shake at room temperature for 0.5- After 1 h, add 100 μL of detection antigen to the activated microspheres, mix well, and shake at room temperature in the dark. After shaking, wash with PBS solution three times, and finally add PBS solution containing 1% BSA for 2 h. -Detection antigen"complex is stored in the preservation solution at 4°C and protected from light for later use.
2、取一定量的“聚合物微球-检测抗原”复合物于1.5mL离心管中，加入待测样本，混合均匀后室温避光震荡反应1h，反应结束后转速3000 rpm离心3 min，并用500 μL的PBS溶液洗涤3次去除未反应抗体，形成“聚合物微球-检测抗原-抗体”复合物。2. Take a certain amount of "polymer microspheres-detection antigen" complex in a 1.5mL centrifuge tube, add the sample to be tested, mix well, shake at room temperature and avoid light for 1 hour, centrifuge at 3000 rpm for 3 minutes after the reaction, and use 500 μL of PBS solution was washed 3 times to remove unreacted antibodies to form a "polymer microsphere-detection antigen-antibody" complex.
3、取“聚合物微球-检测抗原-抗体”复合物于1.5 mL离心管中，加入FITC荧光标记的二抗，混合均匀后室温避光震荡反应1 h，反应结束后转速3000 rpm离心3 min，洗涤去除未反应二抗，形成“聚合物微球-检测抗原-抗体-荧光标记的二抗”复合物。3. Take the "polymer microsphere-detection antigen-antibody" complex in a 1.5 mL centrifuge tube, add FITC fluorescently labeled secondary antibody, mix well, shake at room temperature and avoid light for 1 h, and centrifuge at 3000 rpm for 3 min, wash to remove unreacted secondary antibody, and form a "polymer microsphere-detection antigen-antibody-fluorescent-labeled secondary antibody" complex.
4、将“聚合物微球-检测抗原-抗体-荧光标记的二抗”复合物加入稀释液稀释，用流式细胞仪对其进行荧光强度检测，检测得到抗体是否存在及其浓度。4. Dilute the "polymer microsphere-detection antigen-antibody-fluorescence-labeled secondary antibody" complex by adding diluent, and detect the fluorescence intensity with a flow cytometer to detect the presence and concentration of the antibody.
实施例2：Example 2:
1、聚合物微球与检测抗原偶联：取1.0×106个同一批次生产的羧基聚苯乙烯微球于1.5 mL离心管中，加入定量的磷酸盐缓冲溶液(PBS溶液，Na2HPO4和KH2PO4组成，0.1mol/L,pH7.4)冲洗微球，再加入NaH2PO4(0.1 mol/L,pH6.2)将微球活化，室温避光条件下震荡0.5-1 h，向活化后的微球中加入100 μL检测抗原，混匀后室温避光震荡，震荡结束用PBS溶液洗涤三次，最后加入含有1%BSA、5％瓜尔胶，0.1％羟乙基纤维素，0.5％山梨醇的PBS溶液震荡2h，将“聚合物微球-检测抗原”复合物置于保存液中4℃避光保存备用。1. Coupling of polymer microspheres and detection antigen: Take 1.0×10 6 carboxypolystyrene microspheres produced in the same batch in a 1.5 mL centrifuge tube, add quantitative phosphate buffer solution (PBS solution, Na 2 HPO 4 and KH 2 PO 4 , 0.1mol/L, pH 7.4) to wash the microspheres, then add NaH 2 PO 4 (0.1 mol/L, pH 6.2) to activate the microspheres, shake at room temperature for 0.5- 1 h, add 100 μL of detection antigen to the activated microspheres, mix well, shake at room temperature in the dark, wash with PBS solution three times after shaking, and finally add 1% BSA, 5% guar gum, 0.1% hydroxyethyl The cellulose and 0.5% sorbitol in PBS solution were shaken for 2 hours, and the "polymer microsphere-detection antigen" complex was placed in the preservation solution at 4°C and protected from light for later use.
2、取一定量的“聚合物微球-检测抗原”复合物于1.5mL离心管中，加入待测样本，混合均匀后室温避光震荡反应1h，反应结束后转速3000 rpm离心3 min，并用500 μL的PBS溶液洗涤3次去除未反应抗体，形成“聚合物微球-检测抗原-抗体”复合物。2. Take a certain amount of "polymer microspheres-detection antigen" complex in a 1.5mL centrifuge tube, add the sample to be tested, mix well, shake at room temperature and avoid light for 1 hour, centrifuge at 3000 rpm for 3 minutes after the reaction, and use 500 μL of PBS solution was washed 3 times to remove unreacted antibodies to form a "polymer microsphere-detection antigen-antibody" complex.
3、取“聚合物微球-检测抗原-抗体”复合物于1.5 mL离心管中，加入FITC荧光标记的二抗，混合均匀后室温避光震荡反应1 h，反应结束后转速3000 rpm离心3 min，洗涤去除未反应二抗，形成“聚合物微球-检测抗原-抗体-荧光标记的二抗”复合物。3. Take the "polymer microsphere-detection antigen-antibody" complex in a 1.5 mL centrifuge tube, add FITC fluorescently labeled secondary antibody, mix well, shake at room temperature and avoid light for 1 h, and centrifuge at 3000 rpm for 3 min, wash to remove unreacted secondary antibody, and form a "polymer microsphere-detection antigen-antibody-fluorescent-labeled secondary antibody" complex.
4、将“聚合物微球-检测抗原-抗体-荧光标记的二抗”复合物加入稀释液稀释，用流式细胞仪对其进行荧光强度检测，检测得到抗体是否存在及其浓度。4. Dilute the "polymer microsphere-detection antigen-antibody-fluorescence-labeled secondary antibody" complex by adding diluent, and detect the fluorescence intensity with a flow cytometer to detect the presence and concentration of the antibody.
实施例3：实施例1和实施例2的准确度对比Embodiment 3: the accuracy contrast of embodiment 1 and embodiment 2
准确称取10μg/ML的抗体，分别按照实施例1和实施例2的方法，进行检测，结果为：Accurately weigh 10 μg/ML of the antibody, and perform detection according to the methods of Example 1 and Example 2 respectively, and the results are:
实施例1最终测得的结果是11.3μg/ML,实施例2测得的结果为9.6μg/ML。The final measured result of Example 1 is 11.3 μg/ML, and the measured result of Example 2 is 9.6 μg/ML.
由此可见，实施例1的结果，要高于实际的抗体浓度，原因是二抗加入后，微球中含有少量未封闭的空间被二抗吸附，造成假阳性，使得测试结果偏高。It can be seen that the result of Example 1 is higher than the actual antibody concentration. The reason is that after the secondary antibody is added, a small amount of unclosed space in the microspheres is absorbed by the secondary antibody, resulting in false positives and high test results.
以上实施方式仅用于帮助本领域技术人员更加全面的理解本发明，但并不对本发明做任何限制。翻译局本发明的内容作出的任何本领域等同的替换均属于本发明保护的范围之内。The above embodiments are only used to help those skilled in the art understand the present invention more comprehensively, but do not limit the present invention in any way. Any equivalent replacements in the field made by the translation bureau for the content of the present invention fall within the protection scope of the present invention.
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