CN110095606B - 一种肺癌的筛查试剂盒 - Google Patents
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Abstract
本发明公开了一种肺癌的筛查试剂盒，它包括任选的用于检测非磷酸化BLK表达水平的试剂。本发明还公开了检测非磷酸化BLK表达水平的试剂在制备肺癌筛查用试剂中的用途。本发明试剂盒通过检测非磷酸化BLK的表达水平，能有效判断待检人群患肺癌的风险，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。
Description
技术领域
本发明涉及一种肺癌的筛查试剂盒。
背景技术
肺癌是发病率和死亡率增长最快，对人群健康和生命威胁最大的恶性肿瘤之一。近50年来许多国家都报道肺癌的发病率和死亡率均明显增高，男性肺癌发病率和死亡率均占所有恶性肿瘤的第一位，女性发病率占第二位，死亡率占第二位。非小细胞型肺癌是肺癌中的一类，其约占所有肺癌的80％，约75％的患者发现时已处于中晚期，5年生存率很低。因此，对肺癌进行早期、快速诊断非常重要。
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检查，在出现症状前发现肺癌。如果可以找到血液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。
目前，肺癌的诊断大都依赖于活体检测肺部组织，但是此种检测方法的操作复杂，对患者的肺损伤极大，而且有可能导致肺癌扩散，存在极大的风险。寻找更为简便、创伤小、副作用小的检测方法亟待解决。
非磷酸化BLK，是非磷酸化的B淋巴细胞酪氨酸激酶。现未见有文献报道通过检测非磷酸化BLK来检测肺癌。
发明内容
为了解决上述问题，本发明提供了一种创伤小、副作用小、便于使用的肺癌检测试剂盒。
本发明肺癌的筛查试剂盒，它包括任选的用于检测非磷酸化BLK表达水平的试剂。
其中，所述试剂是用于检测血清和/或肺组织中非磷酸化BLK表达水平的试剂。
其中，所述检测非磷酸化BLK表达水平的试剂是ELISA检测用试剂。
其中，所述检测非磷酸化BLK表达水平的试剂是Western-blot检测方法用试剂。
本发明还提供了检测非磷酸化BLK表达水平的试剂在制备肺癌筛查用试剂中的用途。
其中，所述检测非磷酸化BLK表达水平的试剂是ELISA检测用试剂。
其中，所述检测非磷酸化BLK表达水平的试剂是Western-blot检测方法用试剂。
其中，所述试剂是用于检测血清和/或肺组织中非磷酸化BLK表达水平的试剂。
本发明试剂盒通过检测非磷酸化BLK的表达水平，可以判断待检人群患肺癌的风险，可用于临床肺癌的辅助诊断，本发明试剂盒仅仅只需采集待检样本的血液，就可以筛查其患肺癌的风险，创伤小、副作用小、使用方便，临床应用前景良好。
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。
以下通过实施例形式的具体实施方式，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。
具体实施方式
实施例1 非磷酸化BLK的表达水平与肺癌的关系
一、实验方法
1、临床资料
选取肺癌患者石蜡切片30例，远端对照30例(指肺癌患者的远端正常肺组织石蜡切片，距离癌组织5cm，且经病理确认为正常组织)，基本信息见表1。
表1临床基本信息
*注：其他包括大细胞癌、腺鳞癌、小细胞癌。
对肺癌患者石蜡切片、远端组织对照石蜡切片，进行以非磷酸化BLK 为指标的免疫组化(IHC)。
非磷酸化BLK抗体：购自美国ProteinTech公司，货号10510-1-AP；
EnVisionTM HRP二抗、抗体稀释液、DAB显色剂、抗原修复液、Wash Buffer、免疫组化笔：均购自丹麦Dako公司。
按照如下方法检测非磷酸化BLK的表达水平：
(1)封闭过氧化物酶：取石蜡切片进行脱蜡，脱蜡至水化(脱蜡过程如下：二甲苯I10min→二甲苯II 10min→无水乙醇5min→95％乙醇5min→ 85％乙醇5min→75％乙醇5min)后，用3％H2O2溶液于暗处封闭内源性过氧化物酶，室温孵育15min；
(2)蒸馏水漂洗：切片置于蒸馏水中，摇床上洗5min/次，共三次；
(3)抗原修复：切片放入pH 9.0的EDTA抗原修复溶液，在98℃水浴锅中水浴40min，进行抗原修复；
(4)蒸馏水漂洗：取出切片，自然冷却至室温后，用蒸馏水洗三遍，再用WashBuffer漂洗一次；
(5)孵育一抗：用免疫组化笔将组织圈住，在圈内滴加非磷酸化BLK 抗体工作液(非磷酸化BLK与抗体稀释液1∶200混合)，4℃孵育过夜；
(6)蒸馏水漂洗：用蒸馏水洗三遍，Wash Buffer漂洗一次；
(7)滴加二抗：滴加Dako HRP标记的二抗工作液，室温孵育60min；
(8)蒸馏水漂洗：蒸馏水洗3次，每次5min；
(9)DAB显色：滴加显色底物DAB，在显微镜下观察显色情况，于蒸馏水中终止反应；
(10)苏木素复染：将切片放入苏木素染液中染色2min，1％盐酸酒精分化后，流水冲洗10min；
(11)脱水透明：切片经75％、85％、95％、100％梯度酒精脱水、二甲苯透明，于通风橱中干燥；
(12)封片：用中性快干胶封片，在光学显微镜下观察，DP Controller 图像采集系统采图。采图后，根据肺癌患者和远端组织的肿瘤细胞染色强度及阳性面积给予免疫组化评分。
3、免疫组化评分标准
先按下表规定肿瘤细胞染色强度、阳性面积的评分：
@注：由两名经验丰富的病理医生独立对肿瘤细胞染色强度结果进行评分
再计算肿瘤细胞染色强度评分和阳性面积评分的乘积(0-9分)，规定：
非磷酸化BLK表达阴性：0分；
非磷酸化BLK表达低度阳性：1-3分；
非磷酸化BLK表达高度阳性＞3分。
4、结果分析
采用SPSS17.0肺癌组织组和远端组织对照组进行统计学分析。
二、实验结果
肺癌组织与远端组织对照中非磷酸化BLK的表达水平检测结果见表2。
表2非磷酸化BLK的表达水平
由表2可见，远端正常组织中非磷酸化BLK的阳性表达率为13.3％，高度阳性表达率为0％；肺癌组织中的非磷酸化BLK阳性表达率为100％，高度阳性表达率为73.3％；非磷酸化BLK在肺癌组织中显著升高，与远端正常组织相比，非磷酸化BLK表达水平差异具有统计学意义(P＜0.0001)。
由以上结果可以看出，与癌旁正常肺组织相比，肺癌组织的非磷酸化 BLK表达水平显著升高(P＜0.0001)，说明肺癌与非磷酸化BLK表达水平呈正相关，非磷酸化BLK的高表达会显著提高患肺癌的可能性。由于癌旁正常肺组织的非磷酸化BLK水平可反映正常人肺组织的非磷酸化BLK水平，因此，可以通过检测待检者的非磷酸化BLK的表达水平，将肺癌的易感人群筛查出来。
实施例2 肺癌筛查试剂盒及其使用方法
一、试剂盒组成
检测试剂盒(50人份)：
| 组分 | 体积 |
| 非磷酸化BLK抗体 | 50μl |
| EnVision<sup>TM</sup> HRP二抗 | 10ml |
| 抗体稀释液 | 10ml |
| DAB显色剂 | 10ml |
| 抗原修复液 | 500ml |
| Wash Buffer<sup>*</sup> | 500ml |
| 免疫组化笔 | 1支 |
注：本试剂盒按每张切片滴加各类实验试剂为100μl计，具体用量可根据组织大小适当增减。
二、试剂盒的使用方法
将待检样本肺组织，制备石蜡切片，作为检测标本，按照如下方法来检测非磷酸化BLK的表达水平：
(1)封闭过氧化物酶：取石蜡切片进行脱蜡，脱蜡至水化(脱蜡过程如下：二甲苯I10min→二甲苯II 10min→无水乙醇5min→95％乙醇5min→85％乙醇5min→75％乙醇5min)后，用3％H2O2溶液于暗处封闭内源性过氧化物酶，室温孵育15min；
(2)蒸馏水漂洗：切片置于蒸馏水中，摇床上洗5min/次，共三次；
(3)抗原修复：切片放入pH 9.0的EDTA抗原修复溶液，在98℃水浴锅中水浴40min，进行抗原修复；
(4)蒸馏水漂洗：取出切片，自然冷却至室温后，用蒸馏水洗三遍，再用WashBuffer漂洗一次；
(5)孵育一抗：用免疫组化笔将组织圈住，在圈内滴加非磷酸化BLK 抗体工作液(非磷酸化BLK与抗体稀释液1∶200混合)，4℃孵育过夜；
(6)蒸馏水漂洗：用蒸馏水洗三遍，Wash Buffer漂洗一次；
(7)滴加二抗：滴加Dako HRP标记的二抗工作液，室温孵育60min；
(8)蒸馏水漂洗：蒸馏水洗3次，每次5min；
(9)DAB显色：滴加显色底物DAB，在显微镜下观察显色情况，于蒸馏水中终止反应；
(10)苏木素复染：将切片放入苏木素染液中染色2min，1％盐酸酒精分化后，流水冲洗10min；
(11)脱水透明：切片经75％、85％、95％、100％梯度酒精脱水、二甲苯透明，于通风橱中干燥；
(12)封片：用中性快干胶封片，在光学显微镜下观察，DP Controller 图像采集系统采图。采图后，根据肺癌患者和远端组织的肿瘤细胞染色强度及阳性面积给予免疫组化评分，评分方法见实施例1。
对肺部异常者，可分别取异常部位、正常部位组织，比较非磷酸化BLK 表达水平，进而评价其患肺癌的可能性，作为临床肺癌的辅助诊断手段。
综上，本发明试剂盒通过检测非磷酸化BLK的表达水平，可以筛查待检人群患难肺癌的风险，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 (2)
1.检测非磷酸化BLK表达水平的试剂在制备肺癌筛查用试剂中的用途；所述检测非磷酸化BLK表达水平的试剂中包括BLK抗体，所述抗体为美国ProteinTech公司货号为10510-1-AP的BLK抗体；
当检测到待检者肺组织样品非磷酸化BLK表达水平显著高于正常肺组织时，可判定待检者患肺癌可能性高；
所述BLK为B淋巴细胞酪氨酸激酶。
2.根据权利要求1所述的用途，其特征在于：所述检测非磷酸化BLK表达水平的试剂是免疫组化试剂。
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