CN110988364A - 一种应用流式细胞术检测移植后gvhd相关细胞因子的方法及检测试剂盒 - Google Patents
一种应用流式细胞术检测移植后gvhd相关细胞因子的方法及检测试剂盒 Download PDFInfo
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Abstract
本发明提供了一种应用流式细胞术检测移植后GVHD相关细胞因子的方法及检测试剂盒,在移植后GVHD的早期评估和诊断方面具有重要的临床意义。通过本发明所述的检测方法与试剂盒,能够同时快速均相检测GVHD诊断与预后相关细胞因子IL‑6、IL‑8、ST2、Reg3α、Elafin、TNFR1、可溶性IL‑2R、HGF中的一项或多项不同组合的指标,实现单样本多重检测,节省时间和资源,软件自动分析结果,更加客观准确,而且本发明所述方法简便,检测安全,样本用量少。本发明的应用能够及时快速、长期稳定的评估移植后患者GVHD的风险,提高了患者治疗的准确性和时效性,挽救患者的生命。
Description
技术领域
本发明属于免疫诊断检测领域,具体涉及一种应用流式细胞术检测移植后GVHD相关细胞因子的方法及检测试剂盒。
背景技术
随着医疗技术的发展,移植医学已经广泛应用于各种重大疾病的治疗中。例如:器官移植、骨髓移植以及细胞移植等等。然而在移植治疗中,术后发病率高达50%-80%的术后并发症GVHD(移植物抗宿主反应)一直是困扰医生的难题。GVHD是指由于移植物组织中的免疫活性细胞与免疫受抑制的、组织不相融性抗原受者的组织之间的反应,是移植后患者死亡的主要原因。因此,移植后评估、监测以及抑制GVHD成为治疗的关键。
流式细胞术是近些年发展期起来的新技术,能够对一个样本进行多参数高通量检测,检测速度快、精度高、准确性好。CBA(Cytometric Bead Array)即细胞因子微球检测技术,是一种基于流式细胞检测系统的多重蛋白定量检测方法,它能够同时对单个样品中的多个指标进行检测。CBA集ELISA和细胞流式技术优点为一身,利用微小、分散的颗粒捕获液体待测物,并利用流式细胞仪检测类似“三明治”的颗粒——捕获微球-细胞因子-检测抗体夹心复合物所散发的荧光,从而测定待测物的数量,这种方法对样本需求量更少,检测更快速、更准确。
目前GVHD诊断的主要依据是皮肤、肝脏和胃肠道三大靶器官的临床表现。用于GVHD诊断的方法为活组织检查,诊断过程非常复杂。鉴于目前针对GVHD诊断的手段复杂,特异性不高,研发一种快速且特异性高的评估GVHD的试剂盒具有很大的必要性。研究显示,细胞因子在GVHD发病过程中具有重要意义。因此,可以通过移植后外周血中细胞因子的监测来对病人GVHD发病的风险性进行评估和诊断。
发明内容
通过本发明所述的检测方法与试剂盒,能够同时快速均相检测GVHD诊断与预后相关细胞因子IL-6、IL-8、ST2、Reg3α、Elafin、TNFR1、可溶性IL-2R、HGF八项中的一项或多项不同组合的指标。
针对现有技术的不足,本发明的目的在于提供一种应用流式细胞术检测移植后GVHD相关细胞因子的方法,采用如下技术方案:
应用流式细胞术检测移植后GVHD相关细胞因子的方法,具体包括以下步骤:
一、捕捉微球的制备
将标记有APC荧光素的聚苯乙烯微球与捕获抗体偶联,不同的捕获抗体与不同荧光强度的微球相结合,通过荧光强度识别不同的细胞因子(见图2)。
本发明方法可同时检测8种细胞因子,使用一种微球区别同种荧光素8种不同荧光强度的效果较差,不易区分,为更好地进行结果分析,本发明选用不同大小与颗粒度的2群聚苯乙烯微球为微球1和微球2,大小相同的每种微球有4种不同荧光强度; 制备IL-6、IL-8、TNFR1、ST2捕捉微球使用的是微球1,制备Reg3α、Elafin、可溶性IL-2R、HGF捕捉微球使用的是微球2(见图3)。
二、检测抗体的制备
用荧光素标记检测抗体,所述检测抗体与捕获抗体为配对抗体。
三、标准品的制备
标准品为人IL-6重组蛋白冻干粉、人IL-8重组蛋白冻干粉、人ST2重组蛋白冻干粉、人Reg3α重组蛋白冻干粉、人Elafin重组蛋白冻干粉、人TNFR1重组蛋白冻干粉、人可溶性IL-2R重组蛋白冻干粉、人HGF重组蛋白冻干粉。
四、检测反应体系:捕捉微球25ul+样本或标准品溶液25ul+检测抗体25ul
上述反应体系中,检测抗体上标记PE荧光素,将微球和检测抗体以及样本混合后,就形成了捕获微球-细胞因子-检测抗体这样双抗夹心复合物,通过流式测定PE荧光强度,结合标准曲线,分析软件即可计算出每种细胞因子的浓度。
五、标准曲线制作
将等比稀释的0-5000pg/ml不同浓度的细胞因子标准品蛋白与捕捉微球和检测抗体等体积混合后,室温避光孵育反应,加入清洗液清洗后,将该复合物置于流式细胞仪检测PE通道荧光强度,通过FSC和SSC确定微球群后,通过微球本身APC荧光素的不同强度判定微球,决定被检测细胞因子,通过软件对不同细胞因子浓度分别绘制标准曲线,曲线坐标为细胞因子蛋白浓度和检测平均荧光强度MFI。
六、待测样本检测
通过流式细胞仪检测待测样本复合物PE通道的荧光强度MFI,将荧光强度值带入标准曲线,即可得到待测样本中细胞因子的浓度。
本发明的另一目的在于提供一种应用流式细胞术检测移植后GVHD相关细胞因子的检测试剂盒,技术方案如下:
一种应用流式细胞术检测移植后GVHD相关细胞因子的检测试剂盒,包括微球缓冲液、样本缓冲液、清洗液、校准微球、标准品A、标准品B和至少一种捕捉微球、至少一种检测抗体、至少一种标准品蛋白。其中校准微球用于调整FSC和SSC,得到微球群、标准品A为FITC阳性对照、标准品B为PE阳性对照,二者用于调节通道补偿。
本发明的优点和有益效果:
本发明提供了一种应用流式细胞术检测移植后GVHD相关细胞因子的方法及检测试剂盒,在移植后GVHD的早期评估和诊断方面具有重要的临床意义。通过本发明所述的检测方法与试剂盒,能够同时快速均相检测GVHD诊断与预后相关细胞因子IL-6、IL-8、ST2、Reg3α、Elafin、TNFR1、可溶性IL-2R、HGF中的一项或多项不同组合的指标,实现单样本多重检测,节省时间和资源,软件自动分析结果,更加客观准确,而且本发明所述方法简便,检测安全,样本用量少,仅需25ul即可检测多重指标,且不会造成环境污染和对患者的伤害。
相对于目前传统的活组织检查的诊断手段,本发明具有早期评估临床相关性高,特异性高,动态评估灵敏度好,操作简单等方面的优势。为临床能够准确快速的评估监测GVHD提供了新的方法和材料。本发明的应用能够及时快速、长期稳定的评估移植后患者GVHD的风险,提高了患者治疗的准确性和时效性,挽救患者的生命。
附图说明
图1 为本发明技术原理示意图;
图2 为本发明不同荧光强度的捕捉微球流式细胞仪分析图;
图3 为本发明不同大小(FSC)和颗粒度(SSC)的2群捕捉微球流式细胞分析图;
图4 为本发明方法检测8因子流式细胞仪分析结果图;
图5-图12 依次为本发明IL-6、IL-8、ST2、Reg3α、Elafin、TNFR1、可溶性IL-2R、HGF标准曲线图,其横坐标为IL-6细胞因子浓度(单位pg/mL),纵坐标为平均荧光强度值(MFI),图中各个点所标识的数字,为该检测浓度所对应的MFI。
具体实施方式
一、 缓冲溶液的制备
1.微球缓冲液的制备
本实施例涉及的微球缓冲液是含有体积分数5%的FBS的PBS溶液,其配制方法是:量取50mL FBS,加入950mL pH7.2 0.1M的PBS缓冲液中,加入0.09%NaN3,混合均匀,0.45μm水系过滤膜过滤。
2.样本缓冲液的制备
本实施例涉及的样本缓冲液是含有0.2%BSA(w/v)的PBS溶液,其配制方法是:称取2mgBSA,溶解于1L体积pH7.2 0.1M的PBS缓冲液中,0.45μm水系过滤膜过滤。
3.清洗液的制备
本实施例涉及的清洗液是pH7.2的0.1MPBS溶液,其配制方法是:称取1.482g NaH2PO4•2H2O, 14.8g Na2HPO4•12H2O和8.8gNaCl,加H2O 800mL溶解后,调节PH值至7.2,再加H2O定容至1L,用0.45μm水系过滤膜过滤。
二、 捕捉微球的制备
本发明的所有捕捉微球的制备均为表及有APC荧光素的羧基化聚苯乙烯微球与捕捉抗体的偶联,其方法与反应体系相同,故此实施方式以ST2捕捉微球的制备为例。具体实施方式如下:
1.溶液配制
1.1 bufferA的配制:称取0.96g NaH2PO4•2H2O,加H2O 600mL溶解,调节PH值至6.0,再加H2O定容至800mL,用0.45μm水系过滤膜过滤;
1.2 bufferB的配制:称取甘氨酸7.5g,加H2O 800mL溶解,调节PH值至7.5,再加H2O定容至1000mL,用0.45μm水系过滤膜过滤;
1.3 50mg/mL 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)溶液配制:称取1gEDC,加入50mLH2O溶解,用0.45μm水系过滤膜过滤;
1.4 50mg/mL N-羟基琥珀酰亚胺(NHS)溶液配制:称取1gNHS,加入50mLH2O溶解,用0.45μm水系过滤膜过滤;
2 偶联反应
2.1 取1mL标记有APC荧光素的羧基化聚苯乙烯微球悬浊液,加入离心管中,10000转/分钟离心5分钟,弃上清;
2.2 取80uL的buffer A洗涤微球,10000转/分钟离心5分钟,弃上清,重复两次;
2.3 加入80uL buffer A于离心管管底,超声60秒;
2.4 分别取10uL EDC和NHS溶液,加入到微球悬浊液中,暗室孵育20分钟;
2.5 10000转/分钟离心5分钟,弃上清;
2.6 使用500uL buffer B洗涤微球,10000转/分钟离心5分钟,弃上清,重复两次;
2.7 使用500uL buffer B重悬微球;
2.8 使用500uL buffer B将ST2捕捉抗体配成终浓度为100ug/mL的溶液,加入微球管中;
2.9 将离心管至于旋转混合仪上,室温,避光孵育2小时;
2.10 使用微球缓冲液将标记后的微球抗体偶联物清洗两次,得到捕捉微球;
2.11 使用微球缓冲液对捕捉微球进行保存。
三、检测抗体的制备
本发明的所有检测抗体均为PE荧光素标记,其标记方法、纯化方法与反应体系相同,故此实施方式以PE-ST2检测抗体的制备为例。具体实施方式如下段。
将2mg活化处理的ST2检测抗体(与捕捉抗体为配对抗体)与7mg活化处理过的荧光素PE,混合于pH6.0的0.1MPBS缓冲液中,体积为1.5ml,同时加入苯胺0.137mL苯胺,混匀后,置于旋转混合仪上室温避光孵育2小时;用AKTA蛋白纯化仪进行纯化,用0.5倍柱体积的超声脱气纯化水进行平衡,流速为0.5ml/min,再用约1.5倍柱体积的洗脱缓冲液平衡,流速为0.5-1ml/min,上样,后用洗脱缓冲液洗脱,收集所需峰值附近液体,使用紫外可见分光光度计检测抗体浓度和抗体量,得到检测抗体PE-ST2检测抗体。
七、标准品的制备
标准品为人IL-6重组蛋白冻干粉、人IL-8重组蛋白冻干粉、人ST2重组蛋白冻干粉、人Reg3α重组蛋白冻干粉、人Elafin重组蛋白冻干粉、人TNFR1重组蛋白冻干粉、人可溶性IL-2R重组蛋白冻干粉、人HGF重组蛋白冻干粉。
五、使用检测试剂盒进行GVHD相关细胞因子检测
1.PE标记检测抗体混合
将每个细胞因子的PE标记检测抗体离心后使用移液器取出,在棕色瓶中进行混合。
2. 制备细胞因子标准品
2.1 将每个细胞因子标准品冻干球倒入瓶中,加入2mL样本缓冲液。室温静置15分钟。使用移液器轻轻混匀标准品。此时各因子标准品的浓度均为5000pg:
2.2 取11支流式管,分别标记0,10,20,40,80,156,312.5,625,1250,2500和5000pg。除5000pg管外,其余每管加入200uL样品稀释液;
2.3 取刚溶解好的标准品200uL,加入5000pg管中;
2.4 取刚溶解好的标准品200uL,加入2500pg管中,轻轻吹打混匀;
2.5 从2.4处理好的2500pg管中取200uL,加入1250pg管中,轻轻吹打混匀;
2.6 依此类推,直到10pg管;
2.7 0pg管中加入200uL样品缓冲液;
3. 混合微球
3.1 确定实验管数(包括标准品和样品):待测样本2个,标准品11个,共13管;
3.2 将每个试剂盒中的捕捉微球取出,充分混匀,防止微球沉淀;
3.3 每个实验管中均含有8种微球,每种捕捉微球各取5uL。实验管13个,需要每种捕捉微球量为65uL,将所有8种微球都装入一根流式管中;
3.4 将混合后的微球200g离心5分钟;
3.5 小心吸去上清;
3.6 加入体积为325uL微球缓冲液;
3.7 室温避光孵育30分钟;
4 待测样本准备
检测样本为血清或血浆
血清:标准试管或者有分离胶的试管采集静脉血,4000rpm离心20分钟;
血浆:EDTA抗凝管采集静脉血,4000rpm离心20分钟;
注:每种细胞因子检测范围为10pg-5000pg,如样本浓度过高,可以做样本稀释;
5 标准曲线与样本检测
5.1 将孵育后的微球混匀后,每个实验管中加入25uL;
5.2 标准品管依次加入稀释好的标准品25uL, 如下表所示;
5.3 样本管中每管加入25uL待测样本;
5.4 每管加入PE标记检测抗体25uL,室温避光孵育2.5个小时;
5.6 每管加入0.5mL清洗液,200g离心5分钟;
5.7 小心吸去上清,每管加入150uL清洗液,重悬微球, 准备上机;
6 仪器调整
6.1 取三支流式管,标记A管、B管和C管;
6.2 向三支管中加入25uL校准微球;
6.3 向B管中加入25uL标准品A,为FITC阳性对照;
6.4 向C管中加入25uL标准品B,为PE阳性对照;
6.5 避光孵育30分钟;
6.6 A管中加入375uL清洗液,B管和C管中加入350uL清洗液;
6.7 使用A管,调整FSC和SSC,得到微球群(图3)。使用B管和C管,调节通道补偿;
6.8 每个样本收取微球总数为2400个,每种微球大约300个;
6.9 使用随试剂提供的软件进行分析数据分析。
Claims (4)
1.应用流式细胞术检测移植后GVHD相关蛋白的方法,其特征在于,包括:捕捉微球的制备、检测抗体的制备、缓冲液的制备、标准曲线的制备、待测样品的检测;
所述细胞因子为IL-6、IL-8、ST2、Reg3α、Elafin、TNFR1、可溶性IL-2R、HGF的一种或几种。
2.应用流式细胞术检测移植后GVHD相关细胞因子的检测试剂盒,其特征在于:包含微球缓冲液、样品缓冲液、清洗液、校准微球、标准品A、标准品B和至少一种捕捉微球、至少一种检测抗体、至少一种标准品蛋白;
所述捕捉微球为IL-6捕捉微球、IL-8捕捉微球、ST2捕捉微球、Reg3α捕捉微球、Elafin捕捉微球、TNFR1捕捉微球、可溶性IL-2R捕捉微球、HGF捕捉微球的一种或几种;
所述检测抗体为PE-IL-6检测抗体、PE-IL-8检测抗体、PE-ST2检测抗体、PE-Reg3α检测抗体、PE-Elafin检测抗体、PE-TNFR1检测抗体、PE-可溶性IL-2R检测抗体、PE-HGF检测抗体的一种或几种;
所述标准品蛋白为人IL-6重组蛋白冻干粉、人IL-8重组蛋白冻干粉、人ST2重组蛋白冻干粉、人Reg3α重组蛋白冻干粉、人Elafin重组蛋白冻干粉、人TNFR1重组蛋白冻干粉、人可溶性IL-2R重组蛋白冻干粉、人HGF重组蛋白冻干粉的一种或几种;
所述捕捉微球、检测抗体和标准品蛋白属于同一种细胞因子。
3.根据权利要求1所述的捕捉微球为APC荧光素标记的羧基化聚苯乙烯微球,其特征在于本发明选用不同大小与颗粒度的2群聚苯乙烯微球为微球1和微球2,每种微球有4种不同荧光强度,用于4种捕捉微球的制备。
4.根据权利要求1所述的检测抗体为PE荧光素标记的检测抗体,其检测抗体与只给捕捉微球的捕获抗体为抗体对。
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