CN114113050A - 一种定量检测人趋化因子cx3cl1的化学发光试剂盒 - Google Patents
一种定量检测人趋化因子cx3cl1的化学发光试剂盒 Download PDFInfo
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- CN114113050A CN114113050A CN202111514210.9A CN202111514210A CN114113050A CN 114113050 A CN114113050 A CN 114113050A CN 202111514210 A CN202111514210 A CN 202111514210A CN 114113050 A CN114113050 A CN 114113050A
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- human chemokine
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Abstract
本发明公开了一种定量检测人趋化因子CX3CL1的化学发光试剂盒,包括:包被人趋化因子CX3CL1单克隆抗体的化学发光板,生物素标记的人趋化因子CX3CL1单克隆抗体或多克隆抗体,链霉亲和素标记的酶底物和鲁米诺促发光底物。本发明采用酶促化学发光法,利用化学发光板中的人趋化因子CX3CL1单克隆抗体作为捕获抗体,以生物素标记的人趋化因子CX3CL1单克隆抗体或多克隆抗体作为检测抗体,用鲁米诺酶底物催化发光,利用生物素‑链霉亲和素特异性结合,放大系统检测信号,提高了试剂盒的检测灵敏度和准确性,便于大通量样本检测,同时大大减少了样本使用量,具有高效、便捷的优点,适用于高通量检测人趋化因子CX3CL1。
Description
技术领域
本发明涉及免疫分析技术领域,具体涉及一种定量检测人趋化因子CX3CL1的化学发光试剂盒。
背景技术
趋化因子是由多种细胞分泌的分子量在8-14kDa的可溶性蛋白,目前报道的趋化因子家族有48个成员,根据其蛋白氨基酸序列N端的色氨酸保守性可以分为C、CC、CXC、CX3C四类。趋化因子受体是G蛋白偶联受体(GPCR),趋化因子与其受体结合后,可以激活腺苷酸环化酶、磷脂酶C等经典的GPCR信号通路,调控cAMP、Ca2+水平,还可激活MAPK、PI3K及一些酪氨酸激酶信号通路,在免疫趋化、血管再生、创伤愈合、自身免疫、肿瘤发生等过程中起着重要的作用。
CX3CL1是一种独特的趋化因子,为CX3C-类趋化因子家族中唯一的成员。在人类基因组中,CX3CL1位于16号染色体的长臂内(16q13),编码它的基因由3个外显子组成。与其他趋化因子不同,CX3CL1是一种跨膜蛋白,因此CX3CL1有两种存在形式:膜结合型(mCX3XL1)和分泌型(sCX3CL1),说明它在趋化因子大家族中具有特殊作用,这是迄今为止任何其他趋化因子都无法比拟的。sCX3CL1是由mCX3CL1近膜侧的一对碱基水解位点经蛋白酶的作用水解而来。sCX3CL1对表达CX3CR1的白细胞具有很强的趋化作用,而mCX3CL1则介导其与CX3CR1阳性细胞的黏附功能。因此,它兼有趋化因子和细胞间黏附分子的双重作用。CX3CL1参与多种生理和病理过程,在多种炎症性疾病、肿瘤中发挥着重要作用。
目前检测人趋化因子CX3CL1多采用酶联免疫分析或荧光免疫分析。市面上已有人趋化因子CX3CL1(Fractalkine)检测试剂盒(ELISA方法),用于体外定量分析人血清、血浆、唾液、尿液、母乳、细胞裂解液、细胞培养上清中CX3CL1的含量。其原理是预先包被的抗体和检测相抗体都是CX3CL1多克隆抗体。检测抗体经生物素标记。样品和生物素标记抗体先后加入酶标板孔反应后,PBS或TBS洗涤。随后加入过氧化物酶标记的亲和素反应;经过PBS或TBS的彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的CX3CL1呈正相关。该类试剂盒虽然具有灵敏度高、特异好的优点,但操作步骤繁琐,对于所需设备要求高,不适应大通量检测的需求。
化学发光免疫分析(CLIA)是继放射免疫分析、酶免疫分析和荧光免疫分析之后发展起来的一项新的免疫分析技术。大量的实验结果及临床应用资料显示,化学发光免疫分析技术具有灵敏度高、快速、准确、重复性好、效期长并安全无毒无污染等优点,成为取代放射免疫分析和酶联免疫分析技术的首选。目前未见采用酶促化学发光法定量检测CX3CL1的相关报道。
发明内容
针对现有技术存在的不足,本发明的目的在于提供一种检测灵敏度高、特异性好,操作简便,检测通量高,检测样本量少且不需要太多复杂的辅助设备和试剂的定量检测人趋化因子CX3CL1的化学发光试剂盒。
为解决上述技术问题,本发明提供以下技术方案:
一种定量检测人趋化因子CX3CL1的化学发光试剂盒,包括:包被人趋化因子CX3CL1单克隆抗体的化学发光板,生物素标记的人趋化因子CX3CL1单克隆抗体或多克隆抗体,链霉亲和素标记的酶底物和鲁米诺促发光底物。
优选地,所述化学发光板中,人趋化因子CX3CL1单克隆抗体的浓度为2-10μg/mL。
优选地,所述生物素标记的人趋化因子CX3CL1单克隆抗体或多克隆抗体由缓冲液稀释后得到工作液,二者稀释比例为1:500-1:5000。更优选地,二者稀释比例为1:2000。
优选地,所述链霉亲和素标记的酶底物由缓冲液稀释后得到工作液,二者稀释比例为1:500-1:15000。更优选地,二者稀释比例为1:1000。
其中,缓冲液将组分稀释至工作浓度,可起到确保蛋白物质的活性及效价稳定的作用。优选地,所述缓冲液包括但不限于磷酸盐缓冲液、HEPES缓冲液、MOPS缓冲液、Tris缓冲液、防腐剂或抑菌剂中的任一种或多种。
更优选地,所述防腐剂或抑菌剂包括但不限于酪蛋白、牛血清白蛋白、明胶、牛血清、聚乙二醇、Proclin300、四环素或叠氮钠中的任一种或多种。
优选地,所述链霉亲和素标记的酶底物包括但不限于链霉亲和素标记的辣根过氧化物酶或链霉亲和素标记的碱性磷酸酶。
优选地,所述鲁米诺促发光底物为辣根过氧化物酶或碱性磷酸酶的酶促发光底物。
优选地,所述化学发光试剂盒还包括人趋化因子CX3CL1标准品,为重组蛋白,其氨基酸序列如SEQ ID NO:1所示。
CX3CL1标准品氨基酸序列(SEQ ID NO:1):
MAPISLSWLLRLATFCHLTVLLAGQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAA GG MDESVVLEPEATGESSSLEPTPSSQEAQRALGTSPELPTGVTGSSGTRLPPTPKAQDGGPVGTELFRVPPVSTAATWQSSAPHQPGPSLWAEAKTSEAPSTQDPSTQASTASSPAPEENAPSEGQRVWGQGQSPRPENSLEREEMGPVPAHTDAFQDWGPGSMAHVSVVPVSSEGTPSREPVASGSWTPKAEEPIHATMDPQRLGVLITPVPDAQAATRRQAVGLLAFLGLLFCLGVAMFTYQSLQGCPRKMAGEMAEGLRYIPRSCGSNSYVLVPV
相对于现有技术,本发明具有以下有益效果:
(1)本发明采用酶促化学发光法,利用化学发光板中的人趋化因子CX3CL1单克隆抗体作为捕获抗体,以生物素标记的人趋化因子CX3CL1单克隆抗体或多克隆抗体作为检测抗体,用鲁米诺酶底物催化发光,利用生物素-链霉亲和素特异性结合,放大系统检测信号,提高了试剂盒的检测灵敏度和准确性,便于大通量样本检测,同时大大减少了样本使用量,具有高效、便捷的优点,适用于高通量检测人趋化因子CX3CL1。
(2)相比于市场现有的人趋化因子CX3CL1酶联免疫分析法(ELISA)检测试剂盒,本发明试剂盒检测灵敏度更高,特异性更强,操作简便,对于所需设备也无很高要求,可实现大通量检测,免疫诊断部分可实现半自动化或全自动化批量操作。
附图说明
图1为本发明的人趋化因子CX3CL1化学发光检测试剂盒的标准曲线。
图2为不同浓度PA刺激后LX-2培养上清中CX3CL1水平的变化情况。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例一 定量检测人趋化因子CX3CL1的化学发光检测试剂盒的制备
1、人趋化因子CX3CL1单克隆抗体包被化学发光板的制备
利用磷酸盐缓冲液或碳酸盐缓冲液将人趋化因子CX3CL1单克隆抗体(市售商品)稀释至2-10μg/mL,按照100μL/孔加入化学发光板中,并于4℃放置16-20小时后,取出化学发光板,甩干残液,利用如牛血清白蛋白、酪蛋白、明胶、多糖或聚糖、动物血清等进行封闭处理,按照250μL/孔加样,37℃放置2小时或4℃放置16-20小时,甩干残液,干燥保存于4℃备用。
2、生物素标记的人趋化因子CX3CL1单克隆抗体或人脂多糖结合蛋白(LBP)多克隆抗体的制备
利用0.1M PBS pH7.2配制10mM NHS-DPEG4-Biotin工作溶液。按照一定比例将人趋化因子CX3CL1单克隆抗体或多克隆抗体(市售商品)加入适量的10mM NHS-DPEG4-Biotin工作溶液中,于室温反应1小时,利用葡聚糖凝胶分离纯化或超速离心管14000rpm低温离心10-30min,去除游离生物素。加入0.1%BSA,放置4℃备用。购买商用生物素标记试剂盒操作也可。
3、生物素标记的人趋化因子CX3CL1单克隆抗体或多克隆抗体工作液的制备
(1)稀释液的制备
0.01M PB缓冲液(pH7.4),1%酪蛋白或1%BSA,0.1%proclin300。
(2)工作液的制备
用稀释液稀释生物素标记好的人趋化因子CX3CL1单克隆抗体或多克隆抗体后得到生物素标记的人趋化因子CX3CL1单克隆抗体或人脂多糖结合蛋白(LBP)多克隆抗体工作液,其稀释比例为:
人趋化因子CX3CL1单克隆抗体或多克隆抗体:稀释液=1:500-1:5000,本实施例中的稀释比例为1:2000。
4、链霉亲和素标记的辣根过氧化物酶或碱性磷酸酶
采用戊二醇法,将辣根过氧化物酶或碱性磷酸酶配置成50IU/mL,取适量,加入含1.25%戊二醛的pH6.8的PBS溶液中,混匀至室温下反应过夜。收集反应液,利用PBS(pH7.2)透析4次,取适量SA溶于1mol/L碳酸盐缓冲液(pH9.5)中,与透析后的反应液混匀,于4℃反应,并加入适量0.2mol/L赖氨酸溶液。混匀,室温反应2小时,利用0.05mol/L PBS(pH7.2)透析4次。离心取上清液,至4℃备用。
购买商用链霉亲和素标记试剂盒或直接购买链霉亲和素偶联后的HRP成品,操作也可。
5、链霉亲和素标记的辣根过氧化物酶或碱性磷酸酶工作液的制备
(1)稀释液的制备
0.1M TB缓冲液(pH7.4),1%酪蛋白或1%BSA,0.1%proclin300。
(2)工作液的制备
用稀释液稀释链霉亲和素标记的辣根过氧化物酶或碱性磷酸酶后得到链霉亲和素标记的辣根过氧化物酶工作液或链霉亲和素标记的碱性磷酸酶工作液,其稀释比例为:
链霉亲和素标记的辣根过氧化物酶或碱性磷酸酶:稀释液=1:500-1:15000,本实施例中的稀释比例为1:1000。
6、标准品的制备
人趋化因子CX3CL1标准品抗原为基因重组的全序列的人趋化因子CX3CL1(浓度≥95%)(Gene IDs:6376(Human))。
本发明试剂盒中涉及的人趋化因子CX3CL1标准品的稀释液为含0.5-1%BSA的HEPES或含0.5-1%BSA的PB缓冲液,内含0.1%proclin300。稀释浓度梯度为:0.156ng/mL、0.313ng/mL、0.625ng/mL、1.25ng/mL、2.5ng/mL、5ng/mL、10ng/mL,用于制作检测标准曲线。
7、洗涤液的制备
本发明试剂盒中洗涤液为0.01M PBST溶液。
配制1L的洗涤液:二水磷酸二氢钠9.75g、十二水磷酸氢二钠51g、氯化钠155g、吐温20 10mL,内含0.1%proclin300,使用时利用纯水20倍稀释成工作液。
实施例二 定量检测人趋化因子CX3CL1的化学发光检测试剂盒的使用方法
使用实施例一的试剂盒检测人趋化因子CX3CL1的方法,步骤如下:
1、按照说明书20倍稀释浓缩洗涤液至工作浓度;
2、按照50μL/孔取标准品梯度浓度和血清/血浆样本,加入对应板孔,做好标示;
3、取生物素标记的人趋化因子CX3CL1抗体工作液,按照50μL/孔加入对应板孔;
4、37℃孵育1h,取出,每孔250μL洗涤工作液,洗涤3次,拍干;
5、取链霉亲和素标记的过氧化物酶工作液或链霉亲和素标记的碱性磷酸酶工作液,按照50μL/孔加入对应反应孔,放置37℃反应30min;
6、取出发光板,每孔250μL洗涤液,洗涤5次,拍干;
7、每孔加入100μL鲁米诺发光底物,读取发光值;
8、分别取标准品梯度的浓度值作为x值和发光值作为y值,制作四参数拟合方程标准曲线,获得曲线方程;将待测样本的发光值带入标准曲线,计算出待测样本的浓度值。
实施例三 定量检测人趋化因子CX3CL1的化学发光检测试剂盒的方法学评价
1、标准曲线
本发明试剂盒用校准品浓度作为X值,对应浓度检测的发光值RLU做Y值,以此做四参数拟合方程。如图1所示,所绘制的标准曲线回归方程为y=11205x-93.202,相关系数r2=0.9994。
2、最低检出限
本发明试剂盒在检测标准曲线的同时,检测最低检出限参考品(0.156ng/mL)10次,并通过标准曲线计算最低检出参考品的平均浓度值(x)和标准差(SD)。根据公式:检出限=X±2SD,计算样品的最低检出限,结果如表1所示,本发明试剂盒对血样检测人趋化因子CX3CL1的最低检出限为0.131ng/mL。
表1 人趋化因子CX3CL1化学发光检测试剂盒的最低检出限
3、精密性
同上述第2部分的检测结果,本发明试剂盒利用标准差(SD)/平均浓度值(x)所获得的比值即为精密性值,即:0.024ng/mL÷0.179ng/mL×100%=13.4%,即精密性值<15%。
4、样本检测
(1)样本分组
利用棕榈酸(palmitic acid,PA)模拟高脂环境,刺激人星状细胞LX-2促使其活化,检测不同浓度棕榈酸(0μM,20μM,100μM,200μM,500μM)刺激后,LX-2培养上清中区趋化因子CX3CL1的变化情况。
(2)样本检测结果
利用制备的人趋化因子CX3CL1化学发光检测试剂盒,检测不同浓度PA刺激后LX-2培养上清中CX3CL1水平的变化情况。检测结果如表2和图2所示,随着PA浓度的增加,LX-2培养上清中CX3CL1水平也随之增加,当PA浓度为200μM时,CX3CL1的水平达到最高值,随后PA浓度增加,CX3CL1水平则下降。
表2 不同浓度PA刺激后LX-2培养上清中CX3CL1水平
综上,采用本发明试剂盒检测人趋化因子CX3CL1与目前市面上的ELISA检测试剂盒检测效果相比较,本发明试剂盒具有操作简便、操作灵敏度高、特异性好、检测成本低、样本使用量少,对于设备的要求低等的优点。
最后应当说明的是,以上内容仅用以说明本发明的技术方案,而非对本发明保护范围的限制,本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换,均不脱离本发明技术方案的实质和范围。
序列表
<110> 上海市肿瘤研究所
<120> 一种定量检测人趋化因子CX3CL1的化学发光试剂盒
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 397
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Pro Ile Ser Leu Ser Trp Leu Leu Arg Leu Ala Thr Phe Cys
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His Leu Thr Val Leu Leu Ala Gly Gln His His Gly Val Thr Lys Cys
20 25 30
Asn Ile Thr Cys Ser Lys Met Thr Ser Lys Ile Pro Val Ala Leu Leu
35 40 45
Ile His Tyr Gln Gln Asn Gln Ala Ser Cys Gly Lys Arg Ala Ile Ile
50 55 60
Leu Glu Thr Arg Gln His Arg Leu Phe Cys Ala Asp Pro Lys Glu Gln
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Trp Val Lys Asp Ala Met Gln His Leu Asp Arg Gln Ala Ala Ala Leu
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Thr Arg Asn Gly Gly Thr Phe Glu Lys Gln Ile Gly Glu Val Lys Pro
100 105 110
Arg Thr Thr Pro Ala Ala Gly Gly Met Asp Glu Ser Val Val Leu Glu
115 120 125
Pro Glu Ala Thr Gly Glu Ser Ser Ser Leu Glu Pro Thr Pro Ser Ser
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Gln Glu Ala Gln Arg Ala Leu Gly Thr Ser Pro Glu Leu Pro Thr Gly
145 150 155 160
Val Thr Gly Ser Ser Gly Thr Arg Leu Pro Pro Thr Pro Lys Ala Gln
165 170 175
Asp Gly Gly Pro Val Gly Thr Glu Leu Phe Arg Val Pro Pro Val Ser
180 185 190
Thr Ala Ala Thr Trp Gln Ser Ser Ala Pro His Gln Pro Gly Pro Ser
195 200 205
Leu Trp Ala Glu Ala Lys Thr Ser Glu Ala Pro Ser Thr Gln Asp Pro
210 215 220
Ser Thr Gln Ala Ser Thr Ala Ser Ser Pro Ala Pro Glu Glu Asn Ala
225 230 235 240
Pro Ser Glu Gly Gln Arg Val Trp Gly Gln Gly Gln Ser Pro Arg Pro
245 250 255
Glu Asn Ser Leu Glu Arg Glu Glu Met Gly Pro Val Pro Ala His Thr
260 265 270
Asp Ala Phe Gln Asp Trp Gly Pro Gly Ser Met Ala His Val Ser Val
275 280 285
Val Pro Val Ser Ser Glu Gly Thr Pro Ser Arg Glu Pro Val Ala Ser
290 295 300
Gly Ser Trp Thr Pro Lys Ala Glu Glu Pro Ile His Ala Thr Met Asp
305 310 315 320
Pro Gln Arg Leu Gly Val Leu Ile Thr Pro Val Pro Asp Ala Gln Ala
325 330 335
Ala Thr Arg Arg Gln Ala Val Gly Leu Leu Ala Phe Leu Gly Leu Leu
340 345 350
Phe Cys Leu Gly Val Ala Met Phe Thr Tyr Gln Ser Leu Gln Gly Cys
355 360 365
Pro Arg Lys Met Ala Gly Glu Met Ala Glu Gly Leu Arg Tyr Ile Pro
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Arg Ser Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val
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Claims (9)
1.一种定量检测人趋化因子CX3CL1的化学发光试剂盒,其特征在于,所述化学发光试剂盒包括:包被人趋化因子CX3CL1单克隆抗体的化学发光板,生物素标记的人趋化因子CX3CL1单克隆抗体或多克隆抗体,链霉亲和素标记的酶底物和鲁米诺促发光底物。
2.根据权利要求1所述的化学发光试剂盒,其特征在于,所述化学发光板中,人趋化因子CX3CL1单克隆抗体的浓度为2-10μg/mL。
3.根据权利要求1所述的化学发光试剂盒,其特征在于,所述生物素标记的人趋化因子CX3CL1单克隆抗体或多克隆抗体由缓冲液稀释后得到工作液,二者稀释比例为1:500-1:5000。
4.根据权利要求1所述的化学发光试剂盒,其特征在于,所述链霉亲和素标记的酶底物由缓冲液稀释后得到工作液,二者稀释比例为1:500-1:15000。
5.根据权利要求3或4所述的化学发光试剂盒,其特征在于,所述缓冲液为磷酸盐缓冲液、HEPES缓冲液、MOPS缓冲液、Tris缓冲液、防腐剂或抑菌剂中的任一种或多种。
6.根据权利要求5所述的化学发光试剂盒,其特征在于,所述防腐剂或抑菌剂为酪蛋白、牛血清白蛋白、明胶、牛血清、聚乙二醇、Proclin300、四环素或叠氮钠中的任一种或多种。
7.根据权利要求1所述的化学发光试剂盒,其特征在于,所述链霉亲和素标记的酶底物为链霉亲和素标记的辣根过氧化物酶或链霉亲和素标记的碱性磷酸酶。
8.根据权利要求7所述的化学发光试剂盒,其特征在于,所述鲁米诺促发光底物为辣根过氧化物酶或碱性磷酸酶的酶促发光底物。
9.根据权利要求1所述的化学发光试剂盒,其特征在于,所述化学发光试剂盒还包括人趋化因子CX3CL1标准品,其氨基酸序列如SEQ ID NO:1所示。
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