CN113884670A - 一种免疫学检测的信号放大材料及其制备方法 - Google Patents
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Abstract
本发明提出了一种免疫学检测的信号放大材料,包括偶联至少两个葡聚糖的酪酰胺。对本发明的信号放大材料进行荧光或者生物素标记后可应用于ELISA、IHC,ICC,ISH和FCU检测方法中,可显著提高低丰度靶标的检测灵敏度,具有节约抗体用量,提高检测的效率的有益效果。
Description
技术领域
本发明涉及免疫学检测技术领域,尤其涉及一种用于免疫学检测的信号放大材料及其制备方法。
背景技术
免疫学检测是一种基于抗体与抗原特异性结合的一种实验技术,由于操作简单、效率高、特异性强等优势在基础研究和临床试验中被广泛应用。蛋白质作为细胞中的主要功能分子,其通过显微镜观察疑似病变组织的细胞形态和结构,可直接看出组织发生了怎样的病理变化;通过对生物体蛋白含量变化进行定性定量分析,有助于药物靶标的开发;多种方法结合能较准确地对疾病作出判断。但是对于细胞和组织中低丰度靶标,往往因为检测灵敏度低导致检测失败,传统的检测方法已经无法满足高灵敏度要求。
发明内容
有鉴于此,本发明提出了一种检测灵敏度高,适合低丰度靶标的信号放大材料。
本发明的技术方案是这样实现的:一方面,本发明提供了一种免疫学检测的信号放大材料,包括偶联至少两个葡聚糖的酪酰胺。
在以上技术方案的基础上,优选的,所述葡聚糖的分子量为40kDa-70kDa。
在以上技术方案的基础上,优选的,所述信号放大材料的制备方法包括如下步骤:
S1,配置氧化缓冲液,然后加入葡聚糖,搅拌溶解后得到葡聚糖溶液;
S2,将碘化钠加入葡聚糖溶液中,搅拌直至碘化钠溶解,在25-35℃避光条件下孵育20-40min;
S3,将酪胺溶于碳酸盐缓冲液,然后加入S2孵育后的葡聚糖溶液,25-35℃条件下反应1-3h,反应液经HPLC纯化后得到信号放大材料ploy-TYR。
在以上技术方案的基础上,优选的,所述氧化缓冲液为pH5-7的醋酸钠缓冲液。
在以上技术方案的基础上,优选的,所述葡聚糖:碘化钠:酪胺的质量比为(1-20):(3-5):(1-2)。
另一方面,本发明还提供了一种免疫学检测的信号放大材料在基于HRP试剂的检测方法中的应用。
最后,本发明还提供了一种免疫学检测的信号放大材料在IHC、IF、ICC和ISH检测方法中的应用。
在以上技术方案的基础上,优选的,所述信号放大材料采用荧光或者生物素进行标记。
本发明的一种免疫学检测的信号放大材料相对于现有技术具有以下有益效果:
(1)本发明的信号放大材料ploy-TYR,在酪酰胺上偶联了葡聚糖,葡聚糖作为酪酰胺载体,通过葡聚糖与酪酰胺的偶联,从而增强酪酰胺的活化效率,提高检测灵敏度。
(2)本发明的信号放大材料ploy-TYR,适用于基于HRP试剂的检测方法,并且与免疫学应用中常用的样品类型和荧光成像平台兼容。
(3)对ploy-TYR进行荧光或者生物素标记后可与传统的ELISA、IHC,ICC,ISH和FC应用程序结合使用,可显著提高低丰度靶标的检测灵敏度,具有节约抗体用量,提高检测的效率的优点。
(4)本发明的信号放大材料ploy-TYR应用于ELISA检测方法中,检测灵敏度可以提高60倍。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例二的IHC检测结果图;
图2为本发明实施例三的IF检测结果图。
具体实施方式
下面将结合本发明实施方式,对本发明实施方式中的技术方案进行清楚、完整地描述,显然,所描述的实施方式仅仅是本发明一部分实施方式,而不是全部的实施方式。基于本发明中的实施方式,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式,都属于本发明保护的范围。
葡聚糖是一种水溶性多糖,在临床上用作血浆体积扩张剂、外周血流促进剂等已有50多年的历史,具有体内相容性好、易于储存、价廉易得等优点。分子中含有大量羟基,容易与蛋白质直接或间接相连,制备高分子化合物,广泛用作药物、蛋白质、酶的载体。
酪胺信号放大技术(TSA)是20世纪90年代在传统酶促放大理论基础上发展起来一种新的信号放大技术。它的原理是在辣根过氧化物酶的催化下大量酪胺分子富集,在酪胺上标记荧光或者其它标记物如生物素等用于检测,可以提高。但是传统TSA只有一个分子酪胺,催化能力有限,本发明用葡聚糖作为载体的偶联物(poly-TYR)相比于传统TSA,能够大幅度地提高检测试剂的灵敏度,还可以降低抗体用量。
本发明的技术方案是这样实现的:本发明合成了一种用于免疫学检测的poly-TYR信号放大材料,所述ploy-TYR是偶联至少两个葡聚糖的酪酰胺,其结构式如下:
其中n为大于2的正整数;葡聚糖的分子量为40kDa-70kDa。
所述信号放大材料ploy-TYR的制备方法包括如下步骤:
S1,配置氧化缓冲液:所述氧化缓冲液为pH5.5,0.1mol/L醋酸钠缓冲液;
S2,称取0.5-10mg分子量为40kDa-70kDa的葡聚糖加入1mL氧化缓冲液中,搅拌溶解;
S3,称取2.1mg碘化钠,放入棕色瓶中,加入1mL葡聚糖溶液,轻轻旋转,直到粉末溶解(生成10mmol/L碘化钠),室温避光孵育30min;
S4,称取0.5-1mg酪胺溶于50mmol/L碳酸盐缓冲液,碳酸盐缓冲液pH9.6,包括0.015mol/L Na2CO3和0.035mol/L NaHCO3。
S5,将S3的葡聚糖溶液加入到S4的酪胺溶液中,室温反应2h。
S6,将S5得到的产物经HPLC纯化得到ploy-TYR。
本发明的信号放大材料ploy-TYR适用于基于HRP试剂的检测方法,并且与免疫学应用中常用的样品类型和荧光成像平台兼容。
将本发明的信号放大材料ploy-TYR进行荧光或者生物素标记后可与传统的IHC,ICC,ISH和FC应用程序结合使用,可显著提高检测灵敏度,也有利于节约抗体用量,提高检测的效率。
下面以Elisa检测、IHC检测和IF检测为例验证本发明信号放大材料ploy-TYR的功效。
下面实施例中的实验方法,如无特殊说明,均为常规方法,下述实施例中所用的实验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
实施例一
将本发明合成的ploy-TYR标记生物素后,用于Elisa检测。
以human IL-6蛋白为例,Elisa检测步骤如下:
S1,Ploy-TYR标记生物素:称取5.5mg生物素溶于1mL无菌纯水中,制得5.5mg/mL的溶剂;取13.5μL生物素缓慢加入到1mg ploy-TYR溶液中;室温下避光旋转反应1h。
S2,加入IL-6蛋白样品、生物素标记的Ploy-TYR和标准品到相应的酶标板微孔中,使样品或标准品中的IL-6和固相载体上的抗体结合,洗去未结合的杂蛋白和其他物质;无Ploy-TYR的对比例,在这步中不加Ploy-TYR。
S3,使已结合的IL-6蛋白与生物素标记的抗IL-6的抗体结合,洗去多余的生物素标记抗体;
S4,加入SABC,使SABC和生物素标记的抗体和IL-6蛋白形成复合物,洗去多余的SABC;
S5,加入酶底物显色液,37℃孵育反应,终止酶反应后检测显色强度。
表1 常规未使用ploy-TYR的Elisa检测结果
表2 使用本发明ploy-TYR的Elisa检测结果
表1所示,低丰度蛋白不显色,无法检测出来,说明不使用ploy-TYR的常规ELISA检测方法无法对低丰度蛋白进行检测。
表2所示,≤5pg/mL的低丰度蛋白均可检测出来,表明使用ploy-TYR后,检测灵敏度得到很大的提升,与未使用ploy-TYR的对比例相比,灵敏度提高了60倍。
实施例二
将本发明的ploy-TYR标记生物素后,用于IHC检测,检测步骤如下:
S1,淋巴组织石蜡切片脱蜡至水:100%二甲苯浸泡3次,每次10min;无水乙醇浸泡3次,每次5min;蒸馏水洗5min;
S2,灭活内源性过氧化物酶:将切片浸入装有体积分数为3%的H2O2溶液中,盖上盖子,室温避光10min;
S3,蒸馏水洗:取出切片,用去离子水浸洗3次,每次1min;
S4,抗原修复:根据待检测的抗原,选择EDTA或柠檬酸抗原修复液;
抗原微波修复:用塑料切片架,置于染片缸内,EDTA修复液淹没切片,选择高档(功率100%)处理5min,取出室温放置2min,再放回微波炉低档(功率10%)处理5min,取出染片缸,保持切片浸没在修复液中,室温自然冷却约30min;取出切片,用PBS浸洗3次,每次1min;
S5,正常山羊血清封闭:从染片缸中取出切片,擦净切片背面水分及切片正面组织周围的水分(保持组织呈湿润状态),用组化笔沿着组织边缘画圈,滴加5%正常山羊血清处理,37℃封闭60min;
S6,滴加第一抗体:用滤纸吸去切片上多余的血清,直接滴加第一抗体小鼠抗h.il-6抗体,一抗工作液浓度为0.1-1μg/mL,4℃孵育过夜;
S7,滴加HRP酶联二抗:PBST浸洗切片3次,每次10min;清洗过后甩干切片,滴加HRP酶联羊抗鼠二抗,二抗工作浓度为10μg/mL,37℃孵育45min;
S8,滴加生物素标记的ploy-TYR:PBST浸洗切片3次,每次10min;滴加生物素标记的ploy-TYR 1-2滴,室温孵育15min。无ploy-TYR的对比例,不滴加生物素标记的ploy-TYR;
S9,滴加SABC:PBST浸洗切片3次,每次5min;滴加SABC,37℃孵育30min。使用ploy-TYR的实施例,不添加SABC;无ploy-TYR的对比例,添加SABC;
S10,加DAB显色:PBST浸洗切片3次,每次10min;清洗过后甩干切片,滴加适量DAB溶液,镜下观察,2-5min后迅速终止(去离子水冲洗干净终止),DAB工作液现配现用;
S11,复染:滴加一滴苏木素,室温复染5min,用去离子水冲洗干净,然后在PBS溶液中浸泡5-10min;
S12,脱水:去离子水浸洗切片3次,每次1min;用吹风机将切片吹干;
S13,封片和成像:中性树胶封片,显微镜观察结果,采集图像并分析。
图1结果表明:图1在免疫组化上检测低丰度IL-6蛋白,在淋巴结中的对比结果,其中兔抗人IL-6抗体用量:a和e为1μg/mL;b和f为0.5μg/mL;c和g为0.25μg/mL;d和h为0.1μg/mL。abcd组采用ploy-TYR系统,edgh组采用SABC系统。通过染色结果看,同等抗体用量的情况下,abcd组的染色较深,表现出较强的阳性;edgh组染色较浅,阳性结果不明显。也说明采用ploy-TYR的abcd组的灵敏度较高,即使在0.1μg/mL抗体用量的情况下,也表现出较深的染色效果,说明使用本发明的ploy-TYR具有节约抗体的有益效果。
实施例三
将本发明的ploy-TYR标记FITC后用于IF检测,检测步骤如下:
S1,样本前处理
S11,弃去培养基,在hela细胞上缓慢加入常温TBS,清洗2次,每次5s;
S12,细胞固定:在细胞上覆盖4%中性甲醛固定液(TBS缓冲液配制),置于4℃,固定15min;固定液需足量;
S13,固定液的清洗:去除固定液,使用4℃预冷的TBS缓冲液漂洗3次细胞,每次5min;
S14,通透(可选):加入TBS缓冲液配制的0.3%Triton X-100室温孵育10min,TBS漂洗3次,每次5min。
S2,染色步骤
S21,封闭:用5%空白山羊血清将样本完全覆盖,切片需放置于湿盒内,细胞孔板可直接将孔板密封好,置于37℃恒温恒湿培养箱孵育30min;
S22,一抗孵育:去除封闭液,直接在样本上滴加TBS缓冲液配制的一抗小鼠抗h.il-6抗体工作液,样本需完全覆盖,切片需放置于湿盒内,细胞孔板可直接将孔板密封好,置于4℃孵育过夜;
S23,复温:将样本置于常温,复温15min,去除抗体工作液,用缓冲液TBST洗涤1次,5min;用缓冲液TBS洗涤3次,每次5min;
S24,二抗孵育:在样本上滴加与一抗种属对应的HRP二抗羊抗鼠工作液,样本需完全覆盖,避光,37℃,孵育1小时;去除二抗工作液,用缓冲液TBST洗涤1次,5min;用缓冲液TBS洗涤3次,每次5min;
S25,信号放大:在样本上滴加荧光标记的ploy-TYR,室温反应15min,用缓冲液TBS洗涤3次,每次5min。无ploy-TYR的对比,不滴加荧光标记ploy-TYR;
S26,SABC孵育:在样本上滴加SABC,室温反应30min,用缓冲液TBS洗涤3次,每次5min;
S27,染核:在样本上滴加DAPI工作液,室温避光孵育10min;去除DAPI工作液,用缓冲液TBST洗涤1次,5min;用缓冲液TBS洗涤3次,每次5min;DAPI溶液采用0.01mol/L pH7.2TBS缓冲液配制;
S28,细胞孔板可直接加入抗荧光衰减封片剂后,于荧光显微镜下观察并采集图像;细胞涂片滴加抗荧光衰减封片剂,然后加盖盖玻片封片,再于荧光显微镜下观察并采集图像;细胞爬片可取出,盖在滴加抗荧光衰减封片剂的载玻片上后,于荧光显微镜下观察并采集图像。
结果见图2,图2中左图为未进行信号放大的检测图,右图为是使用本发明ploy-TYR的检测图,右图信号明显强于左图,说明本发明ploy-TYR可显著提高检测灵敏度,且不会降低图像分辨率或增加背景信号,适用于IF检测方法。
以上所述仅为本发明的较佳实施方式而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种免疫学检测的信号放大材料,其特征在于:包括偶联至少两个葡聚糖的酪酰胺。
2.如权利要求1所述的一种免疫学检测的信号放大材料,其特征在于:所述葡聚糖的分子量为40kDa-70kDa。
3.如权利要求1所述的一种免疫学检测的信号放大材料,其特征在于:所述信号放大材料的制备方法包括如下步骤:
S1,配置氧化缓冲液,然后加入葡聚糖,搅拌溶解后得到葡聚糖溶液;
S2,将碘化钠加入葡聚糖溶液中,搅拌直至碘化钠溶解,在25-35℃避光条件下孵育20-40min;
S3,将酪胺溶于碳酸盐缓冲液,然后加入S2孵育后的葡聚糖溶液,25-35℃条件下反应1-3h,反应液经HPLC纯化后得到信号放大材料ploy-TYR。
4.如权利要求3所述的一种免疫学检测的信号放大材料,其特征在于:所述氧化缓冲液为pH5-7的醋酸钠缓冲液。
5.如权利要求3所述的一种免疫学检测的信号放大材料,其特征在于:所述葡聚糖:碘化钠:酪胺的质量比为(1-20):(3-5):(1-2)。
6.如权利要求1所述的一种免疫学检测的信号放大材料在基于HRP试剂的检测方法中的应用。
7.如权利要求1所述的一种免疫学检测的信号放大材料在ELISA、IHC、IF、ICC和ISH检测方法中的应用。
8.如权利要求7所述的一种免疫学检测的信号放大材料在ELISA、IHC、IF、ICC和ISH检测方法中的应用,其特征在于,所述信号放大材料采用荧光或者生物素进行标记。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114736274A (zh) * | 2022-01-25 | 2022-07-12 | 伊莱瑞特(武汉)生物技术有限公司 | 新冠病毒s蛋白总抗体elisa检测试剂盒及其制备方法 |
CN116444424A (zh) * | 2023-06-16 | 2023-07-18 | 广东省大湾区华南理工大学聚集诱导发光高等研究院 | 一种基于聚集诱导发光的酪酰胺荧光材料、免疫组化染色试剂盒及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011059325A2 (en) * | 2009-11-11 | 2011-05-19 | University Of Twente, Institute For Biomedical Technology And Technical Medicine (Mira) | Dextran-hyaluronic acid based hydrogels |
CN104307040A (zh) * | 2014-07-22 | 2015-01-28 | 中国人民解放军第三军医大学第一附属医院 | 一种组织工程用具控释能力的注射式水凝胶及其应用 |
US20160131643A1 (en) * | 2012-09-24 | 2016-05-12 | Nestec S.A. | Microfluidic collaborative enzyme enhanced reactive ceer immunoassay |
CN111830250A (zh) * | 2020-08-04 | 2020-10-27 | 珠海市丽拓生物科技有限公司 | 一种信号放大的酶标二抗的制备方法 |
-
2021
- 2021-08-30 CN CN202111001758.3A patent/CN113884670A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011059325A2 (en) * | 2009-11-11 | 2011-05-19 | University Of Twente, Institute For Biomedical Technology And Technical Medicine (Mira) | Dextran-hyaluronic acid based hydrogels |
US20160131643A1 (en) * | 2012-09-24 | 2016-05-12 | Nestec S.A. | Microfluidic collaborative enzyme enhanced reactive ceer immunoassay |
CN104307040A (zh) * | 2014-07-22 | 2015-01-28 | 中国人民解放军第三军医大学第一附属医院 | 一种组织工程用具控释能力的注射式水凝胶及其应用 |
CN111830250A (zh) * | 2020-08-04 | 2020-10-27 | 珠海市丽拓生物科技有限公司 | 一种信号放大的酶标二抗的制备方法 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114736274A (zh) * | 2022-01-25 | 2022-07-12 | 伊莱瑞特(武汉)生物技术有限公司 | 新冠病毒s蛋白总抗体elisa检测试剂盒及其制备方法 |
CN116444424A (zh) * | 2023-06-16 | 2023-07-18 | 广东省大湾区华南理工大学聚集诱导发光高等研究院 | 一种基于聚集诱导发光的酪酰胺荧光材料、免疫组化染色试剂盒及其应用 |
CN116444424B (zh) * | 2023-06-16 | 2023-09-08 | 广东省大湾区华南理工大学聚集诱导发光高等研究院 | 一种基于聚集诱导发光的酪酰胺荧光材料、免疫组化染色试剂盒及其应用 |
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