CN110716051A - 一种免疫细胞全维分析方法 - Google Patents
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Abstract
本发明公开了一种免疫细胞全维分析方法,包括:取不同的荧光标记的抗体,与待检测样品混合,孵育后,经流式细胞术检测,得检测数据,分析所得检测数据包括中各类免疫细胞的百分比及计数,本发明的方法实现了对免疫细胞进行更加全面的精细免疫分型,所需待测样品少,所需时间短、准确性高、分析全面,可广泛应用于免疫细胞全维分析。
Description
技术领域
本发明属于免疫学技术领域,尤其涉及一种免疫细胞全维分析方 法。
背景技术
免疫系统是覆盖全身的防卫网络,能抵御病原菌侵袭,是身体的 保卫系统。它的主要功能主要包括免疫防御、自我稳定和免疫监视。 机体的免疫功能失调,会表现为免疫反应过强或过弱,导致自身免疫 损伤或对疾病的抵抗力下降。其中免疫细胞是免疫力的主要承担者, 通过各类(亚类)免疫细胞定性、定量的分析,可为疾病做出预警。
随着科学研究的不断进步,科学家发现了更多的细胞表面标志, 可通过对血液样品中各类免疫细胞进行免疫分型和定量分析,能够更 加准确地评价机体的免疫功能,也能够更好地研究某些疾病的发生、 发展以及治疗效果评价。目前,常用于血液样品中的免疫细胞分型和 定量分析的方法主要有:免疫酶标法、普通淋巴细胞分类、较细淋巴 细胞亚群检测。免疫酶标法只是针对某种或类特定的抗原或抗体发生 特异反应,检测面窄、操作复杂、对待检测样品需求量大,费时费力。 普通淋巴细胞分类,普遍应用于淋巴细胞检测,但只能将淋巴细胞分 为T细胞、Tc细胞、Th细胞、B细胞和NK细胞,不能进一步对细胞 进行分型。较细淋巴细胞亚群检测依赖于流式细胞术,采用多色流式 的方法,对淋巴细胞进行较为数量。但是,如果想进一步对细胞亚群 进行免疫分型和定量分析的话,还需要借助于其他的细胞免疫分型方 法,操作复杂,对待测样品的需求量大,并不适合实际应用。所以, 还需要对淋巴细胞免疫分型和定量分析的方法进行研究,以求尽可能 利用简单的方法将淋巴细胞进行较精细地免疫分型和定量分析。
在申请号为:CN201410485865.1,申请日为:20140922,名称为: 一种B淋巴细胞免疫分型的方法和试剂盒的专利中,公开了一种B淋 巴细胞免疫分型的方法和试剂盒,此发明提供的B淋巴细胞免疫分型 的方法,包括:取不同的荧光标记的抗体,与待检测样品混合,孵育 后,经流式细胞术检测,得检测数据,分析所得检测数据;该抗体包 括:抗CD19的抗体、抗IgD的抗体、抗CD38的抗体、抗CD24的抗 体没有全面的对血液样品中的免疫细胞分型和定量分析。
发明内容
本发明的目的就是为了克服上述现有技术存在的缺陷而提供一 种免疫细胞全维分析方法。
本发明的的目的可以通过以下技术方案来实现:
一种免疫细胞全维分析方法,其特征在于,包括T淋巴细胞免疫 分型、B淋巴细胞免疫分型、活化型和抑制型细胞免疫分型、NK细胞 免疫分型、DC细胞免疫分型;
所述免疫细胞全维分析的方法包括:
取不同的荧光标记抗体,与待检测样品混合,孵育处理后,经流 式细胞术检测,得到检测数据,分析所述检测数据;
所述T淋巴细胞亚群免疫分型时,所用抗体包括:
抗CD45的抗体、抗CD3的抗体、抗CD8的抗体、抗CD4的抗体、抗 CD25的抗体、抗CD127的抗体、抗γδ的抗体;
所述B淋巴细胞免疫分型时,所用抗体包括:
抗CD45的抗体、抗CD3的抗体、抗CD19的抗体、抗CD38的抗体、 抗CD27的抗体、抗CD24的抗体、抗IgD+的抗体;
所述活化型和抑制型细胞免疫分型时,所用抗体包括:
抗CD3的抗体、抗CTLA-4的抗体、抗PD-1的抗体、抗NKG2D的抗体、 抗16、56的抗体、抗NKP46的抗体;
所述NK细胞免疫分型时,所用抗体包括:
抗CD45的抗体、抗CD3的抗体、抗CD16、56的抗体;
所述DC细胞免疫分型时,所用抗体包括:
抗LIN的抗体、抗HLA-DR的抗体、抗CD123的抗体、抗CD11c的抗 体;
所述T淋巴细胞免疫分型时,所述分析的方法包括:
细胞表面标志CD45+CD3+SSC-代表总T淋巴细胞;
细胞表面标志CD45+CD3+CD8+代表杀伤性T细胞;
细胞表面标志CD45+CD3+CD4+代表辅助性T细胞;
细胞表面标志CD3+γδ+代表γδT细胞;
细胞表面标志CD4+CD25+CD127+代表调节性CD4+T细胞;
细胞表面标志CD3+CD8+CD28+代表细胞毒T细胞;
细胞表面标志CD3+CD8+CD28-代表抑制性T细胞;
所述B淋巴细胞免疫分型时,所述分析的方法包括:
细胞表面标志CD3-CD19+代表总B淋巴细胞;
细胞表面标志CD3-CD19+CD27+代表记忆B淋巴细胞;
细胞表面标志CD3-CD19+CD27-IgD+代表初始B淋巴细胞;
细胞表面标志CD3-CD19+CD24-CD38+代表浆母细胞;
所述活化型、抑制型细胞免疫分型,所述分析的方法包括:
细胞表面标志CD3+CTLA-4+代表CTLA-4@T;
细胞表面标志CD3+PD-1+代表PD-1@T;
细胞表面标志CD3+NKG2D+代表NKG2D@T;
细胞表面标志CD3-CD16、56+NKG2D+代表NKG2D@NK;
细胞表面标志CD3-CD16、56+NKP46+代表NKP46@NK;
所述NK细胞免疫分型,所述分析的方法包括:
细胞表面标志CD3-CD16、56+代表NK细胞;
细胞表面标志CD3+CD16、56+代表NK-T细胞;
所述DC细胞免疫分型时,所述分析的方法包括:
细胞表面标志LIN-HLA-DR+CD11c+代表髓系DC;
细胞表面标志LIN-HLA-DR+CD123+代表淋巴系DC。
优选的,所述全维分析方法还包括统计所述待测样品中各类免疫 细胞数目的步骤。
其中,更优选的,所述统计所述待测样品中各类免疫细胞数目的 步骤包括:
检测所述待测样品中各类免疫细胞的百分比,通过流式管中预包 被的计数微球,计算后,即得所述待检样品中各类免疫细胞数目。
本发明的有益效果:
实现了对免疫细胞进行更加全面的精细免疫分型,所需待测样品 少,操作简单,所需时间短、准确性高、分析全面,可广泛应用于免 疫细胞全维分析。
附图说明
图1为在CD3/CD8/CD45/CD4抗体标记下T淋巴细胞的分型结果;图 1-A,图1-B,图1-C为T淋巴细胞亚群的分型结果。
图中:R1为总淋巴细胞;R2为杀伤性T淋巴细胞;R3为T淋巴细胞;R4 为辅助性T淋巴细胞。
图2为在CD3/γδ抗体标记下T淋巴细胞的分型结果;图2-A,图 2-B为γδ细胞的分型结果。
图中:R1为淋巴细胞;R2为γδT细胞。
图3为在CD4/CD25/CD127抗体标记下T淋巴细胞的分型结果;图 3-A,图3-B,图3-C为调节性CD4+T细胞的分型结果。
图中:R1为淋巴细胞;R3来源于R1;R4为调节性CD4+T细胞。
图4为在CD3/CD8/CD28抗体标记下T淋巴细胞的分型结果;图4-A, 图4-B,图4-C为细胞毒/抑制性T淋巴细胞的分型结果。
图中:R1为淋巴细胞;R2为T淋巴细胞;R3:细胞毒T细胞;R4 为抑制性T细胞。
图5在CD3/CD45/CD19/IgD/CD27/CD38/CD24抗体标记下B淋巴细 胞免疫分型结果;图5-A,图5-B,图5-C,图5-D,图5-E为B淋巴细胞 的分型结果。
图中:R1为淋巴细胞;R2为B和NK细胞;R3为总B淋巴细胞;R6 为记忆B淋巴细胞;R7为初始B淋巴细胞;R8为浆母细胞。
图6在CD3/CTLA-4/PD-1抗体标记下活化型/抑制型细胞的分型 结果;图6-A,图6-B,图6-C为抑制型细胞的分型结果。
图中:R1为淋巴细胞;R2为CTLA-4@T;R3为PD-1@T。
图7在CD3/CD16+56/NKG2D/NKP46抗体标记下活化型/抑制型细 胞的分型结果;图7-A,图7-B,图7-C,图7-D,图7-E为活化型细 胞的分型结果。
图中:R1淋巴细胞;R3为NK细胞;R4为NKG2D@T细胞;R5 为NKG2D@NK;R6为NKP46@NK细胞。
图8为在CD3/CD16+56抗体标记下NK细胞免疫分型分类结果; 图8-A,图8-B,图8-C为不同荧光标记下的NK细胞的分型结果。 图中:R1淋巴细胞;R2为B和NK细胞;R4为NK细胞;R5为NK-T 细胞。
图9为在LIN/CD123/HLR-DR/CD11c抗体标记下DC细胞免疫分型分类 结果;图9-A,图9-B,图9-C,图9-D为DC细胞的分型结果。
图中:R1为有核细胞;R2来源R1,LIN表达阴性;R3为髓系DC;R4 为淋巴系DC。
具体实施方式
结合附图所示,本发明的技术方案作进一步的描述:
本发明提供的一种用于免疫细胞全维分析的方法中所用到的试 剂和原料均可由市场购得。
本发明中用到的荧光标记FITC、PE、APC、Percp、BV510均为常 见的荧光标记可以由市场购得,各个荧光标记的抗体也可以由市场购 得。
为了使本技术领域的技术人员能够更好地理解本发明的技术方 案,下面结合实施例,进一步阐述本发明:
实验材料:
待测样品:抗凝外用血样品,来源于健康志愿者,为正常人的外用血。 【样本要求】
1.选用EDTA-K2抗凝管静脉采集全血至刻度标识位置。
2.样本要存放于室温且避免剧烈振动摇晃,血液采集后24小时内检 测。
3.样本应无微生物污染、脂血、凝血等情况。若存在上述情况,请在 结果报告时标注。
其它需要准备的材料:
1.流式细胞仪;
2.真空采血管(EDTA-K2抗凝);
3.量程范围适用于10ul、100ul、1000ul的移液器及一次性吸头;
4.漩涡振荡器;
5.溶血素。
实验方法:
【检验方法】
1.流式细胞仪开机预热20min,进行清洗;
2.取10支计数微球管,分别加入50ul混匀的抗凝血,按表1中要 求加入一定量的细胞免疫分型染色液,避光放置20min;
3.加入500μl溶血素后用漩涡振荡器充分混匀,避光放置20min, 待血液呈现透明,溶血成功;
4.上机检测,得到各类免疫细胞分型结果;
检测项目以及染色液量见如下表1:
表1检测项目与染色液加量
检测结果见附图1-9所示的淋巴细胞分类结果。
最后应说明的是:
本领域技术人员可以参考本文内容,实施该方法,实现其应用, 特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是 显而易见的,它们都被视为包括在本发明内。本发明的方法及应用己 经通过较佳的实施例进行了描述,相关人员明显能在不脱离本发明内 容、精神和范围内对本文制各方法和应用进行改动或适当变更与组 合,来实现和应用本发明技术。
Claims (3)
1.一种免疫细胞全维分析方法,其特征在于,包括T淋巴细胞免疫分型、B淋巴细胞免疫分型、活化型和抑制型细胞免疫分型、NK细胞免疫分型、DC细胞免疫分型;
所述免疫细胞全维分析的方法包括:
取不同的荧光标记抗体,与待检测样品混合,孵育处理后,经流式细胞术检测,得到检测数据,分析所述检测数据;
所述T淋巴细胞亚群免疫分型时,所用抗体包括:
抗CD45的抗体、抗CD3的抗体、抗CD8的抗体、抗CD4的抗体、抗CD25的抗体、抗CD127的抗体、抗γδ的抗体;
所述B淋巴细胞免疫分型时,所用抗体包括:
抗CD45的抗体、抗CD3的抗体、抗CD19的抗体、抗CD38的抗体、抗CD27的抗体、抗CD24的抗体、抗IgD+的抗体;
所述活化型和抑制型细胞免疫分型时,所用抗体包括:
抗CD3的抗体、抗CTLA-4的抗体、抗PD-1的抗体、抗NKG2D的抗体、抗CD16、56的抗体、抗NKP46的抗体;
所述NK细胞免疫分型时,所用抗体包括:
抗CD45的抗体、抗CD3的抗体、抗CD16、56的抗体;
所述DC细胞免疫分型时,所用抗体包括:
抗LIN的抗体、抗HLA-DR的抗体、抗CD123的抗体、抗CD11c的抗体;
所述T淋巴细胞免疫分型时,所述分析的方法包括:
细胞表面标志CD45+CD3+SSC-代表总T淋巴细胞;
细胞表面标志CD45+CD3+CD8+代表杀伤性T细胞;
细胞表面标志CD45+CD3+CD4+代表辅助性T细胞;
细胞表面标志CD3+γδ+代表γδT细胞;
细胞表面标志CD4+CD25+CD127+代表调节性CD4+T细胞;
细胞表面标志CD3+CD8+CD28+代表细胞毒T细胞;
细胞表面标志CD3+CD8+CD28-代表抑制性T细胞;
所述B淋巴细胞免疫分型时,所述分析的方法包括:
细胞表面标志CD3-CD19+代表总B淋巴细胞;
细胞表面标志CD3-CD19+CD27+代表记忆B淋巴细胞;
细胞表面标志CD3-CD19+CD27-IgD+代表初始B淋巴细胞;
细胞表面标志CD3-CD19+CD24-CD38+代表浆母细胞;
所述活化型、抑制型细胞免疫分型,所述分析的方法包括:
细胞表面标志CD3+CTLA-4+代表CTLA-4@T;
细胞表面标志CD3+PD-1+代表PD-1@T;
细胞表面标志CD3+NKG2D+代表NKG2D@T;
细胞表面标志CD3-CD16、56+NKG2D+代表NKG2D@NK;
细胞表面标志CD3-CD16、56+NKP46+代表NKP46@NK;
所述NK细胞免疫分型,所述分析的方法包括:
细胞表面标志CD3-CD16、56+代表NK细胞;
细胞表面标志CD3+CD16、56+代表NK-T细胞;
所述DC细胞免疫分型时,所述分析的方法包括:
细胞表面标志LIN-HLA-DR+CD11c+代表髓系DC;
细胞表面标志LIN-HLA-DR+CD123+代表淋巴系DC。
2.如权利要求1所述的一种免疫细胞全维分析方法,其特征在于:所述全维分析方法还包括统计所述待测样品中各类免疫细胞数目的步骤。
3.如权利要求2所述的一种免疫细胞全维分析方法,其特征在于:所述统计所述待测样品中各类免疫细胞数目的步骤包括:
检测所述待测样品中各类免疫细胞的百分比,通过流式管中预包被的计数微球,计算后,即得所述待检样品中各类免疫细胞数目。
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