CN110632311A - Sdpr自身抗体检测试剂在制备肺癌筛查试剂盒中的用途 - Google Patents
Sdpr自身抗体检测试剂在制备肺癌筛查试剂盒中的用途 Download PDFInfo
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Abstract
本发明涉及体外诊断试剂领域，具体涉及SDPR自身抗体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清中SDPR蛋白的自身抗体水平显著高于健康患者。本发明将检测SDPR蛋白自身抗体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SDPR自身抗体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。
自身抗体是指机体对自身器官、细胞或细胞成分产生的抗体。目前，某些蛋白的自身抗体已经成为肺癌的标记物，如：p53、NY-ESO-1、CYFRA等等(Tang Z-M,Ling Z-G,WangC-M,Wu Y-B,Kong J-L(2017)Serum tumor-associated autoantibodies as diagnosticbiomarkers for lung cancer:A systematic review and meta-analysis.PLoS ONE 12(7):e0182117)。
SDPR蛋白(其基因的Ensemble号：ENSG00000168497)是不依赖于钙的磷脂结合蛋白，该蛋白质是蛋白激酶C(PKC)磷酸化的底物，并将聚合酶I和转录物释放因子(PTRF)募集到细胞膜穴样内陷，去除这种蛋白质导致细胞膜穴样内陷丢失，其过度表达导致细胞膜穴样变形和膜管形成。目前尚未见SDPR蛋白自身抗体的相关报道，也未见其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自身抗体类的肺癌标志物，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SDPR蛋白自身抗体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SDPR蛋白自身抗体的试剂为酶联免疫吸附试验用试剂或联免疫分析试剂。
如前述的用途，所述检测SDPR蛋白自身抗体的试剂为western blot试剂。
如前述的用途，所述检测SDPR蛋白自身抗体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SDPR蛋白自身抗体的试剂是检测人血清中SDPR蛋白自身抗体的试剂。
一种肺癌筛查试剂盒，它包括用于检测SDPR蛋白自身抗体的试剂。
如前述的试剂盒，所述检测SDPR蛋白自身抗体的试剂为酶联免疫吸附试验用试剂或酶联免疫分析试剂。
如前述的试剂盒，所述检测SDPR蛋白自身抗体的试剂为western blot试剂。
如前述的试剂盒，所述检测SDPR蛋白自身抗体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SDPR蛋白自身抗体的试剂是检测人血清中SDPR蛋白自身抗体的试剂。
本发明的关键在于，确定了人体血液中SDPR自身抗体的含量与患肺癌的风险显著相关，因此可以通过检测人体血液中SDPR自身抗体的含量来判断患肺癌的风险，至于具体检测人体血液中SDPR自身抗体手段，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SDPR自身抗体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；且能以血清作为检测样品，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。
下文中“SDPR自身抗体”，指的是“SDPR蛋白自身抗体”。
附图说明
图1：肺癌患者(LC)、健康对照(NC)血清浆中SDPR自身抗体水平对比。
图2：肺癌患者(LC)与健康对照(NC)ROC分析。
具体实施方式
实施例1血浆中SDPR自身抗体与肺癌的关系
一、临床资料
选取肺癌患者59例，健康对照29例，基本信息如下：
| 基本信息 | 肺癌患者 | 健康对照 |
| 人数 | 59 | 29 |
| 年龄 | 54.5±7.6 | 42±8.9 |
| 男性比例 | 37(62.7％) | 13(46.7％) |
二、检测原理
HuProtTM人类蛋白质定制芯片上固定有SDPR蛋白(所采用的SDPR蛋白为全长蛋白，蛋白序列号为ENSP00000305675)，加血清孵育后，血清中SDPR自身抗体(主要包括IgG、IgM型抗体，也有一些其它类型的抗体)会结合上去，清洗去除未结合的抗体和其它蛋白质，再用抗人IgM荧光标记二抗(cy5标记，呈现红色)和抗人IgG荧光二抗(cy3标记，呈现绿色)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
本部分所用到的试剂如下：
具体步骤如下：
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)血清样本孵育：封闭完成后，倒尽封闭液，然后迅速加入预先准备好的血清孵育液，每张芯片可孵育14个血清样本，每个血清样本的上样体积为200μL，侧摆摇床20rpm，4℃过夜孵育(血清样本先放在4℃层析柜冻融，加孵育液以1：50比例稀释，得血清孵育液)；
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清样本间无交叉污染。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
肺癌患者血浆中的SDPR自身抗体的平均表达水平为59.7SNR(荧光信号相对定量比值)，健康对照血浆中SDPR自身抗体的平均表达水平为73.6SNR。肺癌组与健康对照组(p<0.05)相比有统计学意义(图1)。肺癌组与健康对照的ROC分析结果特异性为86.2％，敏感性35.6％(图2)，表明SDPR自身抗体可特异地区分肺癌与健康对照。
由以上结果可知，肺癌患者与非肺癌患者的血清中SDPR自身抗体的水平差异显著，通过检测血清中SDPR自身抗体的水平，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)：
二、试剂盒使用方法
同实施例1第三部分——“血清中SDPR自身抗体的检测”。
本发明的试剂盒可以通过检测血清中SDPR自身抗体的水平，可以筛查待检人群患肺癌的风险：若SDPR自身抗体水平低(相对于健康人而言)，则患肺癌的风险高，若SDPR自身抗体水平高，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。
Claims (10)
1.检测SDPR蛋白自身抗体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SDPR蛋白自身抗体的试剂为酶联免疫吸附试验用试剂或联免疫分析试剂。
3.如权利要求1所述的用途，其特征在于，所述检测SDPR蛋白自身抗体的试剂为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SDPR蛋白自身抗体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SDPR蛋白自身抗体的试剂是检测人血清中SDPR蛋白自身抗体的试剂。
6.一种肺癌筛查试剂盒，其特征在于，它包括用于检测SDPR蛋白自身抗体的试剂。
7.如权利要求6所述的试剂盒，其特征在于，所述检测SDPR蛋白自身抗体的试剂为酶联免疫吸附试验用试剂或酶联免疫分析试剂。
8.如权利要求6所述的试剂盒，其特征在于，所述检测SDPR蛋白自身抗体的试剂为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SDPR蛋白自身抗体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SDPR蛋白自身抗体的试剂是检测人血清中SDPR蛋白自身抗体的试剂。
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