CN110596404A - 一种il-6生物素-链霉亲和素免疫层析检测卡 - Google Patents
一种il-6生物素-链霉亲和素免疫层析检测卡 Download PDFInfo
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Abstract
本发明公开了一种IL‑6生物素‑链霉亲和素免疫层析检测卡,属于疾病检测技术领域,包括PVC底板、样品垫、结合垫、包被膜和吸水纸,所述样品垫、结合垫、包被膜和吸水纸从前到后依次设置在PVC底板上,所述包被膜上设有检测T线和质控C线,所述结合垫上设有偶联示踪标记物的抗IL‑6单克隆抗体以及偶联生物素的抗IL‑6单克隆抗体;所述检测T线包被链霉亲和素,所述质控C线包被多克隆抗体。本发明依据双抗体夹心法原理,利用免疫层析技术定量检测人血清、血浆、全血样本中IL‑6的含量,可以准确获知样本中IL‑6的浓度,提高检测灵敏度,尤其是大大提高低端值的检测敏感度。
Description
技术领域
本发明属于疾病检测技术领域,具体地讲,是涉及一种IL-6生物素-链霉亲和素免疫层析检测卡。
背景技术
IL-6是一种炎症因子,是一种功能广泛的多效性细胞因子,机体受炎症刺激后由T细胞、B细胞、单核巨噬细胞及内皮细胞等分泌,是炎症介质网络的关键成分。炎症反应发生后,IL-6率先生成,产生后诱导产生CRP和降钙素原(PCT)生成。如在发生感染、内外伤、外科手术、应激反应、脑死亡、肿瘤产生以及其他情况的急性炎症反应过程中会快速产生。IL-6参与许多疾病的发生和发展,其血液水平与炎症、病毒感染、自身免疫疾病密切相关,它的变化比CRP更早。研究显示,细菌感染后IL-6迅速升高,PCT在2h后增加,而CRP在6h后才迅速增加。
免疫层析法是以胶体金或荧光作为示踪标记物应用于抗原抗体反应的一种免疫标记技术,可以对样本中的指定抗体或抗原进行定性或者定量分析。该方法操作简单,仪器价格不高,可以在医院广泛开展。但因为IL-6的检测灵敏度要求高,达到3pg/mL,当样本中被测物质浓度过低时,使用常规的免疫层析法可能会出现漏检的情况。
生物素-亲和素系统是一种非常有效的生物反应放大系统。生物素与亲和素之间具有高亲和力和多级放大效应,已成为广泛用于微量抗原、抗体定性、定量检测等研究的新技术。生物素-亲和素系统在实际应用中具有巨大的优越性,主要表现在以下几个方面:
1、生物素可与几乎所有生物大分子结合,应用广泛
活化生物素可以在蛋白质交联剂的介导下,与已知的几乎所有生物大分子偶联,包括蛋白质、核酸、多糖、脂类等。偶联后形成的生物素衍生物,不仅可保持生物大分子物质的原有生物活性,而且具有多价性。
交联是指将两个或多个分子通过共价键化学结合的过程。蛋白和多肽上有多个化学基团可用于交联,因此蛋白和多肽可以作为偶联的靶标,用于使用交联方法的研究中。修饰是指连接或裂解化学基团,以改变原始分子的溶解性或其他属性,如亲水性,疏水性等。不同的蛋白或相同的蛋白之间的交联是需要交联剂的。交联剂一般包含具有反应活性的末端,可与位于蛋白分子上的特定功能基团,如伯胺和巯基反应,或羧基与氨基之间形成酰胺键等。根据蛋白的不同基团活化偶联的作用,交联剂一般有氨基-氨基交联剂,巯基-糖类交联剂,巯基-巯基交联剂,羧基-氨基交联剂等。
交联剂用于确定蛋白间的关系以及配体-受体相互作用。具有氨基反应活性的同型双功能的交联剂:琥珀酰亚氨酯或亚氨酸酯、以及具有氨基反应活性和光活化叠氮苯化合物的异型双功能交联剂是这些应用中最常用的交联剂。
2、亲和力高,结合稳定
生物素与亲和素之间的作用是目前已知强度最高的非共价作用,亲和常数(K)为1015mol/L,比抗原与抗体间的亲和力(K=105-11mol/L)至少高1万倍。而且二者的结合稳定性好、专一性强,不受试剂浓度,pH环境,抑或蛋白变性剂等有机溶剂影响。
3、具有多级放大作用,灵敏度高
每个亲和素分子能结合4个分子的生物素,这一特点可以用于构建一个多层次信号放大系统。因此,具有多级放大作用,使其在应用时可极大地提高检测方法的灵敏度。
4、专一性高,非特异性干扰少
由于生物素与亲和素之间的结合具有极高的亲和力,且反应呈高度专一性。因此,BAS的多层次放大作用的提高灵敏度的同时,并不会增加非特异性干扰。而且BAS结合特性不会因反应试剂的高度稀释而受影响,使其有实际应用中可最大限度的降低反应试剂的非特异作用。
5、结合复合物解离常数小,呈不可逆反应性
亲和素结合生物素的亲和常数为抗原-抗体反应的百万倍,二者结合形成复合物的解离常数很小,呈不可逆反应性。而且酸、碱、变性剂、蛋白溶解酶以及有机溶剂均不影响其结合。因此BAS在实际应用中,产物的稳定性高,从而提高测定的精确度。
目前已有一些专利涉及生物素-链霉亲和素系统在免疫层析中的应用,比如专利CN205193076U《一种生物素-亲和素系统快速检测卡》,专利CN109374903A《一种超灵敏IL-6荧光免疫层析测定试剂盒及测定方法》,均采用生物素-亲和素放大系统来提高IL-6的灵敏度,实现方式为:结合垫中有链霉亲和素标记的荧光蛋白和生物素标记的抗人单克隆检测抗体,硝酸纤维素膜上包被抗人单克隆捕获抗体,其信号放大原理为:链霉亲和素标记的荧光蛋白和生物素标记的抗人单克隆检测抗体在反应前结合,抗原再与其中的抗人单克隆检测抗体结合,最后被抗人单克隆捕获抗体捕获,由于生物素与亲和素之间的强作用力,与其他免疫层析卡相比,相同量的抗人单克隆检测抗体结合更多的链霉亲和素标记的荧光蛋白,从而实现信号放大,但当抗原数量较少时,抗人单克隆捕获抗体捕获较难捕获结合物,使得其低端值的检测敏感度不高,且相同量的抗人单克隆检测抗体结合的链霉亲和素标记的荧光蛋白数量不定,虽能检出目标物,但检测结果准确度不高。
发明内容
本发明提供一种IL-6生物素-链霉亲和素免疫层析检测卡,以提高现有技术低端值检测敏感度不高、检测结果准确度不高的问题。
为了实现上述目的,本发明采用的技术方案如下:
一种IL-6生物素-链霉亲和素免疫层析检测卡,包括PVC底板、样品垫、结合垫、包被膜和吸水纸,所述样品垫、结合垫、包被膜和吸水纸从前到后依次设置在PVC底板上,所述包被膜上设有检测T线和质控C线,所述结合垫上设有偶联示踪标记物的抗IL-6单克隆抗体以及偶联生物素的抗IL-6单克隆抗体;所述检测T线包被链霉亲和素,所述质控C线包被多克隆抗体。
本发明依据双抗体夹心法原理,利用免疫层析技术定量检测人血清、血浆、全血样本中IL-6的含量。血清、血浆、全血样本因毛细管作用沿着试剂条向吸水纸端移动,当样品中有IL-6时,样品中的IL-6首先与结合垫中标记好的偶联示踪标记物的抗IL-6单克隆抗体和偶联生物素的抗IL-6单克隆抗体结合,形成双抗体夹心结构;然后继续移动与包被在硝酸纤维素膜上的修饰后的链霉亲和素结合,从而形成链霉亲和素-生物素-抗体-IL-6抗原-荧光微球抗体复合物。T线信号的强弱与样本中IL-6的含量成正比,因而可以准确获知样本中IL-6的浓度。
作为优选地,所述示踪标记物为胶体金、荧光素、荧光微球以及量子点的一种。
作为优选地,所述示踪标记物为荧光微球,示踪标记物偶联抗IL-6单克隆抗体的方法包括如下步骤:
(1)取荧光微球100uL,13000g离心15min去上清,加入pH值为6.0的0.05M MES缓冲液400uL,在超声波清洗仪中重悬荧光微球,13000g离心15min去上清;
(2)加入pH值为6.0的0.05M MES缓冲液200uL,再分别加入40uL NHS和16uL EDC溶液,浓度分别为0.5%和0.2%,混匀,再加入144uL pH值为6.0的0.05M MES缓冲液至总体积为400uL,超声混匀,在振荡器上室温反应1小时;
(3)活化完的微球13000g离心20min去上清;
(4)在离心管中加入400uL pH值为8.0的0.05M MES缓冲液,超声混匀洗涤,13000g离心15min去上清,再加入400uL pH值为8.0的0.05M MES缓冲液,超声重悬,加入0.1mg待标记的蛋白,室温震荡2小时;
(5)在偶联微球中加入50uL 10%BSA进行封闭,超声混匀,室温震荡2小时;
(6)将封闭好的微球10000g离心10min,去上清,沉淀用200uL微球保存液超声混匀,2~8℃保存待用。
作为优选地,所述生物素为长链水溶性生物素。
作为优选地,生物素偶联抗IL-6单克隆抗体的方法包括如下步骤:
(1)用分析天平称取1mg生物素,用0.01M pH值为7.4的PBS缓冲液溶解混匀;
(2)用0.01M pH值为7.4的PBS换成液将待标记抗体稀释至2mg/mL;
(3)将生物素溶液加入到待标记的抗体溶液中,迅速混匀,室温放置30min,生物素与待标记抗体的摩尔比为5:1~20:1;
(4)将标记好的蛋白放入透析袋,用0.01M pH值为7.4的PBS缓冲液透析多余的生物素,每2小时换一次透析液,透析三次,从透析袋中取出纯化好的蛋白,2~8℃保存待用。
作为优选地,所述结合垫的制备方法为:将偶联示踪标记物的IL-6单克隆抗体和偶联生物素的抗IL-6单克隆抗体的混合溶液喷至玻璃纤维载体上,放入37℃烘箱,干燥过夜即可制得结合垫;混合溶液喷至玻璃纤维载体上时,混合溶液喷出的出液量为1~5μL/cm,喷出速度100mm/s。
作为优选地,所述包被膜的制备方法为:用0.01M pH值为7.4的PBS缓冲液将羊抗鼠多克隆抗体稀释到0.8~1.2mg/mL,将链霉亲和素稀释到0.8~1.2mg/mL;在硝酸纤维素膜上进行包被划线,放入37℃烘箱,干燥过夜即可得包被膜;包被划线时,质控C线和检测T线包被时的出液量均为为0.8~1.2μL/cm,速度100mm/s。
作为优选地,所述链霉亲和素为经巯基化修饰的链霉亲和素。
作为优选地,所述样品垫的制备方法为:将样品垫浸泡在样品垫缓冲液中,1小时之后取出,放入37℃烘箱,干燥过夜即可得样品垫;其中,样品垫缓冲液是0.5%Tw-20的0.01M pH值为7.4的PBS缓冲液。
与现有技术相比,本发明具有以下有益效果:
(1)本发明提供的技术方案采用了生物素-链霉亲和素放大系统,提高了检测灵敏度,降低非特异结合,有利于提高检测卡性能,原理为:生物素标记捕获抗体、示踪标记物标记检测抗体和样品中的抗原三者在标记垫上形成复合物后,再通过生物素结合于链霉亲和素包被的硝酸纤维素膜上,由于链霉亲和素与生物素之间亲和力极强,并且具有高度的特异性和稳定性,生物素化抗体分子上连有多个生物素,因此,最终形成的抗体-抗原-生物素化抗体-亲和素复合物,可积聚大量示踪标记物,因而可提高检测灵敏度,尤其是低端值的检测敏感度大大提高;
(2)本发明提供的技术方案采用修饰后的链霉亲和素包被膜替代抗体包被硝酸纤维素膜,一方面增强了链霉亲和素的稳定性,另一方面改变了链霉亲和素的等电点而增强了其在硝酸纤维素膜上的吸附能力,从而有利于提高检测灵敏度,并可避免抗体包被硝酸纤维素膜时容易发生的不稳定现象;
(3)本发明提供的技术方案,示踪物标记的抗IL-6单克隆抗体和偶联生物素抗IL-6单克隆抗体同处一个结合垫,抗原可与两个抗体充分反应,形成三明治结构,再与包被于硝酸纤维素膜上的亲和素结合,有利于提高检测灵敏度;
(4)本发明提供的技术方案,选用长链水溶性生物素,消除了有机基质对反应的影响,同时延展了亲和素与链霉亲和素结合时的三维空间结构,有利于进一步提高检测灵敏度。
(5)本发明优先选择氨基-巯基交联剂,通过交联剂将链霉亲和素分子上的氨基和另一个链霉亲和素分子上的巯基连接在一起,从而形成多聚体。常用的氨基-巯基交联剂包括SPDP(N-琥珀酰亚胺3-(2-吡啶二硫代)丙酸酯)SMCC(4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯)等,这种交联剂一般为一种异-双官能试剂。而本发明优先选择SMCC作为偶联剂。
附图说明
图1为本发明提供的免疫层析检测卡的结构示意图;
图中标号分别为:1、PVC底板;2、吸水纸;3、包被膜;4、质控C线;5、检测T线;6、结合垫;7、样品垫。
具体实施方式
下面结合具体实施例的方式对本发明的权利要求做进一步的详细说明,但并不构成对本发明的任何限制,任何人在本发明权利要求范围内所做的有限次的修改,仍在本发明的权利要求范围之内。
如图1所示,为免疫层析检测卡的结构示意图,包括PVC底板1、样品垫7、结合垫6、包被膜3和吸水纸2,所述样品垫7、结合垫6、包被膜3和吸水纸2从前到后依次设置在PVC底板1上,所述包被膜上设有检测T线5和质控C线4。
以下结合具体实施例来说明本发明方案。
实施例1
制备常规的IL-6荧光免疫层析检测卡,制备过程如下:
1.包被膜3制备:用0.01M pH值为7.4的PBS缓冲液将羊抗鼠IgG抗体稀释到浓度为1.0mg/mL,制得质控C线包被液;将鼠抗人IL-6抗体稀释到浓度为1mg/mL,制得检测T线包被液。喷量为1.0uL/cm,划膜速度为100mm/s,将C线包被液和T线包被液包被于空白NC膜(硝酸纤维素膜)上,形成质控C线4和检测T线5,放入37℃烘箱,干燥过夜即制得包被膜3。
2.结合垫6制备:荧光微球清洗、活化后,按照每0.1mL荧光微球加入鼠抗人IL-6抗体0.1mg的抗体投量进行标记,封闭后保存于荧光稀释液。4.0μL/cm,喷制速度为100mm/s,将荧光微球偶联鼠抗人IL-6抗体喷于玻璃纤维素膜上。放入37℃烘箱,干燥过夜即得结合垫6。
3.样品垫7制备:将样品垫7浸泡在样品垫缓冲液中,1小时之后取出,放入37℃烘箱,干燥过夜即得样品垫7。
4.大板组装:将结合垫6、样品垫7、吸水纸2按照如图1所示的试剂条的结构顺序粘贴到已包被的包被膜3上:结合垫6贴在包被膜3的左端,并覆盖包被膜3左端1~2mm;将样品垫7贴在结合垫6的左端,并覆盖结合垫6左端1~2mm;将吸水纸2贴在包被膜3的右端,并覆盖包被膜3右端1~2mm,将包被膜3连同结合垫6、样品垫7、吸水纸2贴在PVC底板1上,使得样品垫7的左端与PVC底板1左端对齐,吸水纸2右端与PVC底板1右端对齐。
5.试剂卡生产:将步骤4制得的大板裁切成条,宽度为4.0±0.1mm。将试剂条放入塑料卡壳底的凹槽中,保证边缘整齐,扣上塑料卡上盖。将塑料卡放在平板压壳机的传送带上进行压壳。将1个试剂卡和1个干燥剂装入铝箔袋中。将装好试剂卡和干燥剂的铝箔袋用多功能薄膜封口机进行封口。
实施例2
制备IL-6生物素-链霉亲和素免疫层析检测卡,制备过程如下:
1.包被膜3的制备:用0.01M pH值为7.4的PBS缓冲液将羊抗鼠IgG抗体稀释到浓度为1.0mg/mL,制得质控C线包被液;将经巯基化修饰的链霉亲和素稀释到浓度为1mg/mL,制得检测T线包被液。喷量为1.0uL/cm,划膜速度为100mm/s,将C线包被液和T线包被液包被于空白NC膜上,形成质控C线4和检测T线5,放入37℃烘箱,干燥过夜即制得包被膜3。
2.结合垫6制备:将偶联荧光微球的IL-6单克隆抗体和偶联生物素的抗IL-6单克隆抗体的溶液喷至玻璃纤维载体上,放入37℃烘箱,干燥过夜即制得结合垫6。其中,混合溶液喷制的出液量4.0μL/cm,喷制速度为100mm/s;
偶联荧光微球的IL-6单克隆抗体的制备过程如下:
(1)取荧光微球100uL,13000g离心15min去上清,加入pH值为6.0的0.05M MES缓冲液400uL,在超声波清洗仪中重悬荧光微球,13000g离心15min去上清;
(2)加入pH值为6.0的0.05M MES缓冲液200uL,再分别加入40uL NHS和16uL EDC溶液,浓度分别为0.5%和0.2%,混匀,再加入144uL pH值为6.0的0.05M MES缓冲液至总体积为400uL,超声混匀,在振荡器上室温反应1小时;
(3)活化完的微球13000g离心20min去上清;
(4)在离心管中加入400uL pH值为8.0的0.05M MES缓冲液,超声混匀洗涤,13000g离心15min去上清,再加入400uL pH值为8.0的0.05M MES缓冲液,超声重悬,加入0.1mg待标记的蛋白,室温震荡2小时;
(5)在偶联微球中加入50uL 10%BSA进行封闭,超声混匀,室温震荡2小时;
(6)将封闭好的微球10000g离心10min,去上清,沉淀用200uL微球保存液超声混匀,2~8℃保存待用。
生物素偶联抗IL-6单克隆抗体的制备过程如下:
(1)用分析天平称取1mg长链水溶性生物素,用0.01M pH值为7.4的PBS缓冲液溶解混匀;
(2)用0.01M pH值为7.4的PBS缓冲液将待标记抗体稀释至2mg/mL;
(3)将生物素溶液加入到待标记的抗体溶液中,迅速混匀,室温放置30min,生物素与待标记抗体的摩尔比为5:1~20:1;
(4)将标记好的蛋白放入透析袋,用0.01M pH值为7.4的PBS缓冲液透析多余的生物素,每2小时换一次透析液,透析三次,从透析袋中取出纯化好的蛋白,2~8℃保存待用。
3.样品垫制备、大板组装和试剂卡制备过程同正常检测卡。
将实施例1和实施例2制得的免疫层析检测卡进行对比,对比试验如下:
将IL-6纯品用PBST进行稀释,浓度分别为4000pg/mL,2000pg/mL,1000pg/mL,500pg/mL,200pg/mL,100pg/mL,50pg/mL,20pg/mL,10pg/mL,5pg/mL,2pg/mL,0pg/mL。将稀释好的不同浓度的样本各取100ul样本分别加入两种试剂卡中,反应时间为10分钟。然后用时间分辨荧光免疫检测仪进行检测,获取荧光值,检测结果如表1。
表1免疫层析检测卡检测结果
从测试结果可以看出,正常IL-6荧光免疫层析检测卡检测0和2pg/mL的样本时,T值和T/C值差异均不明显;而应用生物素-链霉亲和素系统的IL-6荧光免疫层析检测卡检测0和2pg/mL的样本时,T值和T/C值差异显著,说明该技术在低值端有明显优势。综上所述,本发明提供的技术与传统荧光免疫层析法比较,提高了灵敏度和检出率,检测范围为2~4000pg/ml,且本底更低,性能得以明显提升。
上述实施例仅为本发明的优选实施方案之一,不应当用于限制本发明的保护范围,但凡在本发明的主题设计思想和精神上做出毫无意义的改动或润色,其所解决的技术问题仍然与本发明一致的,均应当包含在本发明的保护范围之内。
Claims (9)
1.一种IL-6生物素-链霉亲和素免疫层析检测卡,包括PVC底板、样品垫、结合垫、包被膜和吸水纸,所述样品垫、结合垫、包被膜和吸水纸从前到后依次设置在PVC底板上,所述包被膜上设有检测T线和质控C线,其特征在于,所述结合垫上设有偶联示踪标记物的抗IL-6单克隆抗体以及偶联生物素的抗IL-6单克隆抗体;所述检测T线包被链霉亲和素,所述质控C线包被多克隆抗体。
2.如权利要求1所述的IL-6生物素-链霉亲和素免疫层析检测卡,其特征在于,所述示踪标记物为胶体金、荧光素、荧光微球以及量子点的一种。
3.如权利要求1所述的IL-6生物素-链霉亲和素免疫层析检测卡,其特征在于,所述示踪标记物为荧光微球,示踪标记物偶联抗IL-6单克隆抗体的方法包括如下步骤:
(1)取荧光微球100uL,13000g离心15min去上清,加入pH值为6.0的0.05M MES缓冲液400uL,在超声波清洗仪中重悬荧光微球,13000g离心15min去上清;
(2)加入pH值为6.0的0.05M MES缓冲液200uL,再分别加入40uL NHS和16uL EDC溶液,浓度分别为0.5%和0.2%,混匀,再加入144uL pH值为6.0的0.05M MES缓冲液至总体积为400uL,超声混匀,在振荡器上室温反应1小时;
(3)活化完的微球13000g离心20min去上清;
(4)在离心管中加入400uL pH值为8.0的0.05M MES缓冲液,超声混匀洗涤,13000g离心15min去上清,再加入400uL pH值为8.0的0.05M MES缓冲液,超声重悬,加入0.1mg待标记的蛋白,室温震荡2小时;
(5)在偶联微球中加入50uL 10%BSA进行封闭,超声混匀,室温震荡2小时;
(6)将封闭好的微球10000g离心10min,去上清,沉淀用200uL微球保存液超声混匀,2~8℃保存待用。
4.如权利要求1所述的IL-6生物素-链霉亲和素免疫层析检测卡,其特征在于,所述生物素为长链水溶性生物素。
5.如权利要求1或4所述的IL-6生物素-链霉亲和素免疫层析检测卡,其特征在于,生物素偶联抗IL-6单克隆抗体的方法包括如下步骤:
(1)用分析天平称取1mg生物素,用0.01M pH值为7.4的PBS缓冲液溶解混匀;
(2)用0.01M pH值为7.4的PBS换成液将待标记抗体稀释至2mg/mL;
(3)将生物素溶液加入到待标记的抗体溶液中,迅速混匀,室温放置30min,生物素与待标记抗体的摩尔比为5:1~20:1;
(4)将标记好的蛋白放入透析袋,用0.01M pH值为7.4的PBS缓冲液透析多余的生物素,每2小时换一次透析液,透析三次,从透析袋中取出纯化好的蛋白,2~8℃保存待用。
6.如权利要求1或5所述的IL-6生物素-链霉亲和素免疫层析检测卡,其特征在于,所述结合垫的制备方法为:将偶联示踪标记物的IL-6单克隆抗体和偶联生物素的抗IL-6单克隆抗体的混合溶液喷至玻璃纤维载体上,放入37℃烘箱,干燥过夜即可制得结合垫;混合溶液喷至玻璃纤维载体上时,混合溶液喷出的出液量为1~5μL/cm,喷出速度100mm/s。
7.如权利要求1所述的IL-6生物素-链霉亲和素免疫层析检测卡,其特征在于,所述包被膜的制备方法为:用0.01M pH值为7.4的PBS缓冲液将羊抗鼠多克隆抗体稀释到0.8~1.2mg/mL,将链霉亲和素稀释到0.8~1.2mg/mL;在硝酸纤维素膜上进行包被划线,放入37℃烘箱,干燥过夜即可得包被膜;包被划线时,质控C线和检测T线包被时的出液量均为为0.8~1.2μL/cm,速度100mm/s。
8.如权利要求1或7所述的IL-6生物素-链霉亲和素免疫层析检测卡,其特征在于,所述链霉亲和素为经巯基化修饰的链霉亲和素。
9.如权利要求1所述的IL-6生物素-链霉亲和素免疫层析检测卡,其特征在于,所述样品垫的制备方法为:将样品垫浸泡在样品垫缓冲液中,1小时之后取出,放入37℃烘箱,干燥过夜即可得样品垫;其中,样品垫缓冲液是0.5%Tw-20的0.01M pH值为7.4的PBS缓冲液。
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