CN112433054A - C9蛋白检测试剂在制备肺癌筛查试剂盒中的用途 - Google Patents
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Abstract
本发明涉及体外诊断试剂领域,具体涉及C9蛋白检测试剂在制备肺癌筛查试剂盒中的用途。本发明首次发现肺癌患者血浆外泌体中C9蛋白的水平显著高于健康人群。本发明将检测C9蛋白的试剂用于制备肺癌筛查试剂盒,能够实现肺癌的有效筛查。
Description
技术领域
本发明涉及体外诊断试剂领域,具体涉及C9蛋白检测试剂在制备肺癌筛查试剂盒中的用途。
背景技术
肺癌是世界上最常见的恶性肿瘤之一,其发病率和死亡率呈逐年上升趋势,目前发病率居世界首位,严重威胁着人类健康和生命。
肺癌是一种善于隐匿的疾病,经常在疾病发展到晚期才表现出临床症状,70~80%的肺癌患者在诊断出患有肺癌症状时已是中、晚期,癌细胞已经扩散,错过了最佳治愈时机,五年生存率低。对于早期的肺癌患者,经过及时治疗可大大提高患者的5年及以上生存率和生存质量。因此肺癌的早期诊断和进行有效的筛查至关重要。
肺癌的筛查,是指对那些没有肺癌相关症状的人群进行常规体检,在出现症状前及时发现肺癌。如果可以找到血浆里面的肺癌分子标志物,用于提示临床医生早期对患者采取相关的治疗措施或者决策具有重要的意义。
C9蛋白(Uniprot编号:P02748)全称Complement component C9,C9蛋白是膜攻击复合物(membrane attack complex,MAC)的组成部分,而MAC的作用在先天性或适应性免疫反应中使靶细胞的质膜形成孔洞。
目前尚未见C9蛋白与肺癌相关的现有技术。
发明内容
本发明的目的在于提供一种新的类的肺癌标志物,以及该标志物的检测试剂在制备肺癌筛查试剂盒中的用途。
本发明的技术方案包括:
检测C9蛋白的试剂在制备肺癌筛查试剂盒中的用途。
如前述的用途,所述检测C9蛋白的试剂为酶联免疫吸附试验用试剂。
如前述的用途,所述检测C9蛋白的试剂为western blot试剂。
如前述的用途,所述检测C9蛋白的试剂为蛋白芯片检测方法用试剂。
如前述的用途,所述检测C9蛋白的试剂是检测人血浆外泌体中C9蛋白的试剂。
一种肺癌筛查试剂盒,它包括用于检测C9蛋白的试剂。
如前述的试剂盒,所述检测C9蛋白的试剂为酶联免疫吸附试验用试剂。
如前述的试剂盒,所述检测C9蛋白的试剂为western blot试剂。
如前述的试剂盒,所述检测C9蛋白的试剂为蛋白芯片检测方法用试剂。
如前述的试剂盒,所述检测C9蛋白的试剂是检测人血浆外泌体中C9蛋白的试剂。
本发明的关键在于,确定了人血浆外泌体中C9的含量与患肺癌的风险显著相关,因此可以通过检测人血浆外泌体中C9的含量来判断患肺癌的风险,至于具体检测人血浆外泌体中C9手段,可以采用现有技术公开的各种手段,本发明实施例具体采用酶联免疫分析方法(蛋白芯片)进行检测,但不仅仅限于该手段,任何能够检测C9含量的方法均可用于肺癌筛查。
本发明提供了一种新的肺癌筛查标记物和一种新的肺癌筛查试剂盒,能够实现肺癌的有效筛查;且能以血浆外泌体作为检测样品,对患者伤害很低。本发明具备良好的应用前景。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过具体实施方式对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1:肺癌患者(LC)、健康对照(NC)血浆外泌体中C9水平对比
图2:肺癌患者(LC)健康对照(NC)ROC分析。
具体实施方式
实施例1血浆中C9与肺癌的关系
一、临床资料
选取肺癌患者80例,健康对照40例,基本信息如下:
基本信息 | 肺癌患者 | 健康对照 |
人数 | 80 | 40 |
年龄 | 57.8±10.9 | 50.1±14.3 |
男性比例 | 62(77.5%) | 17(44.7%) |
二、检测原理
同基本的酶联免疫吸附试验(ELISA)检测方法。
蛋白芯片上固定有C9蛋白C9抗体(特异性识别C9蛋白的LSPIYNLVPVK氨基酸序列(SEQ ID NO.1)),加血浆外泌体蛋白孵育后,若外泌体蛋白中存在C9蛋白,则C9蛋白会结合上去;清洗去除未结合的抗体和其它蛋白质,再用荧光标记的C9抗体结合到C9蛋白上检测,通过荧光扫描仪读取信号,信号的强弱与抗体的亲和力和数量呈正相关。
三、方法
1.血浆外泌体蛋白的制备方法如下:
样品从-80℃取出,4℃,12000g离心15分钟,上清液转移至新的离心管,0.22μM微孔滤膜过滤后,用PTM公司生产的PTM-EVs试剂盒参照说明书去分离外泌体。加入终浓度为8M的Urea以及蛋白酶抑制剂超声裂解,利用BCA试剂盒进行蛋白浓度测定。
2.血浆外泌体C9蛋白检测
试剂如下:
步骤如下:
1)复温:将芯片从-80℃冰箱取出,置于4℃冰箱复温半小时,然后置于室温复温15min;
2)封闭:复温后的芯片,固定14个blocks围栏,固定好之后,向每个block中加入封闭液,并放置侧摆摇床上,室温封闭3hr;
3)外泌体蛋白样本孵育:封闭完成后,倒尽封闭液,然后迅速加入预先准备好的血浆外泌体蛋白孵育液,上样体积为200μL,侧摆摇床20rpm,4℃过夜孵育(血浆外泌体蛋白加孵育液以1:50比例稀释,得血浆外泌体蛋白孵育液);
4)清洗:将芯片及芯片围栏一同取出,吸去样本,然后迅速加入等体积的PBST,如此循环数次,保证在拆除芯片围栏时血浆外泌体蛋白间无交叉污染。拆除芯片围栏后,将芯片置于加有清洗液的芯片清洗盒,水平摇床,室温80rpm,清洗3次,每次10min;
5)二抗孵育:将芯片转移到加入了3mL二抗孵育液的孵育盒中,侧摆摇床40rpm,避光,室温1hr;
6)清洗:将芯片取出(注意不能触及或划破芯片的上表面),置于加有清洗液的芯片清洗盒,水平摇床,室温80rpm,清洗3次,每次10min。完成后用ddH2O清洗2次,每次10min;
7)干燥;
8)扫描:使用晶芯LuxScan 10K微阵列芯片扫描仪扫描;
9)数据提取:将对应GAL文件(记录了芯片中蛋白所在位置)打开,将芯片图像和GAL文件每个阵列整体对齐,按下自动对齐按钮,提取数据并保存。
四、结果
肺癌患者血浆外泌体中的C9的平均表达水平为1.05(蛋白相对定量比值),健康对照血浆外泌体中C9的平均表达水平为0.76。肺癌组与健康对照(p<0.05)相比有统计学意义(图1)。肺癌组与健康对照ROC分析结果特异性为92.3%,敏感性为22.5%(图2);表明血浆外泌体中C9可特异地区分肺癌与肺部良性疾病。
由以上结果可知,肺癌患者与非肺癌患者的血浆外泌体中C9的水平差异显著,通过检测血浆外泌体中C9的水平,能够达到肺癌筛查的目的。
实施例2本发明的检测试剂盒的组成及其使用方法
一、试剂盒组成
检测试剂盒(14人份):
制备血浆外泌体蛋白的试剂需要自备,可以是市售血浆外泌体蛋白提取试剂盒。
二、试剂盒使用方法
同实施例1第三部分。
本发明的试剂盒可以通过检测血浆外泌体中C9的水平,可以筛查待检人群患肺癌的风险:若C9水平高(相对于健康人群而言),则患肺癌的风险高,若C9水平低,则患肺癌的风险低。可用于临床肺癌的辅助诊断,为患者采取相关的治疗措施或者决策提供有效的依据,临床应用前景良好。
SEQUENCE LISTING
<110> 四川大学华西医院
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Claims (10)
1.检测C9蛋白的试剂在制备肺癌筛查试剂盒中的用途。
2.如权利要求1所述的用途,其特征在于,所述检测C9蛋白的试剂为酶联免疫吸附试验用试剂。
3.如权利要求1所述的用途,其特征在于,所述检测C9蛋白的试剂为western blot试剂。
4.如权利要求1所述的用途,其特征在于,所述检测C9蛋白的试剂为蛋白芯片检测方法用试剂。
5.如权利要求1~4任一所述的用途,其特征在于,所述检测C9蛋白的试剂是检测人血浆外泌体中C9蛋白的试剂。
6.一种肺癌筛查试剂盒,其特征在于,它包括用于检测C9蛋白的试剂。
7.如权利要求6所述的试剂盒,其特征在于,所述检测C9蛋白的试剂为酶联免疫吸附试验用试剂。
8.如权利要求6所述的试剂盒,其特征在于,所述检测C9蛋白的试剂为western blot试剂。
9.如权利要求6所述的试剂盒,其特征在于,所述检测C9蛋白的试剂为蛋白芯片检测方法用试剂。
10.如权利要求5~9任一所述的试剂盒,其特征在于,所述检测C9蛋白的试剂是检测人血浆外泌体中C9蛋白的试剂。
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Citations (11)
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