新型表面化学SPR蛋白芯片的构建及其在CITP免疫紊乱诊断中的应用
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摘要
研究背景
表面等离子体共振(Surface Plasmon Resonance, SPR)是当今应用最普遍的非标记光学生物传感器(Label-Free Optical Biosensor)技术,正在被越来越普遍地用于生命科学等多个领域。SPR技术不需要对被测物进行标记的优点使其可以测量生物活性分子在无修饰条件下的反应动力学,适用于高通量生物活性分子特别是小分子的筛选,微量未知物的分析,以及在线样品检测。但其核心技术为光路检测系统、微流控进样系统和SPR芯片。目前改进基质材料的表面处理技术,建立高效和快速的芯片活化方法,并能保持芯片表面物质的稳定性和生物活性是该领域研究的热点,同时与微流控技术相结合可降低样品消耗量,提高检测通量。因此,优化SPR芯片表面的功能,探索建立高通量、简便快速、灵敏度高、特异性强的SPR技术应用于临床检验诊断具有重要的意义。
慢性特发性血小板减少性紫癜(Chronic idiopathic thrombocytopenic purpura, CITP)是一种常见的出血性自身免疫病,在我国的发病率呈上升趋势,严重影响患者的生活质量,但因发病机制不明,还难以找到真正特异且有效的治疗方法。近年免疫调节治疗方式引起了人们的兴趣,可能从源头上改变相关的治疗效果。最近研究表明新型调节性B细胞(Breg)具有负性调节作用,在免疫学领域越来越受重视并被积极研究,我们前期预实验发现Breg细胞可能在CITP患者免疫紊乱中发挥重要作用,CITP患者体内Breg的变化可能与CD4+Th细胞亚群和血小板抗体之间有重要关系,因此需要对该问题进行深入研究,并探索一种简便、快速的检测方法,为CITP的临床诊断与治疗寻找新的靶点与思路。血小板抗体的检测是国内外研究的热点,虽然方法有多种,但因价格太高,或操作复杂,或易至假阳性或假阴性等问题,临床应用较少；CD4+T等相关细胞因子紊乱的检测方法主要为ELISA、流式细胞术和PCR等,都难以高通量快速检测多种因子,限制了它们的临床应用。因此,利用SPR技术优势,探索SPR芯片技术应用于CITP患者的免疫紊乱的动态联合检测,对临床诊断、治疗策略选择、临床效果评价和预后分析以及血小板相容性输注治疗等方面具有非常重要的意义。
研究目的
构建与优化新型表面化学SPR蛋白芯片,建立基于SPR技术高通量、简便快速、灵敏度高、特异性好的新型免疫学检测方法：针对CITP治疗效果差,发病机制复杂以及新型调节性B细胞在自身免疫性疾病中的作用特点,本研究拟分析CITP患者外周血新型调节性B细胞的变化特征、及其对患者血小板抗体与CD4+Th细胞紊乱的调节作用；探索新型SPR芯片技术在CITP免疫紊乱诊断与疗效评价中的应用价值,为CITP免疫紊乱的检测寻找新靶点与新方法,同时也为新型SPR芯片技术应用于其它相关免疫病的临床检验诊断提供参考。
研究内容
1、构建灵敏度高、特异性好的新型表面化学SPR蛋白芯片,进行相关性能的综合评价,并对两种SPR芯片比较分析。
2、研究CITP患者外周血新型调节性B细胞的变化及其与血小板抗体的相关性,并分析它们在CITP患者免疫紊乱中的作用。
3、运用新型表面化学蛋白芯片,建立高通量、非标记的SPR技术筛检CITP患者血小板抗体的新方法,并与固相凝集法和MAIPA法对比分析。
4、研究CITP患者外周血新型调节性B细胞的变化与CD4+T细胞的相关性,分析相关的细胞因子在CITP免疫紊乱中的作用。
5、运用新型表面化学蛋白芯片,探索建立高通量、非标记的SPR技术动态、特异性联合检测CITP患者Breg与CD4+T细胞相关因子紊乱的新方法。
研究方法
1、SPR芯片Poly(OEGMA-co-HEMA)表面的制备
利用二元自组装体系对引发剂表面浓度进行控制,以乙醇为溶剂,配制硫醇引发剂和PEG硫醇混合溶液。将经过UV-ZONE处理的芯片浸泡在上述混合溶液中,放置15小时。取出芯片,用乙醇充分淋洗,氮气吹干。称取2,2’吡啶12.5mg、OEGMA5262.62g和HEMA0.65g,加入溶剂超纯水5ml和甲醇5m1搅拌溶解。加入1mLCuCl2的溶液(0.04mM),通入氮气,排除氧气15min。随后利用注射器缓慢注入1mL抗坏血酸溶液(0.04mM), Cu(II)配合物逐渐被还原为Cu(I)的配合物,反应溶液由通常的淡蓝色变为淡红色。继续无氧处理15min。表面引发聚合反应在氮气环境的手套箱中进行。将有引发剂的芯片,浸泡在反应溶液中。芯片表面将引发单体在聚合于芯片表面,在室温下反应8小时后,将芯片取出,终止反应,并用甲醇和超纯水清洗干净,氮气吹干。基质Poly(OEGMA-co-HEMA)侧链末端羟基活性较差,很难在温和条件中与蛋白质进行偶联反应,所以必须对羟基进行功能化：将表面修饰poly(OEGMA-co-HEMA)的芯片浸泡在含有丁二酸酐(10mg/mL)和DMAP(15mg/mL)的DMF反应溶液中,温室下反应12小时。将芯片取出,用DMF和乙醇充分淋洗,氮气吹干。
2、SPR芯片动态环糊精表面的制备
利用自组装单层技术在芯片表面组装PEG单层作为主链,其中利用稀释剂控制主链PEG的链密度；将芯片浸泡于环糊精溶液中,当足量的环糊精穿入PEG主链后,对线性PEG另一端进行封端。再通过羟基化反应将环糊精的羟基基团转为羧基等活性基团,便于固定生物探针分子。利用SPR技术系统实验参数(链密度、链长度、羧基数量)对于其在生物检测性能的影响,优化出最佳的制备条件。在优化的实验条件下制备基于环糊精分子的传感器基质,并应用该基质检测模型蛋白。
3、新型化学表面SPR芯片的表征方法
(1)AFM表征：基质在干态下的形貌结构由原子力显微镜(AFM,Veeco, Dimension3100)表征,实验采用接触式模型；(2)椭圆偏振表征：膜厚由M-2000V型分光椭圆偏振测量仪测量,测量角度为65、70和75度,测量波长为400nm到800nm。椭圆偏振仪数据用特定的模型拟合得到膜的厚度,其中SAMs和Poly(OEGMA)采用Cauchy模型,(An, Bn)分别为(1.45,0.01)和(1.46,0.01)。样品厚度值为三个位置测量值的平均,报道结果为平均值±标准差。
4、新型化学表面SPR芯片性能评价
(1)抗污效果测试：按照仪器操作手册将芯片(羧基功能化芯片)装载后,设定工作温度25度,流速2μL/s,通入PBS缓冲溶液(pH=7.4),得到稳定基线后注入特定浓度的蛋白质溶液(PBS缓冲液稀释),最后通入PBS缓冲溶液。
(2)蛋白检测能力测试：按照仪器的操作要求设定点样矩阵参数,将30uL点样样品添加384孔板中。设定温度为25度,湿度为50%。用接触方式将蛋白样品点样于SPR芯片表面。点样后芯片放置于25度,湿度为75%的恒温恒湿箱中2小时；将蛋白质芯片安装于SPR系统中,通入PBS缓冲溶液(pH=7.4),流速2μL/s,得到稳定基线后,通入封闭试剂乙醇胺(10mmol/L, pH=8.5)7min,封闭多余的活性位点。注入特定浓度的蛋白质溶液(PBS缓冲液稀释),利用甘氨酸溶液(10mM, pH=2.0)可以洗掉结合的蛋白,从而达到再生的目的。
5、标本采集及细胞分离培养
分别采集激素治疗前后的CITP患者和正常对照组静脉血各5mL,肝素钠抗凝,密度梯度离心法分离PBMC,用含10%胎牛血清的RPMI1640培养液调整PBMC密度为1×109/L。其中1mL细胞悬液,用于荧光抗体染色,FCM测定Th1、Th17、Th22以及Breg细胞亚群；另取1mL细胞悬液加佛波酯PMA(终浓度为50μg/L)、离子霉素(终浓度为1μmol/L)置37℃温箱中培养过夜,收集上清置-70℃冻存待ELISA及SPR芯片技术检测细胞因子。
6、FCM法检测Th1、Th17、Th22及Breg细胞亚群
取5支试管,分别取上述淋巴细胞分层液分离单个核细胞,PBS洗2次计数,各取106个单个核细胞,其中第1支试管作为同型对照；第2支试管加入FITC-CD4抗体、PE-IFN-y抗体各5μL；第3支试管加入FITC-CD4抗体、PE-IL-17A抗体各5gL；第4支试管加入FITC-CD4抗体、PE-IL-22抗体各5μL；第5支试管分别加入CD5-FITC、CD1d-PE、PerCP-CD19抗体各5ul,同时设立同型对照,加入IgG1-FITC、IgG1-PE、IgG1-PerCP抗体各10μL,然后将标本与抗体充分混匀后,加入破膜剂,室温避光孵育1h。取出后用2ml红细胞裂解液裂解10min,离心后弃上清,用PBS洗2遍,上流式细胞仪检测,相关检测结果运用软件分析比较。
7、ELISA法检测PBMC培养液上清相关细胞因子的水平
取上述各组按要求冻存的PBMC培养上清,用ELISA法检测各组IL-10、TGF-p1、IFN-γ、IL-17, IL-22水平,按试剂盒说明书操作,每个样本和标准品均设3个复孔,酶联仪测定450nm波长吸光度(A450),各标准品均绘制相应的标准曲线,根据检测的吸光度值在各个标准曲线上确定对应的细胞因子浓度。
8、新型化学表面SPR芯片检测条件的摸索
影响SPR芯片表面固定的因素：主要是离子强度,溶液的pH值及蛋白抗原的浓度等。本研究是在一定的离子强度下,将血小板抗原进行倍比稀释后,分别点样芯片,根据不浪费样品,又能保证达到一定的固定量的原则,确定血小板抗原的稀释度。同时我们选用了不同pH值(分别为4.0,4.5,5.0,5.5,6.0)的10mmol/L醋酸盐缓冲溶液作为血小板的固定液,探索pH值对血小板抗原固定的影响,确定最佳pH值。芯片的表面再生原理是利用一定浓度的酸、碱、或者是高离子强度的溶液脉冲来洗脱抗原与抗体间的结合,从而达到传感芯片的再生。关于采用再生试剂中我们尝试采用了10mM NaOH,1:300稀释的磷酸、pH=2.0的甘氨酸作为再生试剂,确定最佳的再生试剂类型。
9、血小板在SPR芯片表面固定的方法
(1)SPR芯片活化：先室温下分别溶解0.3939克EDC(0.4M)和0.430克NHS(0.1M),配制成10mLEDC/NHS活化液,将SPR芯片置于活化液中活化30min,流水冲洗；(2)血小板抗原点样：用pH值为6.0的10mM乙酸盐与甘油配制10%的甘油点样液,将通用型血小板抗原用点样液作5倍稀释处理后,取0.5μL混合液点样于芯片表面相应的位置,室温下固定1小时；(3)芯片表面封闭：将点样好的SPR芯片,固定后,安装在SPR分析仪上,用PBS液调选最佳共振角(由最小角至最大角调试,再确定最佳的共振角约为70至100之间),基线平稳后,用封闭液乙醇胺处理10min,封闭芯片未反应的活化表面。完成后用再生液处理,去除物理吸附的物质和封闭液,然后可上样检测。
10、SPR芯片技术检测血小板抗体的性能比较
SPR技术性能分析：运用上述探索的条件及固定的SPR芯片,将阳性对照血清、阴性对照血清、患者样本等作相应的检测,分析SPR技术的稳定性、敏感度、特异性,SPR分析仪检测的数据运用BIAevaluation软件分析；(2)对比研究：运用SPR技术、固相疑集法及MAIPA法同时对40例CITP患者进行血小板抗体筛选,比较三种方法的差异性,并分析它们的性能。
11、SPR芯片技术检测血小板抗体及临床应用
对CITP患者采用SPR技术作配合性试验,选择配合型血小板输注,并跟综分析临床疗效。具体检测方法：将已知的O型通用血小板(抗原)固定在芯片上(芯片准备的方法按上述进行),检测患者血清中是否有相关抗体；同样将供者的血小板洗涤后稀释,取微量(0.5μL)点样于芯片上,检测血小板抗体阳性患者的血清是否存在SPR信号的改变,若无信号改变,说明它们是配型相合,否则为配型不相合。运用该技术对10例血小板抗体阳性患者选择配合的血小板进行输注,并进行临床跟综分析,研究患者临床血小板输注的有效性。
12、SPR芯片技术检测CITP患者PBMC培养液上清细胞因子的水平
取上述冻存PBMC培养上清,采用SPR芯片技术检测上清中IFN-y、IL-17、 IL-22、IL-10水平,具体操作是取出前述研制好的SPR新型芯片,活化处理后,分别将抗-IFN-γ、抗-IL-17A、抗-IL-22、抗-IL-10的抗体点样于芯上,固定60分钟后上机检测,同时点样PBS作为空白对照(由于该芯片的性能分析已在前面研究了,在此不再重复叙述)；上述细胞因子的标准品配制成不同的浓度,上机检测的信号响应值(RU)绘出它们相应的标准曲线,根据标准曲线可计算出相应的细胞因子的含量。
13、建立CITP特异性细胞因子-SPR芯片检测平台
该研究探索性研究,为CITP患者细胞因子的特异性检测进行摸索：先将CD4抗体固定在芯片上,捕获CD4+T细胞,芯片上刺激培养细胞,然后在芯片上动态检测细胞因子的变化,并与正常对照组比较分析,该方法的建立将有助于CITP患者的细胞免疫紊乱更准确特异的分析。我们将以检测CD4+T细胞分泌IFN-γ为例进行方法学研究。
14、统计学分析方法
各组实验结果均用x±s表示,采用SPSS16.0软件分析,完全随机设计的两本均数比较,采用两样本t检验；多样本均数间两两比较采用方差分析(ANOVA);两变量的相关程度用Spearman相关分析法；以P<0.05为差异有统计学意义。
研究结果
1、基于初步掌握SIP动力学基础上,我们充分利用表面引发聚合反应技术活性可控的优点,在纳米尺度上调控分子基质poly的相关参数(链密度、厚度),搭建了一个基于高分子poly(OEGMA-co-HEMA)的生物传感器基质平台,实现“零”蛋白非特异性吸附和较高蛋白固定能力的高度统一,解决了生物传感器的共性问题。
2、利用自组装单层技术在芯片表面组装PEG单层作为主链,其中利用稀释剂控制主链PEG的链密度；将芯片浸泡于环糊精溶液中,当足量的环糊精穿人PEG主链后,对线性PEG另一端进行封端；通过羧基化反应将环糊精的羟基基团转为羧基等活性基团,便于固定生物探针分子,成功构建了新型的SPR芯片环糊精表面。
3、实验结果表明,本项目研发的芯片具有很好的抗蛋白质非特异性吸附能力；但是,随着羧基化的程度的增加,基质的抗蛋白非特异性能力有所下降,主要由于羧基修饰后芯片表面带电荷所致。静电吸附一方面使得芯片出现非特异性吸附,但从另一个角度看来,静电吸附在固定蛋白(特别浓度比较低的蛋白溶液)起重要作用：有利于蛋白质在芯片表面富集,提高蛋白质的固定效率。
4、本项目采用模型蛋白人IgG/山羊抗人IgG系统验证两种芯片检测蛋白质相互作用的应用效果。实验结果表明：两种芯片均较高的固定蛋白质的能力,而且能够保持蛋白质生物活性,但动态的环糊精表面比poly(OEGMA-co-HEMA)基质的灵敏度更高,相同浓度的蛋白信号响应值更大。
5、FCM检测结果显示,与对照组比较,CITP患者外周血中CD19+B、 CD5+CD19+B细胞亚群的比例均升高,差异有统计学意义(P<0.05)；而Breg的比例降低,差异有统计学意义(P<0.05)。
6、ELISA法测定结果显示,与健康人对照组比较,CITP患者PBMC培养液相关细胞因子IL-10、TGF-β1的表达水平均降低,差异有统计学意义(P<0.05); CITP患者外周血中Breg的比例与PBMC培养液上清中IL-10的水平呈正相关(P<0.05),与TGF-β1水平无相关性(P>0.05)；调节性B细胞的变化与患者血小板抗体的水平呈负相关性(P<0.05)。
7、采用乙酸缓冲溶液稀释血小板,分别稀释5倍,10倍,20倍,50倍,100倍。结果表明随着点样的浓度增加检测分析物(含血小板抗体血清)信号值增加,稀释到10倍后,点样物的浓度对检测信号影响不明显；分别采用系列pH值(pH=4.0,4.5,5.0,5.5,6.0)的乙酸缓冲溶液作为点样液稀释血小板。通入分析物(含血小板抗体血清)结果表明在pH=4.5的乙酸缓冲溶液时具有较强的检测信号。在NaOH的缓冲溶液中,高分子链部分水解,导致基线往下漂移,而采用1：300稀释的磷酸基线没有回到原始基线水平即再生彻底。在采用pH=2.0的甘氨酸再生后基线基本回到初始值水平,而且再生后探针仍然保持较高的生物活性。
8、在同一张芯片上点样试剂血小板抗原与非特异性NS1抗原,其中点样一个为空白对照点,其余各点为样本检测点,将此点样好的芯片在SPR仪上分别检测血小板抗体阳性对照血清与NS1抗体阳性对照血清,结果表明SPR芯片能特异检测血小板抗体。对40例CITP患者,分别用SPR芯片技术、固相凝集法和MAIPA法检测,经McNemr检验,SPR芯片技术与固相凝集法比较(P>0.05),一致性为92.5%。SPR芯片技术与MAIPA法比较(P>0.05),SPR法的灵敏度为96.3%,特异性为92.3%,总一致性为95%,三种方法差异均无统计学意义。
9、将ABO同型的供者血小板样本,经洗涤后稀释,按照上述血小板点样的条件与方法,点样于SPR芯片上,然后根据前面的检测方法,分析患者的血清样本与供者机采血小板的相容性。我们对SPR技术检测的其中10位血小板抗体阳性患者,进行交叉配合试验,从多位血小板供者中选择配合型血小板输注,经过临床随访分析,其中8例患者1h血小板增加值CCI>7.5,24hCCI>4.5,为输注有效,并且病人状态良好,另外2例因有其它基础疾病。
10、FCM检测结果表明：CITP患者糖皮质激素治疗前,血小板计数均数为28×109/L,外周血Th1、Th17、Th22细胞百分率明显上升,调节性B细胞(Breg)的比例降低,与正常对照比较差别有统计学意义(P<0.05)；经过治疗4天后,血小板计数上升,均数为71×109/L, CITP患者外周血Thl、Th17、Th22细胞百分率明显下降,调节性B细胞(Breg)的比例上升,与正常对照组比较,它们均无显著性差异(P>0.05)。CITP患者外周血Th1、Th17、Th22细胞比例变化与血小板数成负相关性(P<0.05)；调节性B细胞(Breg)的比例变化与血小板数成正相关性(P<0.05)。
11、ELISA法测定结果显示,与正常对照组比较,CITP患者糖皮质激素治疗前外周血PBMC细胞培养上清液IFN-y、IL-17、IL-22水平升高,IL-10的表达水平均降低,差别有统计学意义(P<0.05)；糖皮质激素治疗后,与正常对照组比较,均无显著性差异；CITP患者Breg细胞的比例变化与PBMC培养上清中细胞因子IL-10表达水平成正相关(P<0.05),IL-10水平与IFN-γ、IL-17、IL-22水平也呈负相关性(P<0.05)。
12、SPR芯片技术检测显示：CITP患者PBMC培养液中分泌的相关细胞因子IL-10、IFN-γ、IL-17、IL-22水平,结果与ELISA法比较,无显著性差异(P>0.05)。
13、成功建立特异性细胞因子-SPR芯片检测平台：运用SPR新型表面化学蛋白芯片先捕获CD4+T细胞,再研究分析芯片上细胞的活性与纯度均大于90%,CD4细胞与IFN-y与相应抗体均有良好的特异性反应,动态分析它们在芯片表面培养刺激产生的IFN-y水平,检测到信号的时间比试管培养刺激的时间更短,特异性更好,该方法具有更进一探索研究的价值。
研究结论
1、利用表面引发聚合反应技术活性可控的优点,在纳米尺度上调控高分子的相关参数,搭建了一个基于Poly(OEGMA-co-HEMA)的SPR生物传感器的化学表面基质,实现了抗蛋白质非特异性吸附的效果,降低了背景噪音。
2、利用分子自组装技术构建了SPR芯片动态性环糊精表面,利用环糊精在聚乙二醇链上的自由转动和滑动的能力,实现动态型的生物传感器表面,从而提高了探针的空间自适配功能,提高了其空间的选择性,实现了动态型的生物传感器表面的制备,为蛋白分子的检测提供了新平台。
3、FCM法分析表明：CITP患者Breg细胞的比例下降,且与IL-10的水平呈正相关,与血小板抗体PAIgG的水平呈负相关性。该研究为CITP的发病机制提供了新的思路。
4、新型的SPR芯片技术高通量、快速、灵敏地检测CITP患者的血小板抗体的水平与血小板配型输注,经临床随访分析效果良好,表明新型SPR芯片技术可初步应用于CITP患者血小板抗体的检测与配型。
5、研究表明CITP患者Breg、Th1、Th17、Th22细胞及其相关细胞因子IL-10、IFN-γ、IL-17、IL-22分泌紊乱在CITP发病中发挥重要作用,该研究为CITP的临床诊断与疗效评价提供了新靶点。
6、新型SPR蛋白芯片技术可用于CITP患者细胞因子的联合检测,高通量、快速地分析其免疫紊乱的特征。建立了新型SPR芯片捕获CD4+T细胞、特异性分析细胞因子IFN-γ变化的平台,更有利于CITP I临床诊断的准确性,具有重要的研发价值。
7、该研究为CITP免疫紊乱的临床诊断与疗效评价提供了新靶点和新方法,新型环糊精表面SPR芯片在高通量、快速、特异、联合检测血小板抗体和细胞紊乱中具有重要意义。
Research background
Surface plasmon resonance (SPR) is a free labeled optical biosensor technology, which is more and more widely used in many fields today, such as life sciences, which do not need to lable the analyte and measure the kinetics of molecular biological activity in non-modified conditions, be suitable for the high throughput screening of bioactive molecules, especially for the small molecules, the trace analysis of the unknown materials and the clinical samples online.
Chronic idiopathic thrombocytopenic purpura(CITP) is a kind of common bleeding autoimmune disease, the incidence is on the rise in China, which seriously affect the life quality of the CITP patients, but the mechanism is unknown, it is difficult to find really specific and effective method for treatment. Many studies show that the pathogenic CD4+Th cell proliferation rising, apoptosis reducing, activity increasing and the Thl,Th17, Th22cell differentiation imbalance.Recently, a new regulatory B cells has been paid more and more attention and has been actively studied in the field of immunology. So far there are no related reports in CITP patients, we will study the role of Breg in immune disorders of patients with CITP, and to explore the relationship between Breg and CD4+Th cell subsets and platelet antibody, and we will look for one new ideas for the clinical diagnosis and treatment of CITP.
Therefore, we will construct a novel SPR chemistry chip, and explore the application of SPR technology in the detection of immune disorders for CITP patients. SPR technology has a very important significance for the choice of treatment strategy effective evaluation and prognosis and platelet compatibility.
Research purpose
To construct and optimize the novel chemical surface chip for SPR technology, and to establish the new detection method based on SPR technology, which is simple and rapid, high sensitivity and specificity. As the treatment effect for CITP patients is poor, whose pathogenesis is complex and unknown to the role of new regulatory B cells in autoimmune diseases, we will analyse the characters of the regulatory B cells in the peripheral blood of patients with CITP and its effect on CD4+Th cell immune disorder in this study. We plan to explore its application value in clinical diagnosis and treatment of CITP patients, which will provide a new target and a novel assay for the immune disorder of CITP.
Research contents
This study intends to carry out the following work:
1Construct a novel surface chemistry SPR protein chip, which is quick and simple with high sensitivity and specificity, and evaluate the methodological character.
2Analyse the changes of regulatory B cells and its correlation with platelet antibody in the peripheral blood of patients with CITP.
3Using the novel SPR protein chip, to establish a new assay with high throughput and free lable for platelet antibody screening and match in CITP patients, and to compare the characters with the MAIPA and SPA assays.
4Study the correlation between the changes of regulatory B cells and CD4+T cell and related cytokines in the peripheral blood of patients with CITP, to analyse the role of them for CITP immune disorder.
5Using the novel SPR protein chip, to establish a new assay with high throughput and free lable for detecting the special cytokines in CITP patients.
Research methods
1Preparation for poly (OEGMA-co-HEMA) matrix
Using binary self-assembly system to control the concentration of initiator surface. Specific operation process is as follows:ethanol as solvent, mixed mercaptan initiator and PEG mercaptan, a total concentration of1mM, the SPR chips treated with UV-ZONE are immersed in the mixed solution for15hours, then chips are taken out, washed with ethanol fully, blown dry with nitrogen. According to2,2'pyridine (tendency)12.5mg (0.8mM),2.62g OEGMA526(5mM), HEMA0.65g (5mM), dissolved them in5mL ultrapure water and5mL methanol. Add1mL CuCl2solution (0.04mM), nitrogen gas is piped in, eliminate oxygen for15min. Then slowly inject1mL ascorbic acid(AscA) solution (0.04mM), Cu (II) complexes are gradually turned into Cu (I) complexes, reaction solution is changed from blue to pink. Continue to anaerobic treatment for15min, polymerization reaction is processed in the glove box of nitrogen gas environment on the chip surface. Chip surface agent will trigger polymerization reaction on the chip surface at room temperature for8hours, then remove chips and terminate reaction, using methanol and ultrapure water clean them, blow dry with nitrogen for further analysis, such as elliptical polarization measurement of film thickness, etc. The hydroxyl activity is poor at Poly (OEGMA-co-HEMA) matrix chain end, which is difficult to couple reaction with the protein under mild conditions, so we must have functionalization for hydroxyl groups, such as functional hydroxy. Hydroxy functional experiment operation is as follows:the surface modified poly (OEGMA-co-HEMA) chips are soaked in DMF reaction solution with succinic anhydride (10mg/mL)and DMAP(15mg/mL), react under room tempture for12hours. Remove chips and wash them with DMF and ethanol fully, blow dry by nitrogen.
2Preparation for dynamic cyclodextrin surface
(1) Using self-assembly monolayer technique, PEG monolayer is assembled on chip surface as the main chain, we use thinner to control the density of PEG main chain, then soake the chips in cyclodextrin solution, when plenty of cyclodextrin went into the PEG main chain, stopping the linear PEG at the other end.(2)By hydroxylation reaction, cyclodextrin hydroxyl groups are turned into active carboxyl group, which is advantageous for the immobilized biological probe. Using system experimental parameters (chain density, chain length and carboxyl numbers) of SPR technology for their influence on biological detection performance, we optimize the best conditions.(3) Under the optimization experimental conditions, we have prepared for the novel sensors based on cyclodextrin molecule matrix, and applied the chip matrix to detect protein model.
3Characterization methods for SPR chips
(1)AFM characterization:Characterization of matrix morphology structure is analysed by atomic force microscope (AFM,Veeco,Dimension3100). The experiment is done by the contact model.(2) Elliptic polarization characterization:film thickness is measured by M-2000V spectral elliptic polarization measuring instrument (J.A.Woollam Co, inc). Measurement angle is65,70,65degrees, wavelength varies from400nm to800nm. From elliptic polarization meter data, we get the thickness of the film with a specific model, including SAMs and Poly (OEGMA) using Cauchy Model,(An, Bn)=(1.45,0.01) and (1.46,0.01), respectively. Sample thickness are measured as the average values for the three position and reported the results for x±s values.
4Performance test
(1)Anti-fouling effect test:According to the instrument manuals, carboxyl functional chip will be loaded, set the working temperature at25℃, flow velocity at2μL/s, ran in PBS buffer solution (pH=7.4), after a stable baseline appear, inject specific concentrations protein solution (PBS dilution), finally inject PBS buffer solution.(2)Protein detection ability test:According to the operational requirements, set parameters of instrument for sample and add30μL sample in384well plates, set temperature at25℃,50%humidity, protein sample will be pointed on the SPR chip surface by contacted way. After sample chip was placed in the box of25℃temperature and75%humidity, keep constant temperature and humidity for2hours, then install the protein chip in SPR system, ran in PBS solution (pH=7.4) and at2μ L/s flow velocity, after a stable baseline is seen, ethanol is run into the chip for7min, closed the extra active site, then a specific concentration protein solution(PBS dilution) is injected, finally glycine solution (lOmM, pH=2.0) can wash out the combination of proteins, so as to achieve the goal of regeneration.
5Collect specimens and separate PBMC from CITP patients
Extraction of5mL venous blood with heparin sodium anti-coagulation from CITP patients and control group, PBMC is separated by density gradient centrifugation assay, and adjusted in RPMI1640medium containing10%fetal bovine serum,PBMC density is1×109/L.1mL suspension cell is used for detecting CD4+T and Breg subsets with fluorescent antibody staining by FCM,2mL cell suspension is incubated in phorbolester PMA (final concentration50μg/L) and ionomycin (final concentration1μmol/L) at37℃overnight, then collecting the supernatant and saving them at-70℃for detection of cytokines.
6Determination of Thl, Th17,Th22subsets and Breg by FCM
Take five tubes for detection, respectively take the mononuclear cells using lymphocytes stratified separation, wash twice by PBS,106mononuclear cells for each tube, No.1tube as isotype controls, No.2tube including5μL FITC-CD4antibodies and5μL PE-IFN-y, No.3tube including5μL FITC-CD4antibody and5μL IL-17A antibody, No.4tube including5μL FITC-CD4antibody and5μL PE-IL-22antibody, No.5tube including CD5-FITC, CDld-PE, PerCP-CD19antibodies5u L, respectively. The specimen after thoroughly incorporated with antibody, incubated at room temperature away from light for1h. Take2mL cracking fluid for red blood cells to act for10mins, after the centrifugal supernatant is removed, then wash2times by PBS and detect them by FCM, the related analysis and comparison is done using the software.
7Detection of IFN-γ, IL-17, IL-22and IL-10cytokines by ELISA
Take out the PBMC culture supernatant, we detect IFN-y, IL-17, IL-22and IL-10levels by ELISA method, and do it according to the reagent operation manual, providing three holes for each sample and standard, read the OD value at450nm.
8The conditions for platelet immobilized on SPR chip surface
The influencing factors mainly are the ionic strength, pH value of solution and the concentration of the protein. In this study, under certain ionic strength, we dilute platelet antigen and point them on the novel SPR chips, respectively. According to not wasting samples, and making sure that the immobilized platelets reach a certain amounts, to determine the platelet antigen dilution degrees. At the same time, we choose the different pH value (4.0,4.0,4.5,5.5,6.0)10mM acetate buffer solution as stationary liquid for platelets, to explore the influence of pH value for platelet antigen, to find the optimum pH value. The principle of chip regeneration is to use a certain concentration of acid, alkali, or high ionic strength of solution to wash away the combination of antigen and antibody, so as to achieve the regeneration of the sensor chip. On the regenerating reagent, we try to adopt the10mM NaOH,1:300diluted phosphoric acid and pH=2.0glycine as regenerative reagent, finally to determine the best regenerative reagent types.
9Platelets immobilization on the SPR chip surface
Platelet antigens are immobilized:According to the explored conditions, amino coupling method is used to immobilize corresponding general-purpose platelet antigen on the SPR sensor chip surface for detecting the corresponding platelet antibody. Method as follows:(1) Activation for the SPR chip:First,0.3939g EDC (0.4M) and0.430g NHS (0.1M) were dissolved at room temperature, then make up10mL EDC/NHS activation fluid mixture, the SPR chip is activated in the activation fluid for30min.(2) Point platelet antigen:10%glycerol fluid is made in10mM acetate (pH=6.0) and glycerin, the universal platelet antigen is diluted for five times with it,0.5μL mixture is pointed on the chip surface, and immobilized at room temperature for1hour.(3) The surface of the chip is enclosed:after chip immobilization, which will be installed in the SPR analyzer, inject PBS solution to choose optimum resonance angle (debugging from minimum angle to maximum angle, then choose the optimum resonance angle between70and100), when baseline is stationary, treated for10min with closed fluid ethanolamine, enclosing unreacted activated surface, removing physical adsorption material and sealing fluid by injecting PBS, then testing the samples.
10The performance analysis of SPR technology
(1) Using the exploring conditions and the fixed SPR chips, to detect the positive control, negative control and patient samples serum correspondingly, analyse the stability, sensitivity and specificity of SPR technology, data from SPR analyzer are analysed using BIAevaluation software.(2) Comparative study:detect platelet antibody for40CITP patients using SPR technology,SPA assay and MAIPA assay, compare with the differences of three methods, and to analyze their performance.
11Application for platelet antibody detection by the novel SPR chips
For CITP patients, screen platelet antibody by SPR technology, and choose compatible platelets for transfusion. The known type O general platelets are immobilized on a chip (preparation for chip according to the above), to detect whether there are relevant antibody in CITP patients serum. Also the platelets supplied by blood center are washed and diluted, then0.5u L platelet is pointed on the chip, to detect SPR signal change in serum for platelet antibody positive patients, if no signal change, show that they are in matches, or matching statements do not agree. Using the SPR technology for10CITP patients with platelet antibody positive to choose cooperated platelets for infusion, and to analyze the clinical effectiveness of platelet transfusion according to the long-term follow up.
12Application of cytokines detection by the novel SPR chip
(1) First method:take out the PBMC culture supernatant, using SPR chip technology to detect IFN-y, IL-17, IL-22and IL-10levels. The ways as follows: after SPR chip is activated, anti-IFN-y, anti-IL-17, anti-IL-22, anti-IL-10mAb are immobilized on the SPR chip for60minutes, respectively. At the same time pointing PBS as blank control (due to performance analysis of the chip has been ae studied), standard cytokines are diluted into different concentrations, detecting their corresponding signal value and make up standard curves, according to the standard curves, the concentrations of the corresponding cytokines can be calculated.(2) Second method:for exploratory research, for detecting the specific cytokines in CITP patient, First the anti-CD4antibody is immobilized on the chip, capturing CD4+T cells, then culture cells on the chip, and have the dynamic detection of cytokines, and comparing with normal control group, this method will help to analyse the cellular immunity disorders for CITP patients more accurately. We will study to detect IFN-gama from CD4+T secretion as an example.
13Statistical processing
Experimental results show as x±s, analysed by the SPSS16.0software, using two samples t test, muti-samples ANOVA test and spearman correlation analysis for the relation degree between the two variables, the difference was statistically significan with P<0.05.
Research results
1The construction of novel SPR matrix:For the preliminary master in SIP dynamics, we made full use of the controllable polymerization technology, and adapt the poly matrix related parameters at the nanoscale molecular, built a cyclodextrin matrix platform and Poly(OEGMA-co-HEMA) matrix, and achieved a high unity between the "zero" protein nonspecific adsorption and the high protein fixed ability, solved the common problems of biological sensors.
2(1)For the novel chip, protein nonspecific adsorption amount is lower than the detection sensitivity of SPR. The experimental results showed that the objective of the research and development of the chip had good ability to resist protein nonspecific adsorption.(2)This project adapt the model protein IgG/goat anti-human IgG system to verify application effect for two kinds of chips in the detection of protein interactions. The experimental results showed that both chips had the ability of high immobilization protein, and can maintain protein biological activity,but the cyclodextrin matrix platform was more sensitive than the Poly (OEGMA-co-HEMA) matrix.
3FCM results showed that the proportion of peripheral blood CD19+B, CD5+CD19+B cells were increased in CITP patients compared with control group, the difference was statistically significant (P<0.05), but the proportion of CD1d (hi)CD5+CD19+B(Breg) was reduced, the difference was statistically significant (P<0.05).
4ELISA results showed that, compared with healthy controls, the expression levels of IL-10and TGF-betal from PBMC cultures supernatan were reduced in CITP patients, the difference was statistically significant (P<0.05). The proportions of Breg positively correlated with IL-10levels from PBMC culture supernatant (P<0.05), but the proportions of Breg were of no correlation with TGF-betal level (P>0.05). The changes of regulatory B cells had negative correlation with the changes of platelet related antibody(PAIgG) in CITP patients.
5Before glucocorticoid treatment in CITP patients, the platelet counts were (5-37) x109/L, mean count was28x109/L, the percentage of peripheral blood Thl, Th17, Th22subsets increased obviously, the difference was statistically significant compared with normal controls (P<0.05). The proportion of regulatory B cells (Breg) was reduced, the difference was statistically significant (P<0.05). After glucocorticoid treatment for four days, the platelet counts rised, mean counts were71×109/L, compared with normal control group, the percentages of peripheral blood Thl, Thl7, Th22subsets decreased obviously, they had no significant difference (P>0.05). The percentages of Thl, Th17, Th22subsets had negative correlations with the platelet counts (P<0.05), but the proportion of Breg had positive correlation with the platelet counts (P<0.05).
6According to the ELISA results, compared with normal control group, before glucocorticoid therapy in CITP patients, IFN-y, IL-17, IL-22levels from PBMC culture supernatant rised, the expression of IL-10level was lower, the difference was statistically significant (P<0.05), but after glucocorticoid treatment, compared with normal control group, there was no significant difference. The ratio of Breg cells in CITP patients showed positive correlation with the change of IL-10level in PBMC culture supernatant(P<0.05), the changes between IL-10and IFN-y, IL-17, IL-22levels showed a negative correlation,respectively (P<0.05).
7Detection conditions for platelet anibody by the novel SPR chips in CITP patients:(1) Platelet diluted times:Using acetic acid buffer solution to dilute platelets, respectively for5times,10times,20times,50times,100times, results showed that with the concentration of pointed analyte increasing (including platelet antibody in serum) signal value increased, after20times dilution, SPR detection signal is not obvious, so10times dilution was pointed concentration for sample platelets.(2) Series of pH value (pH=4.0,4.5,5.0,5.5,6.0) acetic acid buffer solution were used as platelets pointing sample dilution, respectively, the results showed that the acetic acid buffer solution at pH=4.5had more strong signal.(3) In NaOH buffer solution, partially hydrolyzed polymer chain, lead to the baseline drift down, but using1:300diluted phosphoric acid, baseline did not return to the original baseline. Using the pH=2.0glycine regeneration, baseline return to the initial value, after regeneration the probe still maintained high biological activity.
8Detection limit for platelet antibody:Platelet reagent antigen was pointed on the same chip, and selected the corresponding seven areas, one for blank control point, the other6points for testing, standard controls were taken from positive serum5,10,20,30,40,60uL, the corresponding concentration was50ng/ml,100ng/ml,200 ng/ml,300ng/ml,400ng/ml,600ng/ml, respectively, at2u L/s the velocity of sample testing, processing by the BIAevaluation analysis software, results showed that had good sensitivity and minimum detection limit was about50ng/ml.
9Platelet antibody specific detection:reagent platelet antigen and nonspecific NS1antigen were pointed on the same chip, one spot for blank control, the other each point for sample testing, using platelet antibody positive controls and NS1antibody positive serum, respectively, results showed that, when platelet antibody positive controls run into the SPR chips, strong signal was seen at the platelet antigen spot, but had weak signal at nonspecific NS1antigen spot and control spot. This study suggests that the chip specificity is better, the novel SPR chip can detect specific platelet antibody.
10Platelet antibody screening was analysed by SPR chip technology,SPA assay and MAIPA method for40CITP patients, test results were shown that, by McNemr test, there was no statistically significant difference between SPR technology and SPA assay (P>0.05). There was also no statistically significant difference between SPR technology and MAIPA assay (P>0.05), the sensitivity, specificity and total consistency for SPR method were91%,97.9%,97.2%, respectively. In the40CITP patients,26of them platelet antibody were positive by SPR assay. Of the10platelet antibody positive CITP patients, by SPR technology, we choose compatible platelets transfusion from a number of donor platelet, after clinical follow-up analysis, of which8cases of patients, the platelet added value1h CCI>7.5,24h CCI>4.5, got effective infusion and they were in good condition, the other2cases were invalid for other basic diseases.
11Results for detection of related cytokines by the novel SPR chip in CITP patients:cytokines IL-10, IFN-y, IL-17, IL-22levels form PBMC culture fluid in CITP patients were detected by the novel SPR chip, the results showed that, compared with ELISA, there were no significant difference(P>0.05), but we can assayed all the cytokines in one time test, which was more simple and convenient.
12Another application of SPR model for specific cytokines detection, CD4cells were captured on the surface of SPR protein chip, and then we analysed cytokines.The activity and purity of CD4+T cells are more than90%, by the dynamic analysis method, IFN-gama levels from CD4+T cells culture stimulation were detected on the surface of the chip, results showed tha the detected signal time was shorter than those of stimulation in vitro, and had better specificity.
Research conclusion
1This project has used controllable surface activity by polymerization technology to regulate the related parameters of polymer on nanometer scale, and built a novel chemistry surface SPR biosensor chip based on oligomeric ethylene glycol methyl methacrylate(Poly(OEGMA-co-HEMA)),which implemented the resistant protein nonspecific adsorption effect, and reduced background noise.
2The project has built another novel dynamic cyclodextrin SPR chip surface. Through the chemical modification of cyclodextrins in polyethylene glycol (PEG) chain on dextrin free rotation and sliding, freedomly taking advantage of cyclodextrins rotation and sliding ability in polyethylene glycol (PEG) chain, which realized the biosensor surface with the dynamic state, can improve the space adaptation and the space selectivity of the probe for detecting protein molecules.
3This project has shown that the proportion of Breg is reduced, the proportion of the Breg positively correlated with IL-10levels from PBMC culture supernatant, the changes of regulatory B cells and platelet antibody show a negative correlation in CITP patients, which may provide a new idea for pathognisis of CITP.
4We have established a new type of SPR microarray to analyse platelet antibody and the compatibility between the serum of CITP patients and platelets from donors, and to cross matching test. By clinical follow-up analysis, showing effective infusion and good conditions,which showed that the novel SPR technology can provide preliminary application for platelet antibody screening and matches.
5The percentages of peripheral blood Thl, Th17, Th22subsets increased obviously in CITP patients, and showed negative correlation with the platelet counts. The proportion of Breg is reduced, and show positive correlation with the platelet counts, IFN-y, IL-17, IL-22levels are reduced, IL-10levels are rised, but show a negative correlation between IL-10with IFN-y, IL-17, IL-22level, which may provide a new target for clinical diagnosis and treatment of CITP.
6This kind of novel SPR protein chip can be used to detect the cytokines from PBMC secretion, to study the CITP immune disorders with high-throughput and rapid analysis. Besides, a new special SPR chip platform was established for capturing CD4+T cells to analyse the related cytokine IFN-gama in dynamic detection, the real situation of immune disorders is more evident, which is better in favor of clinical diagnosis and treatment for CITP.
7This study provides a new target and the new detection method for clinical diagnosis and treatment in CITP immune disorders. This novel cyclodextrin chemical surface SPR chip has important significance in the detection of platelet antibody and the cytokines disorder for CITP patients with high-throughput and rapid analysis.
表面等离子体共振(Surface Plasmon Resonance, SPR)是当今应用最普遍的非标记光学生物传感器(Label-Free Optical Biosensor)技术,正在被越来越普遍地用于生命科学等多个领域。SPR技术不需要对被测物进行标记的优点使其可以测量生物活性分子在无修饰条件下的反应动力学,适用于高通量生物活性分子特别是小分子的筛选,微量未知物的分析,以及在线样品检测。但其核心技术为光路检测系统、微流控进样系统和SPR芯片。目前改进基质材料的表面处理技术,建立高效和快速的芯片活化方法,并能保持芯片表面物质的稳定性和生物活性是该领域研究的热点,同时与微流控技术相结合可降低样品消耗量,提高检测通量。因此,优化SPR芯片表面的功能,探索建立高通量、简便快速、灵敏度高、特异性强的SPR技术应用于临床检验诊断具有重要的意义。
慢性特发性血小板减少性紫癜(Chronic idiopathic thrombocytopenic purpura, CITP)是一种常见的出血性自身免疫病,在我国的发病率呈上升趋势,严重影响患者的生活质量,但因发病机制不明,还难以找到真正特异且有效的治疗方法。近年免疫调节治疗方式引起了人们的兴趣,可能从源头上改变相关的治疗效果。最近研究表明新型调节性B细胞(Breg)具有负性调节作用,在免疫学领域越来越受重视并被积极研究,我们前期预实验发现Breg细胞可能在CITP患者免疫紊乱中发挥重要作用,CITP患者体内Breg的变化可能与CD4+Th细胞亚群和血小板抗体之间有重要关系,因此需要对该问题进行深入研究,并探索一种简便、快速的检测方法,为CITP的临床诊断与治疗寻找新的靶点与思路。血小板抗体的检测是国内外研究的热点,虽然方法有多种,但因价格太高,或操作复杂,或易至假阳性或假阴性等问题,临床应用较少；CD4+T等相关细胞因子紊乱的检测方法主要为ELISA、流式细胞术和PCR等,都难以高通量快速检测多种因子,限制了它们的临床应用。因此,利用SPR技术优势,探索SPR芯片技术应用于CITP患者的免疫紊乱的动态联合检测,对临床诊断、治疗策略选择、临床效果评价和预后分析以及血小板相容性输注治疗等方面具有非常重要的意义。
研究目的
构建与优化新型表面化学SPR蛋白芯片,建立基于SPR技术高通量、简便快速、灵敏度高、特异性好的新型免疫学检测方法：针对CITP治疗效果差,发病机制复杂以及新型调节性B细胞在自身免疫性疾病中的作用特点,本研究拟分析CITP患者外周血新型调节性B细胞的变化特征、及其对患者血小板抗体与CD4+Th细胞紊乱的调节作用；探索新型SPR芯片技术在CITP免疫紊乱诊断与疗效评价中的应用价值,为CITP免疫紊乱的检测寻找新靶点与新方法,同时也为新型SPR芯片技术应用于其它相关免疫病的临床检验诊断提供参考。
研究内容
1、构建灵敏度高、特异性好的新型表面化学SPR蛋白芯片,进行相关性能的综合评价,并对两种SPR芯片比较分析。
2、研究CITP患者外周血新型调节性B细胞的变化及其与血小板抗体的相关性,并分析它们在CITP患者免疫紊乱中的作用。
3、运用新型表面化学蛋白芯片,建立高通量、非标记的SPR技术筛检CITP患者血小板抗体的新方法,并与固相凝集法和MAIPA法对比分析。
4、研究CITP患者外周血新型调节性B细胞的变化与CD4+T细胞的相关性,分析相关的细胞因子在CITP免疫紊乱中的作用。
5、运用新型表面化学蛋白芯片,探索建立高通量、非标记的SPR技术动态、特异性联合检测CITP患者Breg与CD4+T细胞相关因子紊乱的新方法。
研究方法
1、SPR芯片Poly(OEGMA-co-HEMA)表面的制备
利用二元自组装体系对引发剂表面浓度进行控制,以乙醇为溶剂,配制硫醇引发剂和PEG硫醇混合溶液。将经过UV-ZONE处理的芯片浸泡在上述混合溶液中,放置15小时。取出芯片,用乙醇充分淋洗,氮气吹干。称取2,2’吡啶12.5mg、OEGMA5262.62g和HEMA0.65g,加入溶剂超纯水5ml和甲醇5m1搅拌溶解。加入1mLCuCl2的溶液(0.04mM),通入氮气,排除氧气15min。随后利用注射器缓慢注入1mL抗坏血酸溶液(0.04mM), Cu(II)配合物逐渐被还原为Cu(I)的配合物,反应溶液由通常的淡蓝色变为淡红色。继续无氧处理15min。表面引发聚合反应在氮气环境的手套箱中进行。将有引发剂的芯片,浸泡在反应溶液中。芯片表面将引发单体在聚合于芯片表面,在室温下反应8小时后,将芯片取出,终止反应,并用甲醇和超纯水清洗干净,氮气吹干。基质Poly(OEGMA-co-HEMA)侧链末端羟基活性较差,很难在温和条件中与蛋白质进行偶联反应,所以必须对羟基进行功能化：将表面修饰poly(OEGMA-co-HEMA)的芯片浸泡在含有丁二酸酐(10mg/mL)和DMAP(15mg/mL)的DMF反应溶液中,温室下反应12小时。将芯片取出,用DMF和乙醇充分淋洗,氮气吹干。
2、SPR芯片动态环糊精表面的制备
利用自组装单层技术在芯片表面组装PEG单层作为主链,其中利用稀释剂控制主链PEG的链密度；将芯片浸泡于环糊精溶液中,当足量的环糊精穿入PEG主链后,对线性PEG另一端进行封端。再通过羟基化反应将环糊精的羟基基团转为羧基等活性基团,便于固定生物探针分子。利用SPR技术系统实验参数(链密度、链长度、羧基数量)对于其在生物检测性能的影响,优化出最佳的制备条件。在优化的实验条件下制备基于环糊精分子的传感器基质,并应用该基质检测模型蛋白。
3、新型化学表面SPR芯片的表征方法
(1)AFM表征：基质在干态下的形貌结构由原子力显微镜(AFM,Veeco, Dimension3100)表征,实验采用接触式模型；(2)椭圆偏振表征：膜厚由M-2000V型分光椭圆偏振测量仪测量,测量角度为65、70和75度,测量波长为400nm到800nm。椭圆偏振仪数据用特定的模型拟合得到膜的厚度,其中SAMs和Poly(OEGMA)采用Cauchy模型,(An, Bn)分别为(1.45,0.01)和(1.46,0.01)。样品厚度值为三个位置测量值的平均,报道结果为平均值±标准差。
4、新型化学表面SPR芯片性能评价
(1)抗污效果测试：按照仪器操作手册将芯片(羧基功能化芯片)装载后,设定工作温度25度,流速2μL/s,通入PBS缓冲溶液(pH=7.4),得到稳定基线后注入特定浓度的蛋白质溶液(PBS缓冲液稀释),最后通入PBS缓冲溶液。
(2)蛋白检测能力测试：按照仪器的操作要求设定点样矩阵参数,将30uL点样样品添加384孔板中。设定温度为25度,湿度为50%。用接触方式将蛋白样品点样于SPR芯片表面。点样后芯片放置于25度,湿度为75%的恒温恒湿箱中2小时；将蛋白质芯片安装于SPR系统中,通入PBS缓冲溶液(pH=7.4),流速2μL/s,得到稳定基线后,通入封闭试剂乙醇胺(10mmol/L, pH=8.5)7min,封闭多余的活性位点。注入特定浓度的蛋白质溶液(PBS缓冲液稀释),利用甘氨酸溶液(10mM, pH=2.0)可以洗掉结合的蛋白,从而达到再生的目的。
5、标本采集及细胞分离培养
分别采集激素治疗前后的CITP患者和正常对照组静脉血各5mL,肝素钠抗凝,密度梯度离心法分离PBMC,用含10%胎牛血清的RPMI1640培养液调整PBMC密度为1×109/L。其中1mL细胞悬液,用于荧光抗体染色,FCM测定Th1、Th17、Th22以及Breg细胞亚群；另取1mL细胞悬液加佛波酯PMA(终浓度为50μg/L)、离子霉素(终浓度为1μmol/L)置37℃温箱中培养过夜,收集上清置-70℃冻存待ELISA及SPR芯片技术检测细胞因子。
6、FCM法检测Th1、Th17、Th22及Breg细胞亚群
取5支试管,分别取上述淋巴细胞分层液分离单个核细胞,PBS洗2次计数,各取106个单个核细胞,其中第1支试管作为同型对照；第2支试管加入FITC-CD4抗体、PE-IFN-y抗体各5μL；第3支试管加入FITC-CD4抗体、PE-IL-17A抗体各5gL；第4支试管加入FITC-CD4抗体、PE-IL-22抗体各5μL；第5支试管分别加入CD5-FITC、CD1d-PE、PerCP-CD19抗体各5ul,同时设立同型对照,加入IgG1-FITC、IgG1-PE、IgG1-PerCP抗体各10μL,然后将标本与抗体充分混匀后,加入破膜剂,室温避光孵育1h。取出后用2ml红细胞裂解液裂解10min,离心后弃上清,用PBS洗2遍,上流式细胞仪检测,相关检测结果运用软件分析比较。
7、ELISA法检测PBMC培养液上清相关细胞因子的水平
取上述各组按要求冻存的PBMC培养上清,用ELISA法检测各组IL-10、TGF-p1、IFN-γ、IL-17, IL-22水平,按试剂盒说明书操作,每个样本和标准品均设3个复孔,酶联仪测定450nm波长吸光度(A450),各标准品均绘制相应的标准曲线,根据检测的吸光度值在各个标准曲线上确定对应的细胞因子浓度。
8、新型化学表面SPR芯片检测条件的摸索
影响SPR芯片表面固定的因素：主要是离子强度,溶液的pH值及蛋白抗原的浓度等。本研究是在一定的离子强度下,将血小板抗原进行倍比稀释后,分别点样芯片,根据不浪费样品,又能保证达到一定的固定量的原则,确定血小板抗原的稀释度。同时我们选用了不同pH值(分别为4.0,4.5,5.0,5.5,6.0)的10mmol/L醋酸盐缓冲溶液作为血小板的固定液,探索pH值对血小板抗原固定的影响,确定最佳pH值。芯片的表面再生原理是利用一定浓度的酸、碱、或者是高离子强度的溶液脉冲来洗脱抗原与抗体间的结合,从而达到传感芯片的再生。关于采用再生试剂中我们尝试采用了10mM NaOH,1:300稀释的磷酸、pH=2.0的甘氨酸作为再生试剂,确定最佳的再生试剂类型。
9、血小板在SPR芯片表面固定的方法
(1)SPR芯片活化：先室温下分别溶解0.3939克EDC(0.4M)和0.430克NHS(0.1M),配制成10mLEDC/NHS活化液,将SPR芯片置于活化液中活化30min,流水冲洗；(2)血小板抗原点样：用pH值为6.0的10mM乙酸盐与甘油配制10%的甘油点样液,将通用型血小板抗原用点样液作5倍稀释处理后,取0.5μL混合液点样于芯片表面相应的位置,室温下固定1小时；(3)芯片表面封闭：将点样好的SPR芯片,固定后,安装在SPR分析仪上,用PBS液调选最佳共振角(由最小角至最大角调试,再确定最佳的共振角约为70至100之间),基线平稳后,用封闭液乙醇胺处理10min,封闭芯片未反应的活化表面。完成后用再生液处理,去除物理吸附的物质和封闭液,然后可上样检测。
10、SPR芯片技术检测血小板抗体的性能比较
SPR技术性能分析：运用上述探索的条件及固定的SPR芯片,将阳性对照血清、阴性对照血清、患者样本等作相应的检测,分析SPR技术的稳定性、敏感度、特异性,SPR分析仪检测的数据运用BIAevaluation软件分析；(2)对比研究：运用SPR技术、固相疑集法及MAIPA法同时对40例CITP患者进行血小板抗体筛选,比较三种方法的差异性,并分析它们的性能。
11、SPR芯片技术检测血小板抗体及临床应用
对CITP患者采用SPR技术作配合性试验,选择配合型血小板输注,并跟综分析临床疗效。具体检测方法：将已知的O型通用血小板(抗原)固定在芯片上(芯片准备的方法按上述进行),检测患者血清中是否有相关抗体；同样将供者的血小板洗涤后稀释,取微量(0.5μL)点样于芯片上,检测血小板抗体阳性患者的血清是否存在SPR信号的改变,若无信号改变,说明它们是配型相合,否则为配型不相合。运用该技术对10例血小板抗体阳性患者选择配合的血小板进行输注,并进行临床跟综分析,研究患者临床血小板输注的有效性。
12、SPR芯片技术检测CITP患者PBMC培养液上清细胞因子的水平
取上述冻存PBMC培养上清,采用SPR芯片技术检测上清中IFN-y、IL-17、 IL-22、IL-10水平,具体操作是取出前述研制好的SPR新型芯片,活化处理后,分别将抗-IFN-γ、抗-IL-17A、抗-IL-22、抗-IL-10的抗体点样于芯上,固定60分钟后上机检测,同时点样PBS作为空白对照(由于该芯片的性能分析已在前面研究了,在此不再重复叙述)；上述细胞因子的标准品配制成不同的浓度,上机检测的信号响应值(RU)绘出它们相应的标准曲线,根据标准曲线可计算出相应的细胞因子的含量。
13、建立CITP特异性细胞因子-SPR芯片检测平台
该研究探索性研究,为CITP患者细胞因子的特异性检测进行摸索：先将CD4抗体固定在芯片上,捕获CD4+T细胞,芯片上刺激培养细胞,然后在芯片上动态检测细胞因子的变化,并与正常对照组比较分析,该方法的建立将有助于CITP患者的细胞免疫紊乱更准确特异的分析。我们将以检测CD4+T细胞分泌IFN-γ为例进行方法学研究。
14、统计学分析方法
各组实验结果均用x±s表示,采用SPSS16.0软件分析,完全随机设计的两本均数比较,采用两样本t检验；多样本均数间两两比较采用方差分析(ANOVA);两变量的相关程度用Spearman相关分析法；以P<0.05为差异有统计学意义。
研究结果
1、基于初步掌握SIP动力学基础上,我们充分利用表面引发聚合反应技术活性可控的优点,在纳米尺度上调控分子基质poly的相关参数(链密度、厚度),搭建了一个基于高分子poly(OEGMA-co-HEMA)的生物传感器基质平台,实现“零”蛋白非特异性吸附和较高蛋白固定能力的高度统一,解决了生物传感器的共性问题。
2、利用自组装单层技术在芯片表面组装PEG单层作为主链,其中利用稀释剂控制主链PEG的链密度；将芯片浸泡于环糊精溶液中,当足量的环糊精穿人PEG主链后,对线性PEG另一端进行封端；通过羧基化反应将环糊精的羟基基团转为羧基等活性基团,便于固定生物探针分子,成功构建了新型的SPR芯片环糊精表面。
3、实验结果表明,本项目研发的芯片具有很好的抗蛋白质非特异性吸附能力；但是,随着羧基化的程度的增加,基质的抗蛋白非特异性能力有所下降,主要由于羧基修饰后芯片表面带电荷所致。静电吸附一方面使得芯片出现非特异性吸附,但从另一个角度看来,静电吸附在固定蛋白(特别浓度比较低的蛋白溶液)起重要作用：有利于蛋白质在芯片表面富集,提高蛋白质的固定效率。
4、本项目采用模型蛋白人IgG/山羊抗人IgG系统验证两种芯片检测蛋白质相互作用的应用效果。实验结果表明：两种芯片均较高的固定蛋白质的能力,而且能够保持蛋白质生物活性,但动态的环糊精表面比poly(OEGMA-co-HEMA)基质的灵敏度更高,相同浓度的蛋白信号响应值更大。
5、FCM检测结果显示,与对照组比较,CITP患者外周血中CD19+B、 CD5+CD19+B细胞亚群的比例均升高,差异有统计学意义(P<0.05)；而Breg的比例降低,差异有统计学意义(P<0.05)。
6、ELISA法测定结果显示,与健康人对照组比较,CITP患者PBMC培养液相关细胞因子IL-10、TGF-β1的表达水平均降低,差异有统计学意义(P<0.05); CITP患者外周血中Breg的比例与PBMC培养液上清中IL-10的水平呈正相关(P<0.05),与TGF-β1水平无相关性(P>0.05)；调节性B细胞的变化与患者血小板抗体的水平呈负相关性(P<0.05)。
7、采用乙酸缓冲溶液稀释血小板,分别稀释5倍,10倍,20倍,50倍,100倍。结果表明随着点样的浓度增加检测分析物(含血小板抗体血清)信号值增加,稀释到10倍后,点样物的浓度对检测信号影响不明显；分别采用系列pH值(pH=4.0,4.5,5.0,5.5,6.0)的乙酸缓冲溶液作为点样液稀释血小板。通入分析物(含血小板抗体血清)结果表明在pH=4.5的乙酸缓冲溶液时具有较强的检测信号。在NaOH的缓冲溶液中,高分子链部分水解,导致基线往下漂移,而采用1：300稀释的磷酸基线没有回到原始基线水平即再生彻底。在采用pH=2.0的甘氨酸再生后基线基本回到初始值水平,而且再生后探针仍然保持较高的生物活性。
8、在同一张芯片上点样试剂血小板抗原与非特异性NS1抗原,其中点样一个为空白对照点,其余各点为样本检测点,将此点样好的芯片在SPR仪上分别检测血小板抗体阳性对照血清与NS1抗体阳性对照血清,结果表明SPR芯片能特异检测血小板抗体。对40例CITP患者,分别用SPR芯片技术、固相凝集法和MAIPA法检测,经McNemr检验,SPR芯片技术与固相凝集法比较(P>0.05),一致性为92.5%。SPR芯片技术与MAIPA法比较(P>0.05),SPR法的灵敏度为96.3%,特异性为92.3%,总一致性为95%,三种方法差异均无统计学意义。
9、将ABO同型的供者血小板样本,经洗涤后稀释,按照上述血小板点样的条件与方法,点样于SPR芯片上,然后根据前面的检测方法,分析患者的血清样本与供者机采血小板的相容性。我们对SPR技术检测的其中10位血小板抗体阳性患者,进行交叉配合试验,从多位血小板供者中选择配合型血小板输注,经过临床随访分析,其中8例患者1h血小板增加值CCI>7.5,24hCCI>4.5,为输注有效,并且病人状态良好,另外2例因有其它基础疾病。
10、FCM检测结果表明：CITP患者糖皮质激素治疗前,血小板计数均数为28×109/L,外周血Th1、Th17、Th22细胞百分率明显上升,调节性B细胞(Breg)的比例降低,与正常对照比较差别有统计学意义(P<0.05)；经过治疗4天后,血小板计数上升,均数为71×109/L, CITP患者外周血Thl、Th17、Th22细胞百分率明显下降,调节性B细胞(Breg)的比例上升,与正常对照组比较,它们均无显著性差异(P>0.05)。CITP患者外周血Th1、Th17、Th22细胞比例变化与血小板数成负相关性(P<0.05)；调节性B细胞(Breg)的比例变化与血小板数成正相关性(P<0.05)。
11、ELISA法测定结果显示,与正常对照组比较,CITP患者糖皮质激素治疗前外周血PBMC细胞培养上清液IFN-y、IL-17、IL-22水平升高,IL-10的表达水平均降低,差别有统计学意义(P<0.05)；糖皮质激素治疗后,与正常对照组比较,均无显著性差异；CITP患者Breg细胞的比例变化与PBMC培养上清中细胞因子IL-10表达水平成正相关(P<0.05),IL-10水平与IFN-γ、IL-17、IL-22水平也呈负相关性(P<0.05)。
12、SPR芯片技术检测显示：CITP患者PBMC培养液中分泌的相关细胞因子IL-10、IFN-γ、IL-17、IL-22水平,结果与ELISA法比较,无显著性差异(P>0.05)。
13、成功建立特异性细胞因子-SPR芯片检测平台：运用SPR新型表面化学蛋白芯片先捕获CD4+T细胞,再研究分析芯片上细胞的活性与纯度均大于90%,CD4细胞与IFN-y与相应抗体均有良好的特异性反应,动态分析它们在芯片表面培养刺激产生的IFN-y水平,检测到信号的时间比试管培养刺激的时间更短,特异性更好,该方法具有更进一探索研究的价值。
研究结论
1、利用表面引发聚合反应技术活性可控的优点,在纳米尺度上调控高分子的相关参数,搭建了一个基于Poly(OEGMA-co-HEMA)的SPR生物传感器的化学表面基质,实现了抗蛋白质非特异性吸附的效果,降低了背景噪音。
2、利用分子自组装技术构建了SPR芯片动态性环糊精表面,利用环糊精在聚乙二醇链上的自由转动和滑动的能力,实现动态型的生物传感器表面,从而提高了探针的空间自适配功能,提高了其空间的选择性,实现了动态型的生物传感器表面的制备,为蛋白分子的检测提供了新平台。
3、FCM法分析表明：CITP患者Breg细胞的比例下降,且与IL-10的水平呈正相关,与血小板抗体PAIgG的水平呈负相关性。该研究为CITP的发病机制提供了新的思路。
4、新型的SPR芯片技术高通量、快速、灵敏地检测CITP患者的血小板抗体的水平与血小板配型输注,经临床随访分析效果良好,表明新型SPR芯片技术可初步应用于CITP患者血小板抗体的检测与配型。
5、研究表明CITP患者Breg、Th1、Th17、Th22细胞及其相关细胞因子IL-10、IFN-γ、IL-17、IL-22分泌紊乱在CITP发病中发挥重要作用,该研究为CITP的临床诊断与疗效评价提供了新靶点。
6、新型SPR蛋白芯片技术可用于CITP患者细胞因子的联合检测,高通量、快速地分析其免疫紊乱的特征。建立了新型SPR芯片捕获CD4+T细胞、特异性分析细胞因子IFN-γ变化的平台,更有利于CITP I临床诊断的准确性,具有重要的研发价值。
7、该研究为CITP免疫紊乱的临床诊断与疗效评价提供了新靶点和新方法,新型环糊精表面SPR芯片在高通量、快速、特异、联合检测血小板抗体和细胞紊乱中具有重要意义。
Research background
Surface plasmon resonance (SPR) is a free labeled optical biosensor technology, which is more and more widely used in many fields today, such as life sciences, which do not need to lable the analyte and measure the kinetics of molecular biological activity in non-modified conditions, be suitable for the high throughput screening of bioactive molecules, especially for the small molecules, the trace analysis of the unknown materials and the clinical samples online.
Chronic idiopathic thrombocytopenic purpura(CITP) is a kind of common bleeding autoimmune disease, the incidence is on the rise in China, which seriously affect the life quality of the CITP patients, but the mechanism is unknown, it is difficult to find really specific and effective method for treatment. Many studies show that the pathogenic CD4+Th cell proliferation rising, apoptosis reducing, activity increasing and the Thl,Th17, Th22cell differentiation imbalance.Recently, a new regulatory B cells has been paid more and more attention and has been actively studied in the field of immunology. So far there are no related reports in CITP patients, we will study the role of Breg in immune disorders of patients with CITP, and to explore the relationship between Breg and CD4+Th cell subsets and platelet antibody, and we will look for one new ideas for the clinical diagnosis and treatment of CITP.
Therefore, we will construct a novel SPR chemistry chip, and explore the application of SPR technology in the detection of immune disorders for CITP patients. SPR technology has a very important significance for the choice of treatment strategy effective evaluation and prognosis and platelet compatibility.
Research purpose
To construct and optimize the novel chemical surface chip for SPR technology, and to establish the new detection method based on SPR technology, which is simple and rapid, high sensitivity and specificity. As the treatment effect for CITP patients is poor, whose pathogenesis is complex and unknown to the role of new regulatory B cells in autoimmune diseases, we will analyse the characters of the regulatory B cells in the peripheral blood of patients with CITP and its effect on CD4+Th cell immune disorder in this study. We plan to explore its application value in clinical diagnosis and treatment of CITP patients, which will provide a new target and a novel assay for the immune disorder of CITP.
Research contents
This study intends to carry out the following work:
1Construct a novel surface chemistry SPR protein chip, which is quick and simple with high sensitivity and specificity, and evaluate the methodological character.
2Analyse the changes of regulatory B cells and its correlation with platelet antibody in the peripheral blood of patients with CITP.
3Using the novel SPR protein chip, to establish a new assay with high throughput and free lable for platelet antibody screening and match in CITP patients, and to compare the characters with the MAIPA and SPA assays.
4Study the correlation between the changes of regulatory B cells and CD4+T cell and related cytokines in the peripheral blood of patients with CITP, to analyse the role of them for CITP immune disorder.
5Using the novel SPR protein chip, to establish a new assay with high throughput and free lable for detecting the special cytokines in CITP patients.
Research methods
1Preparation for poly (OEGMA-co-HEMA) matrix
Using binary self-assembly system to control the concentration of initiator surface. Specific operation process is as follows:ethanol as solvent, mixed mercaptan initiator and PEG mercaptan, a total concentration of1mM, the SPR chips treated with UV-ZONE are immersed in the mixed solution for15hours, then chips are taken out, washed with ethanol fully, blown dry with nitrogen. According to2,2'pyridine (tendency)12.5mg (0.8mM),2.62g OEGMA526(5mM), HEMA0.65g (5mM), dissolved them in5mL ultrapure water and5mL methanol. Add1mL CuCl2solution (0.04mM), nitrogen gas is piped in, eliminate oxygen for15min. Then slowly inject1mL ascorbic acid(AscA) solution (0.04mM), Cu (II) complexes are gradually turned into Cu (I) complexes, reaction solution is changed from blue to pink. Continue to anaerobic treatment for15min, polymerization reaction is processed in the glove box of nitrogen gas environment on the chip surface. Chip surface agent will trigger polymerization reaction on the chip surface at room temperature for8hours, then remove chips and terminate reaction, using methanol and ultrapure water clean them, blow dry with nitrogen for further analysis, such as elliptical polarization measurement of film thickness, etc. The hydroxyl activity is poor at Poly (OEGMA-co-HEMA) matrix chain end, which is difficult to couple reaction with the protein under mild conditions, so we must have functionalization for hydroxyl groups, such as functional hydroxy. Hydroxy functional experiment operation is as follows:the surface modified poly (OEGMA-co-HEMA) chips are soaked in DMF reaction solution with succinic anhydride (10mg/mL)and DMAP(15mg/mL), react under room tempture for12hours. Remove chips and wash them with DMF and ethanol fully, blow dry by nitrogen.
2Preparation for dynamic cyclodextrin surface
(1) Using self-assembly monolayer technique, PEG monolayer is assembled on chip surface as the main chain, we use thinner to control the density of PEG main chain, then soake the chips in cyclodextrin solution, when plenty of cyclodextrin went into the PEG main chain, stopping the linear PEG at the other end.(2)By hydroxylation reaction, cyclodextrin hydroxyl groups are turned into active carboxyl group, which is advantageous for the immobilized biological probe. Using system experimental parameters (chain density, chain length and carboxyl numbers) of SPR technology for their influence on biological detection performance, we optimize the best conditions.(3) Under the optimization experimental conditions, we have prepared for the novel sensors based on cyclodextrin molecule matrix, and applied the chip matrix to detect protein model.
3Characterization methods for SPR chips
(1)AFM characterization:Characterization of matrix morphology structure is analysed by atomic force microscope (AFM,Veeco,Dimension3100). The experiment is done by the contact model.(2) Elliptic polarization characterization:film thickness is measured by M-2000V spectral elliptic polarization measuring instrument (J.A.Woollam Co, inc). Measurement angle is65,70,65degrees, wavelength varies from400nm to800nm. From elliptic polarization meter data, we get the thickness of the film with a specific model, including SAMs and Poly (OEGMA) using Cauchy Model,(An, Bn)=(1.45,0.01) and (1.46,0.01), respectively. Sample thickness are measured as the average values for the three position and reported the results for x±s values.
4Performance test
(1)Anti-fouling effect test:According to the instrument manuals, carboxyl functional chip will be loaded, set the working temperature at25℃, flow velocity at2μL/s, ran in PBS buffer solution (pH=7.4), after a stable baseline appear, inject specific concentrations protein solution (PBS dilution), finally inject PBS buffer solution.(2)Protein detection ability test:According to the operational requirements, set parameters of instrument for sample and add30μL sample in384well plates, set temperature at25℃,50%humidity, protein sample will be pointed on the SPR chip surface by contacted way. After sample chip was placed in the box of25℃temperature and75%humidity, keep constant temperature and humidity for2hours, then install the protein chip in SPR system, ran in PBS solution (pH=7.4) and at2μ L/s flow velocity, after a stable baseline is seen, ethanol is run into the chip for7min, closed the extra active site, then a specific concentration protein solution(PBS dilution) is injected, finally glycine solution (lOmM, pH=2.0) can wash out the combination of proteins, so as to achieve the goal of regeneration.
5Collect specimens and separate PBMC from CITP patients
Extraction of5mL venous blood with heparin sodium anti-coagulation from CITP patients and control group, PBMC is separated by density gradient centrifugation assay, and adjusted in RPMI1640medium containing10%fetal bovine serum,PBMC density is1×109/L.1mL suspension cell is used for detecting CD4+T and Breg subsets with fluorescent antibody staining by FCM,2mL cell suspension is incubated in phorbolester PMA (final concentration50μg/L) and ionomycin (final concentration1μmol/L) at37℃overnight, then collecting the supernatant and saving them at-70℃for detection of cytokines.
6Determination of Thl, Th17,Th22subsets and Breg by FCM
Take five tubes for detection, respectively take the mononuclear cells using lymphocytes stratified separation, wash twice by PBS,106mononuclear cells for each tube, No.1tube as isotype controls, No.2tube including5μL FITC-CD4antibodies and5μL PE-IFN-y, No.3tube including5μL FITC-CD4antibody and5μL IL-17A antibody, No.4tube including5μL FITC-CD4antibody and5μL PE-IL-22antibody, No.5tube including CD5-FITC, CDld-PE, PerCP-CD19antibodies5u L, respectively. The specimen after thoroughly incorporated with antibody, incubated at room temperature away from light for1h. Take2mL cracking fluid for red blood cells to act for10mins, after the centrifugal supernatant is removed, then wash2times by PBS and detect them by FCM, the related analysis and comparison is done using the software.
7Detection of IFN-γ, IL-17, IL-22and IL-10cytokines by ELISA
Take out the PBMC culture supernatant, we detect IFN-y, IL-17, IL-22and IL-10levels by ELISA method, and do it according to the reagent operation manual, providing three holes for each sample and standard, read the OD value at450nm.
8The conditions for platelet immobilized on SPR chip surface
The influencing factors mainly are the ionic strength, pH value of solution and the concentration of the protein. In this study, under certain ionic strength, we dilute platelet antigen and point them on the novel SPR chips, respectively. According to not wasting samples, and making sure that the immobilized platelets reach a certain amounts, to determine the platelet antigen dilution degrees. At the same time, we choose the different pH value (4.0,4.0,4.5,5.5,6.0)10mM acetate buffer solution as stationary liquid for platelets, to explore the influence of pH value for platelet antigen, to find the optimum pH value. The principle of chip regeneration is to use a certain concentration of acid, alkali, or high ionic strength of solution to wash away the combination of antigen and antibody, so as to achieve the regeneration of the sensor chip. On the regenerating reagent, we try to adopt the10mM NaOH,1:300diluted phosphoric acid and pH=2.0glycine as regenerative reagent, finally to determine the best regenerative reagent types.
9Platelets immobilization on the SPR chip surface
Platelet antigens are immobilized:According to the explored conditions, amino coupling method is used to immobilize corresponding general-purpose platelet antigen on the SPR sensor chip surface for detecting the corresponding platelet antibody. Method as follows:(1) Activation for the SPR chip:First,0.3939g EDC (0.4M) and0.430g NHS (0.1M) were dissolved at room temperature, then make up10mL EDC/NHS activation fluid mixture, the SPR chip is activated in the activation fluid for30min.(2) Point platelet antigen:10%glycerol fluid is made in10mM acetate (pH=6.0) and glycerin, the universal platelet antigen is diluted for five times with it,0.5μL mixture is pointed on the chip surface, and immobilized at room temperature for1hour.(3) The surface of the chip is enclosed:after chip immobilization, which will be installed in the SPR analyzer, inject PBS solution to choose optimum resonance angle (debugging from minimum angle to maximum angle, then choose the optimum resonance angle between70and100), when baseline is stationary, treated for10min with closed fluid ethanolamine, enclosing unreacted activated surface, removing physical adsorption material and sealing fluid by injecting PBS, then testing the samples.
10The performance analysis of SPR technology
(1) Using the exploring conditions and the fixed SPR chips, to detect the positive control, negative control and patient samples serum correspondingly, analyse the stability, sensitivity and specificity of SPR technology, data from SPR analyzer are analysed using BIAevaluation software.(2) Comparative study:detect platelet antibody for40CITP patients using SPR technology,SPA assay and MAIPA assay, compare with the differences of three methods, and to analyze their performance.
11Application for platelet antibody detection by the novel SPR chips
For CITP patients, screen platelet antibody by SPR technology, and choose compatible platelets for transfusion. The known type O general platelets are immobilized on a chip (preparation for chip according to the above), to detect whether there are relevant antibody in CITP patients serum. Also the platelets supplied by blood center are washed and diluted, then0.5u L platelet is pointed on the chip, to detect SPR signal change in serum for platelet antibody positive patients, if no signal change, show that they are in matches, or matching statements do not agree. Using the SPR technology for10CITP patients with platelet antibody positive to choose cooperated platelets for infusion, and to analyze the clinical effectiveness of platelet transfusion according to the long-term follow up.
12Application of cytokines detection by the novel SPR chip
(1) First method:take out the PBMC culture supernatant, using SPR chip technology to detect IFN-y, IL-17, IL-22and IL-10levels. The ways as follows: after SPR chip is activated, anti-IFN-y, anti-IL-17, anti-IL-22, anti-IL-10mAb are immobilized on the SPR chip for60minutes, respectively. At the same time pointing PBS as blank control (due to performance analysis of the chip has been ae studied), standard cytokines are diluted into different concentrations, detecting their corresponding signal value and make up standard curves, according to the standard curves, the concentrations of the corresponding cytokines can be calculated.(2) Second method:for exploratory research, for detecting the specific cytokines in CITP patient, First the anti-CD4antibody is immobilized on the chip, capturing CD4+T cells, then culture cells on the chip, and have the dynamic detection of cytokines, and comparing with normal control group, this method will help to analyse the cellular immunity disorders for CITP patients more accurately. We will study to detect IFN-gama from CD4+T secretion as an example.
13Statistical processing
Experimental results show as x±s, analysed by the SPSS16.0software, using two samples t test, muti-samples ANOVA test and spearman correlation analysis for the relation degree between the two variables, the difference was statistically significan with P<0.05.
Research results
1The construction of novel SPR matrix:For the preliminary master in SIP dynamics, we made full use of the controllable polymerization technology, and adapt the poly matrix related parameters at the nanoscale molecular, built a cyclodextrin matrix platform and Poly(OEGMA-co-HEMA) matrix, and achieved a high unity between the "zero" protein nonspecific adsorption and the high protein fixed ability, solved the common problems of biological sensors.
2(1)For the novel chip, protein nonspecific adsorption amount is lower than the detection sensitivity of SPR. The experimental results showed that the objective of the research and development of the chip had good ability to resist protein nonspecific adsorption.(2)This project adapt the model protein IgG/goat anti-human IgG system to verify application effect for two kinds of chips in the detection of protein interactions. The experimental results showed that both chips had the ability of high immobilization protein, and can maintain protein biological activity,but the cyclodextrin matrix platform was more sensitive than the Poly (OEGMA-co-HEMA) matrix.
3FCM results showed that the proportion of peripheral blood CD19+B, CD5+CD19+B cells were increased in CITP patients compared with control group, the difference was statistically significant (P<0.05), but the proportion of CD1d (hi)CD5+CD19+B(Breg) was reduced, the difference was statistically significant (P<0.05).
4ELISA results showed that, compared with healthy controls, the expression levels of IL-10and TGF-betal from PBMC cultures supernatan were reduced in CITP patients, the difference was statistically significant (P<0.05). The proportions of Breg positively correlated with IL-10levels from PBMC culture supernatant (P<0.05), but the proportions of Breg were of no correlation with TGF-betal level (P>0.05). The changes of regulatory B cells had negative correlation with the changes of platelet related antibody(PAIgG) in CITP patients.
5Before glucocorticoid treatment in CITP patients, the platelet counts were (5-37) x109/L, mean count was28x109/L, the percentage of peripheral blood Thl, Th17, Th22subsets increased obviously, the difference was statistically significant compared with normal controls (P<0.05). The proportion of regulatory B cells (Breg) was reduced, the difference was statistically significant (P<0.05). After glucocorticoid treatment for four days, the platelet counts rised, mean counts were71×109/L, compared with normal control group, the percentages of peripheral blood Thl, Thl7, Th22subsets decreased obviously, they had no significant difference (P>0.05). The percentages of Thl, Th17, Th22subsets had negative correlations with the platelet counts (P<0.05), but the proportion of Breg had positive correlation with the platelet counts (P<0.05).
6According to the ELISA results, compared with normal control group, before glucocorticoid therapy in CITP patients, IFN-y, IL-17, IL-22levels from PBMC culture supernatant rised, the expression of IL-10level was lower, the difference was statistically significant (P<0.05), but after glucocorticoid treatment, compared with normal control group, there was no significant difference. The ratio of Breg cells in CITP patients showed positive correlation with the change of IL-10level in PBMC culture supernatant(P<0.05), the changes between IL-10and IFN-y, IL-17, IL-22levels showed a negative correlation,respectively (P<0.05).
7Detection conditions for platelet anibody by the novel SPR chips in CITP patients:(1) Platelet diluted times:Using acetic acid buffer solution to dilute platelets, respectively for5times,10times,20times,50times,100times, results showed that with the concentration of pointed analyte increasing (including platelet antibody in serum) signal value increased, after20times dilution, SPR detection signal is not obvious, so10times dilution was pointed concentration for sample platelets.(2) Series of pH value (pH=4.0,4.5,5.0,5.5,6.0) acetic acid buffer solution were used as platelets pointing sample dilution, respectively, the results showed that the acetic acid buffer solution at pH=4.5had more strong signal.(3) In NaOH buffer solution, partially hydrolyzed polymer chain, lead to the baseline drift down, but using1:300diluted phosphoric acid, baseline did not return to the original baseline. Using the pH=2.0glycine regeneration, baseline return to the initial value, after regeneration the probe still maintained high biological activity.
8Detection limit for platelet antibody:Platelet reagent antigen was pointed on the same chip, and selected the corresponding seven areas, one for blank control point, the other6points for testing, standard controls were taken from positive serum5,10,20,30,40,60uL, the corresponding concentration was50ng/ml,100ng/ml,200 ng/ml,300ng/ml,400ng/ml,600ng/ml, respectively, at2u L/s the velocity of sample testing, processing by the BIAevaluation analysis software, results showed that had good sensitivity and minimum detection limit was about50ng/ml.
9Platelet antibody specific detection:reagent platelet antigen and nonspecific NS1antigen were pointed on the same chip, one spot for blank control, the other each point for sample testing, using platelet antibody positive controls and NS1antibody positive serum, respectively, results showed that, when platelet antibody positive controls run into the SPR chips, strong signal was seen at the platelet antigen spot, but had weak signal at nonspecific NS1antigen spot and control spot. This study suggests that the chip specificity is better, the novel SPR chip can detect specific platelet antibody.
10Platelet antibody screening was analysed by SPR chip technology,SPA assay and MAIPA method for40CITP patients, test results were shown that, by McNemr test, there was no statistically significant difference between SPR technology and SPA assay (P>0.05). There was also no statistically significant difference between SPR technology and MAIPA assay (P>0.05), the sensitivity, specificity and total consistency for SPR method were91%,97.9%,97.2%, respectively. In the40CITP patients,26of them platelet antibody were positive by SPR assay. Of the10platelet antibody positive CITP patients, by SPR technology, we choose compatible platelets transfusion from a number of donor platelet, after clinical follow-up analysis, of which8cases of patients, the platelet added value1h CCI>7.5,24h CCI>4.5, got effective infusion and they were in good condition, the other2cases were invalid for other basic diseases.
11Results for detection of related cytokines by the novel SPR chip in CITP patients:cytokines IL-10, IFN-y, IL-17, IL-22levels form PBMC culture fluid in CITP patients were detected by the novel SPR chip, the results showed that, compared with ELISA, there were no significant difference(P>0.05), but we can assayed all the cytokines in one time test, which was more simple and convenient.
12Another application of SPR model for specific cytokines detection, CD4cells were captured on the surface of SPR protein chip, and then we analysed cytokines.The activity and purity of CD4+T cells are more than90%, by the dynamic analysis method, IFN-gama levels from CD4+T cells culture stimulation were detected on the surface of the chip, results showed tha the detected signal time was shorter than those of stimulation in vitro, and had better specificity.
Research conclusion
1This project has used controllable surface activity by polymerization technology to regulate the related parameters of polymer on nanometer scale, and built a novel chemistry surface SPR biosensor chip based on oligomeric ethylene glycol methyl methacrylate(Poly(OEGMA-co-HEMA)),which implemented the resistant protein nonspecific adsorption effect, and reduced background noise.
2The project has built another novel dynamic cyclodextrin SPR chip surface. Through the chemical modification of cyclodextrins in polyethylene glycol (PEG) chain on dextrin free rotation and sliding, freedomly taking advantage of cyclodextrins rotation and sliding ability in polyethylene glycol (PEG) chain, which realized the biosensor surface with the dynamic state, can improve the space adaptation and the space selectivity of the probe for detecting protein molecules.
3This project has shown that the proportion of Breg is reduced, the proportion of the Breg positively correlated with IL-10levels from PBMC culture supernatant, the changes of regulatory B cells and platelet antibody show a negative correlation in CITP patients, which may provide a new idea for pathognisis of CITP.
4We have established a new type of SPR microarray to analyse platelet antibody and the compatibility between the serum of CITP patients and platelets from donors, and to cross matching test. By clinical follow-up analysis, showing effective infusion and good conditions,which showed that the novel SPR technology can provide preliminary application for platelet antibody screening and matches.
5The percentages of peripheral blood Thl, Th17, Th22subsets increased obviously in CITP patients, and showed negative correlation with the platelet counts. The proportion of Breg is reduced, and show positive correlation with the platelet counts, IFN-y, IL-17, IL-22levels are reduced, IL-10levels are rised, but show a negative correlation between IL-10with IFN-y, IL-17, IL-22level, which may provide a new target for clinical diagnosis and treatment of CITP.
6This kind of novel SPR protein chip can be used to detect the cytokines from PBMC secretion, to study the CITP immune disorders with high-throughput and rapid analysis. Besides, a new special SPR chip platform was established for capturing CD4+T cells to analyse the related cytokine IFN-gama in dynamic detection, the real situation of immune disorders is more evident, which is better in favor of clinical diagnosis and treatment for CITP.
7This study provides a new target and the new detection method for clinical diagnosis and treatment in CITP immune disorders. This novel cyclodextrin chemical surface SPR chip has important significance in the detection of platelet antibody and the cytokines disorder for CITP patients with high-throughput and rapid analysis.
引文
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7. Jian-Cheng XU, Fang LI, et al. Polarization and apoptosis of T cell subsets in idiopathic thrombocytopenic purpura..Int J Lab Hematol.2007;29 (3):177-84.
8. Matsushita T, Horikawa M, Iwata Y, et al. Regulatory B cells (B10 cells) and regulatory T cells have independent roles in controlling experimental autoimmune encephalomyelitis initiation and late-phase immunopathogenesis.J Immunol. 2010;185(4):2240-52.
9. Maseda D, Smith SH, DiLillo DJ, et al. Regulatory B10 cells differentiate into antibody-secreting cells after transient IL-10 production in vivo. J Immunol. 2012;188(3):1036-48.
10. Matsushita T, Tedder TF. Identifying regulatory B cells (B10 cells) that produce IL-10 in mice.Methods Mol Biol.2011;677:99-111.
11. Bub CB, Martinelli BM, Avelino TM,et al. Platelet antibody detection by flow cytometry:an effective method to evaluate and give transfusional support in platelet refractoriness. Rev Bras Hematol Hemoter.2013;35(4):252-5.
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13. Seefeld TH, Zhou WJ, Corn RM. Rapid microarray detection of DNA and proteins in microliter volumes with surface plasmon resonance imaging measurements. Langmuir.2011;27(10):6534-40.
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26. Matharu Z, Bandodkar AJ, Sumana G, et al.Low density lipoprotein detection based on antibody immobilized self-assembled monolayer:investigations of kinetic and thermodynamic properties. J Phys Chem B.2009;113(43):14405-12.
27. Buhl A,Metzger JH,Heegaard NH,et al. Novel biosensor-based analytic device for the detection of anti-double stranded DNA antibodies[J].Clin Chem,2007;53(2):334-341.
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