CN113092757B - 一种肺癌肝转移早期诊断试剂盒及制备使用方法 - Google Patents
一种肺癌肝转移早期诊断试剂盒及制备使用方法 Download PDFInfo
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Abstract
本发明提供了一种肺癌肝转移早期诊断试剂盒及制备使用方法,涉及医学临床诊断领域,包括:抗体预包被酶标板、ELA2‑DNA标准品、样品稀释液、Anti‑DNA‑POD抗体、ABTS溶液、ABTS终止溶液和洗涤液,其中,抗体预包被酶标板由纯化抗人ELA2抗体包被制成;ELA2‑DNA标准品中含有多个肺癌肝转移患者血清;样品稀释液包括磷酸盐缓冲液和调节酸碱度的盐酸;洗涤液为PBST溶液。通过采集肺癌患者外周血标本,特异性检测血清中NETs成分蛋白弹性蛋白酶(ELA2)‑DNA含量,筛查患者是否发生肝转移,组分简单、易操作。
Description
技术领域
本发明涉及医学临床诊断领域,具体而言,涉及一种肺癌肝转移早期诊断试剂盒及制备使用方法。
背景技术
肺癌是一种发病率高、恶性程度高、预后差的恶性疾病,大部分患者被诊断患有肺癌时,往往已经到晚期,癌细胞发生了远处转移,这也是导致肺癌死亡率高的主要原因。肝脏是肺癌常见的转移部位,约有30%的肺癌患者会出现肝转移,且临床上肝转移早期常呈隐匿经过,检出肝转移对正确分期、制订合理治疗方案和判断预后具有重要意义。因此,建立一个准确、灵敏的检测方法,监测肺癌肝转移是亟待解决的问题。
目前诊断肺癌患者是否发生肝转移的手段主要有超声、CT及MRI等影像学检查,但由于转移瘤病灶分布及大小等,在临床工作中易被误诊、漏诊。另外,肝穿刺活检也可明确诊断肝转移瘤和寻找原发癌,但这种有创操作有引起腹腔内出血的可能。因此目前缺少一种准确、灵敏的诊断肺癌肝转移的方法。
发明内容
本发明旨在至少解决现有技术或相关技术中存在的技术问题之一,公开了一种肺癌肝转移早期诊断试剂盒及制备使用方法,基于中性粒细胞胞外诱捕网的理论基础,建立一种操作简便、特异性高的无创检测方法(试剂诊断),通过采集肺癌患者外周血标本,检测血清中弹性蛋白酶(ELA2)-DNA水平,筛查患者是否发生肝转移。
本发明的第一方面公开了一种肺癌肝转移早期诊断试剂盒,包括:抗体预包被酶标板、ELA2-DNA标准品、样品稀释液、Anti-DNA-POD抗体、ABTS溶液、ABTS终止溶液和洗涤液,其中,抗体预包被酶标板由纯化抗人ELA2抗体包被制成;ELA2-DNA标准品中含有多个肺癌肝转移患者混合血清;样品稀释液包括磷酸盐缓冲液和调节酸碱度的盐酸;洗涤液为PBST溶液。
根据本发明公开的肺癌肝转移早期诊断试剂盒,优选地,样品稀释液具体包括:NaCl、KCl、Na2HPO4和KH2PO4、HCl和双蒸水。
根据本发明公开的肺癌肝转移早期诊断试剂盒,优选地,ABTS溶液具体包括:ABTS二胺盐溶液和二硫酸钾溶液;ABTS终止溶液具体包括:十二烷基硫酸钠和去离子水;PBST溶液为含0.05%Tween20的1×磷酸盐缓冲液。
根据本发明公开的肺癌肝转移早期诊断试剂盒,优选地,预包被酶标板为96孔聚苯乙烯透明微孔板。
本发明的第二方面公开了一种肺癌肝转移早期诊断试剂盒的制备方法,包括:纯化抗人ELA2抗体用0.01mol/L pH 9.5的碳酸盐缓冲液稀释成5μg/ml,按每孔100μl的量包被ELISA微孔板,4℃过夜;
包被步骤完成后,去除ELISA微孔板内的包被液,按每孔160μl的量加入1%BSA封闭液,4℃封闭过夜;
封闭步骤完成后,去除ELISA微孔板内的封闭液后制成抗体预包被酶标板;
收集肺癌肝转移患者血液样本,在4℃温度条件下以3000rmp的速率离心10min,取上清,制成血清样本;
将血清样本进行5倍稀释,制成稀释样本;
清洗预包被酶标板,每孔加入100μl稀释样本,设置阴性孔,封板后在25℃温度条件下,以250rmp的速率震荡,孵育120min;
孵育完成后洗板并拍干;
将Anti-DNA-POD抗体用样品稀释液20倍稀释,加入反应孔,每孔100μl,封板后在25℃温度条件下,以300rmp的速率震荡,孵育120min;
反应完成后洗板并拍干;
每孔加入100μl ABTS溶液,设置空白孔,封板后在25℃温度条件下,以250rmp的速率震荡,孵育20~30min;
待显色均匀,在每孔加入100μl ABTS终止溶液终止反应,并于405nm处测定OD值;
根据ELA2-DNA含量数值,将多个肺癌肝转移患者的血清混合;
另取一酶标板,将纯化的1μg/ml的小牛胸腺DNA作为检测混合血清样本中游离DNA的标准品,使用TE缓冲液稀释1μg/ml小牛胸腺DNA,调整体积为50μl加入酶标板,再向每孔加入50μl的2×PicaGreen试剂,使终体系为100μl;
使用TE缓冲液将混合的血清样本分别做0倍稀释、5倍稀释、25倍稀释,向酶标板中每孔加入50μl的混合血清样本,再向每孔加入50μl的2×PicaGreen试剂,使终体系为100μl;
避光于室温下放置2min~5min,使用多功能酶标仪选择蓝色激发光,检测样品中游离DNA含量;
对小牛胸腺DNA浓度与OD值进行一元一次方程回归拟合标准曲线,回归方程的相关系数R2>0.9500;将混合血清样本的OD值代入标准曲线计算出混合血清样本中的游离DNA浓度,并调整为200ng/ml,制成ELA2-DNA标准品。
根据本发明公开的肺癌肝转移早期诊断试剂盒的制备方法,优选地,还包括:称取8g NaCl、0.2g KCl、1.44g Na2HPO4和0.24g KH2PO4,溶于900ml双蒸水中,用盐酸调pH值至7.4,加水定容至1L,121℃、0.1MPa高压灭菌20min后,制成样品稀释液,室温贮藏备用。
根据本发明公开的肺癌肝转移早期诊断试剂盒的制备方法,优选地,还包括:将7mmol/L ABTS二胺盐溶液和4.9mmol/L二硫酸钾(K2S2O8)溶液等体积混合,避光放置12h,用无水乙醇在分光光度计A734nm下调节OD值至0.7,制成ABTS溶液,避光储存备用。
根据本发明公开的肺癌肝转移早期诊断试剂盒的制备方法,优选地,还包括:将1g十二烷基硫酸钠(SDS)溶解在100ml去离子水中,制成ABTS终止溶液,储存备用。
根据本发明公开的肺癌肝转移早期诊断试剂盒的制备方法,优选地,还包括:配制含0.05%Tween20的1×磷酸盐缓冲液,制成洗涤液。
本发明的第三方面公开了一种肺癌肝转移早期诊断试剂盒的使用方法,包括:
取出抗体预包被酶标板恢复至室温,洗板拍干;
将标准品使用样品稀释液按浓度200ng/ml、100ng/ml、50ng/ml、25ng/ml、12.5ng/ml、6.25ng/ml、3.125ng/ml、0ng/ml进行稀释备用;
将血清样本使用样品稀释液进行5倍稀释备用;
抗体预包被酶标板中每孔加入100μl梯度浓度标准品或血清样本,设置阴性孔,封板后在25℃温度条件下,以250rmp的速率震荡,孵育120min;
洗板3次并拍干;
将Anti-DNA-POD抗体用样品稀释液20倍稀释,加入反应孔,每孔100μl,封板后在25℃温度条件下,以300rmp的速率震荡,孵育120min;
洗板3次并拍干;
每孔加入100μl ABTS溶液,设置空白孔,封板后在25℃温度条件下,以250rmp的速率震荡,孵育20min~30min;
待显色均匀后,每孔加入100μl ABTS终止溶液终止反应,并于405nm处测定OD值;
绘制标准曲线,样本中的ELA2-DNA含量通过对应OD值由标准曲线换算出相应的浓度数据;
根据浓度数据确定是否出现肺癌肝转移。
本发明的有益效果至少包括:基于中性粒细胞胞外诱捕网的理论基础,建立了一种操作简便、特异性高的无创检测方法,通过采集肺癌患者外周血标本,特异性检测血清中NETs成分蛋白弹性蛋白酶(ELA2)-DNA含量,筛查患者是否发生肝转移,组分简单、易操作。
附图说明
图1示出了根据本发明的一个实施例的检测混合血清样本中游离DNA的标准曲线。
图2示出了根据本发明的一个实施例的ELA2-DNA ELISA检测方法的标准曲线。
图3示出了根据本发明的一个实施例的肺癌患者不转移组和肝转移组中ELA2-DNA的浓度示意图。
图4示出了根据本发明的一个实施例的血清中ELA2-DNA含量预测肺癌肝转移的ROC曲线。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点,下面结合附图和具体实施方式对本发明进行进一步的详细描述。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是,本发明还可以采用其他不同于在此描述的其他方式来实施,因此,本发明并不限于下面公开的具体实施例的限制。
中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)是由DNA与组蛋白、髓过氧化物酶、ELA2、乳铁蛋白等形成的不规则网状结构复合物。当肺癌患者发生肝转移时,肝脏合成补体C3a,补体C3a能够与中性粒细胞上受体结合活化,促使中性粒细胞释放大量NETs到肝脏以及外周血中;肿瘤细胞通过表面受体CCDC25,与NETs高特异性和高亲和力结合,被NETs所捕获,从而转移到肝脏。
目前常用的液态样本中NETs的检测方法包括可溶性胞外游离DNA(cell-freeDNA,cfDNA)检测和NETs成分蛋白-DNA复合物检测,例如组蛋白-DNA、髓过氧化物酶-DNA、乳铁蛋白-DNA。
cfDNA检测:cfDNA是双链和高度碎裂的DNA,和组蛋白等作为NETs的关键组成部分,可以用来评估NETs水平。但是不能排除其他形式产生的cfDNA的干扰。技术手段为荧光染色法、免疫组化和共聚焦显微镜法。
NETs成分蛋白-DNA复合物检测:NETs包含DNA和组蛋白、髓过氧化物酶、ELA2、乳铁蛋白等蛋白质,故可检测蛋白-DNA复合物,从而特异性地反映样本中NETs的含量。现已有检测组蛋白-DNA、髓过氧化物酶-DNA、乳铁蛋白-DNA的理论报道,技术手段为ELISA检测法。
根据本发明的一个实施例,本发明的试剂盒组成为:抗体预包被酶标板、样品稀释液、标准品、Anti-DNA-POD(过氧化物酶)抗体、ABTS溶液、ABTS终止溶液、洗涤液。检测原理为:将抗人ELA2抗体包被在酶标板上;分别加入梯度稀释的标准品和预稀释的样本,标准品和样本中的人ELA2-DNA会与酶标板上的抗人ELA2抗体充分结合;洗板后加入Anti-DNA-POD抗体,该抗体会与ELA2-DNA复合物中的DNA发生特异性结合;洗板后加入ABTS溶液,该溶液是氧化物酶相关的ELISA反应显色底物,酶与酶底物反应最终产生一种水溶性的蓝绿色产物。加入ABTS终止溶液终止反应,在405nm处测定OD值。
试剂盒中标准品制备原理为:应用上述双抗体夹心ELISA方法,检测42例确诊肺癌肝转移患者血清ELA2-DNA复合物。筛选出其中检测OD值大于1.0的4例患者血清(即ELA2-DNA含量较高的血清)进行混合,应用PicaGreen特异性结合DNA染料,以1μg/ml的小牛胸腺DNA作为标准品进行绝对定量,测定混合血清中的cfDNA浓度。将血清中cfDNA的浓度等量于ELA2-DNA浓度,并调整混合血清浓度为200ng/ml,作为本次申请发明专利试剂盒中的ELA2-DNA标准品。
根据本发明的又一个实施例,提供了试剂盒制备方法:
1.抗体预包被酶标板的制备
1.1抗体的包被:用纯化抗人ELA2抗体包被96孔聚苯乙烯透明微孔板(ELISA微孔板)。抗人ELA2抗体用0.01mol/L pH 9.5的碳酸盐缓冲液(包被液)稀释成5μg/ml,按每孔100μl的量包被ELISA微孔板,4℃过夜。
1.2微孔板封闭:甩弃ELISA微孔板内的包被液,在吸水纸上轻轻拍干。按每孔160μl的量加入1%BSA封闭液,4℃封闭过夜。甩弃ELISA微孔板内的封闭液,在吸水纸上用力拍干后备用。
2.样品稀释液:配制1×磷酸盐缓冲液,称取8g NaCl、0.2g KCl、1.44g Na2HPO4和0.24g KH2PO4,溶于900ml双蒸水中,用盐酸调pH值至7.4,加水定容至1L,121℃、0.1MPa高压灭菌20min后,室温贮藏备用。
3.ABTS溶液:将7mmol/L ABTS二胺盐溶液和4.9mmol/L二硫酸钾(K2S2O8)溶液等体积混合,避光放置12h,用无水乙醇在分光光度计A734nm下调节OD值至0.7,避光储存备用。
4.ABTS终止溶液:配制1%SDS溶液,将1g十二烷基硫酸钠(SDS)溶解在100ml去离子水中,储存备用。
5.洗涤液:PBST溶液,即含0.05%Tween20的1×磷酸盐缓冲液。
6.标准品的制备
6.1样本制备:收集2019年7月至2021年1月承德医学院附属医院收治的42例肺癌肝转移患者血液样本到生化管中,4℃条件下3000rmp离心10min,取上清。将血清样品进行5倍稀释(即20μl的血清样本和80μl样品稀释液)备用。
6.2抗体预包被酶标板使用前洗板3次并拍干。
6.3加血清样本:抗体预包被酶标板中每孔加入100μl稀释样本,设置阴性孔(100μl样品稀释液),封板后25℃、震荡(250rmp)孵育120min。
6.4洗板3次并拍干。
6.5将Anti-DNA-POD抗体用样品稀释液20倍稀释,加入反应孔,每孔100μl,封板后25℃、震荡(300rmp)孵育120min。
6.6洗板3次并拍干。
6.7每孔加入100μl ABTS溶液,设置空白孔(100μl ABTS溶液),封板后25℃、震荡(250rmp)孵育20~30min。
6.8观察显色(蓝绿色)均匀即可每孔加入100μl ABTS终止溶液终止反应,并于405nm处测定OD值。
6.9根据实验结果,将多个ELA2-DNA含量较高的肺癌肝转移患者的血清混合,进行下一步实验。
6.10另取一酶标板。将纯化的1μg/ml的小牛胸腺DNA作为检测混合血清样本中游离DNA的标准品,使用TE缓冲液稀释1μg/ml小牛胸腺DNA,调整体积为50μl加入酶标板,(按照表1添加),再向每孔加入50μl的2×PicaGreen试剂,使终体系为100μl。
表1小牛胸腺DNA孔中的各成分:
6.11使用TE缓冲液将混合的血清样本分别做0倍稀释、5倍稀释、25倍稀释,向酶标板中每孔加入50μl的混合血清样本,再向每孔加入50μl的2×PicaGreen试剂,使终体系为100μl。
6.12避光于室温下放置2~5min,使用多功能酶标仪选择蓝色激发光(激发波长~480nm,发射波长~520nm)检测样品中游离DNA含量。
6.13对小牛胸腺DNA浓度与OD值进行一元一次方程回归拟合标准曲线(如图1所示),回归方程的相关系数R2=0.9995。将混合血清样本的OD值代入标准曲线计算出混合血清样本中的游离DNA浓度,并调整为200ng/ml。
将上述游离DNA浓度为200ng/ml的混合血清样本,作为本次申请发明专利试剂盒中的ELA2-DNA标准品。
根据本发明的又一个实施例,试剂盒使用方法包括:
1.实验前30min,拿出试剂盒,恢复至室温,加入标准品或样本前,洗板3次并拍干。
2.标准品准备:将标准品使用样品稀释液按浓度200ng/ml、100ng/ml、50ng/ml、25ng/ml、12.5ng/ml、6.25ng/ml、3.125ng/ml、0ng/ml进行稀释备用。
3.血清样本准备:将血清样本使用样品稀释液进行5倍稀释备用。
4.加样:抗体预包被酶标板中每孔加入100μl梯度浓度标准品或血清样本,设置阴性孔(100μl样品稀释液),封板后25℃、震荡(250rmp)孵育120min。
5.洗板3次并拍干。
6.将Anti-DNA-POD抗体用样品稀释液20倍稀释,加入反应孔,每孔100μl,封板后25℃、震荡(300rmp)孵育120min。
7.洗板3次并拍干。
8.每孔加入100μl ABTS溶液,设置空白孔(100μl ABTS溶液),封板后25℃、震荡(250rmp)孵育20~30min。
9.观察显色(蓝绿色)均匀即可每孔加入100μl ABTS终止溶液终止反应,并于405nm处测定OD值。
10.绘制标准曲线,样本中的ELA2-DNA含量可通过对应OD值由标准曲线换算出相应的浓度。
检测肺癌患者血清中ELA2-DNA的水平,当含量>93.431ng/ml时,即可初步诊断发生肺癌肝转移。
根据本发明的又一个实施例,还公开了使用试剂盒预测肺癌肝转移的实验数据:
实验材料:
样本来源和制备:血清样本收集于2019年7月至2021年1月收治的42例肺癌肝转移患者及62例不转移患者,急性重症感染的患者不纳入本研究。患者均已签署知情同意书。
实验过程:
参见上述实施例公开的试剂盒使用方法。
实验结果:
1.绘制标准曲线
以标准品ELA2-DNA梯度浓度为横坐标,以405nm处的OD值为纵坐标,拟合标准曲线,R2=0.9898,如图2所示。
2.肺癌患者不转移组和肝转移组中ELA2-DNA的含量
不转移组血清中ELA2-DNA含量为70.857±33.499ng/ml,转移组中ELA2-DNA含量为121.169±30.854ng/ml,差异具有统计学意义(t=-7.755,P<0.001),如图3所示。
3.血清ELA2-DNA含量对肺癌肝转移的预测价值
检测血清中ELA2-DNA含量,预测肺癌肝转移的ROC曲线下面积为0.8552(95%CI:0.784~0.926,P<0.001),如图4所示。截断值为93.431ng/ml,对应的灵敏度为90.476%,特异度为75.806%。
根据上述实施例,cfDNA检测试剂盒是基于cfDNA和组蛋白等作为NETs关键组成部分的理论基础,用来评估NETs水平,但是cfDNA检测试剂盒不能排除其他形式产生的cfDNA的干扰。与cfDNA检测试剂盒相比,本发明能够特异性检测NETs成分蛋白ELA2-DNA含量。其他NETs成分蛋白-DNA复合物检测目前只限于理论阶段,尚无商品化试剂盒,检测步骤复杂。本发明建立的ELA2-DNA检测试剂盒组分简单、易操作。本发明建立ELA2-DNA的ELISA试剂盒,采用双抗体夹心法,以抗人中性粒细胞ELA2抗体作为捕获抗体,加入偶联过氧化物酶的Anti-DNA抗体,准确、灵敏地反映患者血清中ELA2-DNA复合物含量,对肺癌患者肝转移进行早期诊断。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。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 (9)
1.一种肺癌肝转移早期诊断试剂盒,其特征在于,包括:抗体预包被酶标板、ELA2-DNA标准品、样品稀释液、Anti-DNA-POD抗体、ABTS溶液、ABTS终止溶液和洗涤液,其中,所述抗体预包被酶标板由纯化抗人ELA2抗体包被制成;所述ELA2-DNA标准品是多个肺癌肝转移患者的混合血清;所述样品稀释液包括磷酸盐缓冲液和调节酸碱度的盐酸;所述洗涤液为PBST溶液;
应用PicaGreen特异性结合DNA染料,以1μg/ml的小牛胸腺DNA作为标准品进行绝对定量,测定混合血清中的cfDNA浓度;将血清中cfDNA的浓度等量于ELA2-DNA浓度,并调整混合血清浓度为200ng/ml,作为ELA2-DNA标准品,所述cfDNA为游离DNA。
2.根据权利要求1所述的肺癌肝转移早期诊断试剂盒,其特征在于,所述样品稀释液具体包括:NaC1、KCl、Na2HPO4和KH2PO4、HCl和双蒸水。
3.根据权利要求1所述的肺癌肝转移早期诊断试剂盒,其特征在于,所述ABTS溶液具体包括:ABTS二胺盐溶液和二硫酸钾溶液;所述ABTS终止溶液具体包括:十二烷基硫酸钠和去离子水;所述PBST溶液为含0.05%Tween20的1×磷酸盐缓冲液。
4.根据权利要求1至3中任一项所述的肺癌肝转移早期诊断试剂盒,其特征在于,所述预包被酶标板为96孔聚苯乙烯透明微孔板。
5.一种肺癌肝转移早期诊断试剂盒的制备方法,其特征在于,包括:纯化抗人ELA2抗体用0.01mol/L pH 9.5的碳酸盐缓冲液稀释成5μg/ml,按每孔100μl的量包被ELISA微孔板,4℃过夜;包被步骤完成后,去除ELISA微孔板内的包被液,按每孔160μl的量加入1%BSA封闭液,4℃封闭过夜;封闭步骤完成后,去除所述ELISA微孔板内的封闭液后制成抗体预包被酶标板;收集肺癌肝转移患者血液样本,在4℃温度条件下以3000rmp的速率离心10min,取上清,制成血清样本;将所述血清样本进行5倍稀释,制成稀释样本;清洗所述预包被酶标板,每孔加入100μl所述稀释样本,设置阴性孔,封板后在25℃温度条件下,以250rmp的速率震荡,孵育120min;孵育完成后洗板并拍干;将Anti-DNA-POD抗体用样品稀释液20倍稀释,加入反应孔,每孔100μl,封板后在25℃温度条件下,以300rmp的速率震荡,孵育120min;反应完成后洗板并拍干;每孔加入100μlABTS溶液,设置空白孔,封板后在25℃温度条件下,以250rmp的速率震荡,孵育20~30min;待显色均匀,在每孔加入100μl ABTS终止溶液终止反应,并于405nm处测定OD值;根据ELA2-DNA含量数值,将多个肺癌肝转移患者的血清混合;另取一酶标板,将纯化的1μg/ml的小牛胸腺DNA作为检测混合血清样本中游离DNA的标准品,使用TE缓冲液稀释1μg/ml小牛胸腺DNA,调整体积为50μl加入酶标板,再向每孔加入50μl的2×PicaGreen试剂,使终体系为100μl;使用TE缓冲液将混合的血清样本分别做0倍稀释、5倍稀释、25倍稀释,向酶标板中每孔加入50μl的混合血清样本,再向每孔加入50μl的2×PicaGreen试剂,使终体系为100μl;避光于室温下放置2min~5min,使用多功能酶标仪选择蓝色激发光,检测样品中游离DNA含量;对小牛胸腺DNA浓度与OD值进行一元一次方程回归拟合标准曲线,回归方程的相关系数R2>0.9500;将混合血清样本的OD值代入标准曲线计算出混合血清样本中的游离DNA浓度,并调整为200ng/ml,制成ELA2-DNA标准品。
6.根据权利要求5所述的肺癌肝转移早期诊断试剂盒的制备方法,其特征在于,还包括:称取8g NaC1、0.2g KCl、1.44g Na2HPO4和0.24g KH2PO4,溶于900ml双蒸水中,用盐酸调pH值至7.4,加水定容至1L,121℃、0.1MPa高压灭菌20min后,制成样品稀释液,室温贮藏备用。
7.根据权利要求5所述的肺癌肝转移早期诊断试剂盒的制备方法,其特征在于,还包括:将7mmol/L ABTS二胺盐溶液和4.9mmol/L二硫酸钾K2S2O8溶液等体积混合,避光放置12h,用无水乙醇在分光光度计A734nm下调节OD值至0.7,制成ABTS溶液,避光储存备用。
8.根据权利要求5所述的肺癌肝转移早期诊断试剂盒的制备方法,其特征在于,还包括:将1g十二烷基硫酸钠SDS溶解在100ml去离子水中,制成ABTS终止溶液,储存备用。
9.根据权利要求5所述的肺癌肝转移早期诊断试剂盒的制备方法,其特征在于,还包括:配制含0.05%Tween20的1×磷酸盐缓冲液,制成洗涤液。
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