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CN114544955B - Gasp-2检测试剂在制备肺癌早期诊断和易感性检测试剂盒中的用途 - Google Patents

Gasp-2检测试剂在制备肺癌早期诊断和易感性检测试剂盒中的用途 Download PDF

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CN114544955B
CN114544955B CN202011351233.8A CN202011351233A CN114544955B CN 114544955 B CN114544955 B CN 114544955B CN 202011351233 A CN202011351233 A CN 202011351233A CN 114544955 B CN114544955 B CN 114544955B
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周清华
梁好
许峰
邓汉宇
范雪娇
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West China Hospital of Sichuan University
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Abstract

本发明公开了G蛋白偶联受体相关分选蛋白‑2(GASP‑2)检测试剂在制备肺癌早期诊断和易感性检测试剂盒中的用途，属于癌症诊断领域。肺癌患者血清中，GASP‑2的含量显著高于正常人。本发明的肺癌早期诊断试剂盒，通过检测GASP‑2含量，可快速准确诊断肺癌，有利于提高肺癌早诊率，从而极大的改善肺癌病人的预后，减少肺癌的疾病负担。

Description

GASP-2检测试剂在制备肺癌早期诊断和易感性检测试剂盒中 的用途
技术领域
本发明属于癌症诊断领域。
背景技术
肺癌是全世界发病率和死亡率增长最快的恶性肿瘤，每年新发肺癌200多万人。肺癌是我国发病率和死亡率居第一位的恶性肿瘤，我国每年新发肺癌接近80万人，死亡近70万人。我国肺癌发病率占全世界肺癌发病率的37.9％，死亡率占全世界肺癌死亡的39.4％。美国SEER数据库显示，超过一半以上的肺癌病人在确诊时都处于局部晚期或伴有远处转移。
目前对于肺癌的早期诊断的技术尚未成熟，目前临床常用的肺癌相关肿瘤标记物CEA、NSE等抗原，在肺癌的早期诊断中的价值有限。因此，探索肺癌早期诊断的试剂盒能够极大地提高肺癌早诊率，从而极大的改善肺癌病人的预后，减少肺癌的疾病负担。
G蛋白偶联受体相关分选蛋白-2(G-protein coupled receptor-associatedsorting protein 1，GASP-2，Uniprot：Q96D09)是一种可通过抑制肌生成抑制素(myostatin)来调节肌生成的蛋白。目前尚未见关于其与肺癌关系的报道。
发明内容
本发明要解决的问题是：提供一种新的肺癌早期诊断试剂盒。
本发明的技术方案如下：
检测GASP-2的试剂在制备肺癌早期诊断和易感性检测(可同时或分别进行肺癌早期诊断和易感性检测)试剂盒中的用途。
如前述的用途，所述检测GASP-2的试剂是ELISA检测试剂。
如前述的用途，所述检测GASP-2的试剂是Western blot试剂。
如前述的用途，所述检测GASP-2的试剂是放射性免疫检测试剂。
如前述的用途，所述检测GASP-2的试剂是蛋白芯片检测试剂。
一种肺癌早期诊断试剂盒，所述试剂盒包括检测GASP-2的试剂。
如前述的试剂盒，所述检测GASP-2的试剂是ELISA检测试剂。
如前述的试剂盒，所述检测GASP-2的试剂是Western blot试剂。
如前述的试剂盒，所述检测GASP-2的试剂是放射性免疫检测试剂。
如前述的试剂盒，所述检测GASP-2的试剂是蛋白芯片检测试剂。
发明人通过研究发现，在肺癌患者血清GASP-2含量(31.55±5.43ng/ml)显著高于正常人血清中GASP-2含量(4.75±1.26ng/ml)。通过检测血清中GASP-2的含量，可以有效识别肺癌患者。如果将血清GASP-2浓度的CUT-OFF值定为：12.87ng/ml时，诊断肺癌的敏感度为95.1％，特异度为96.0％。研究还发现，血清内GASP-2含量高的非肺癌人群患肺癌风险较高。
本发明基于前述发现提供的试剂盒可以快速准确诊断肺癌，筛查识别肺癌易感人群，有利于提高肺癌早诊率，从而极大的改善肺癌病人的预后，减少肺癌的疾病负担，临床应用价值较高。
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。尤其需要了解的是，本发明的关键点是检测血清中GASP-2的检测，并不拘泥与检测的具体手段，但凡能检测GASP-2的方法均能用于肺癌早期诊断和易感性检测。
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。
附图说明
图1对照组与高危人群组间血清GASP-2浓度变化趋势。横坐标单位：年。
图2高危人群患肺癌组与未患肺癌组人群间血清GASP-2浓度的变化趋势。横坐标单位：年。
具体实施方式
实施例1肺癌诊断试剂盒(ELISA)
1.组成
(1)预包被板：孔内壁及底部包被了抗人GASP-2兔IgG抗体的九十六孔板。
(2)酶标抗体：(30倍浓缩)HRP标记抗人GASP-2兔IgG抗体。
(3)标准品：GASP-2。
(4)缓冲液：含1％BSA、0.05％吐温20的BPS。
(5)显色剂：TMB底物液。
(6)终止液：1N硫酸。
2.使用
(1)将血清样本(标准品)0.1mL加入预包被板孔内，37℃孵育30min，倒掉孔内液体，拍打3次，减少液体附着；
(2)加0.15mL缓冲液洗涤未结合到预包被板的蛋白，重复5次；
(3)加入酶标抗体0.1mL，37℃孵育30min，倒掉酶标抗体，加0.15mL缓冲液洗涤5次；
(4)加显色剂0.1mL，37℃孵育10min；
(5)加终止液0.05mL；
(6)结果判定：肉眼观察颜色深浅，颜色越深，阳性程度越强；或测OD值(450nm)。
可提前用梯度稀释标准品按前述步骤制备标准曲线，并在正式检测样本时调整样本稀释度，使浓度落在线性区间(浓度与OD值的曲线)内。
以下用实验例的形式对本发明的有益效果做进一步说明。
实验例1临床样本检测
1.方法
对肺癌病人980名和正常人1010名采集血清样本，使用实施例1的试剂盒检测血清中GASP-2的含量。
其中：肺癌病人年龄60.8±9.0岁；按性别分，男性，624例，女性：356例；按类型分，腺癌：639例，肺腺癌：341例。正常人年龄58.6±7.8岁；按性别分，男性：612例，女性：398例；按TNM分期来分，I+II期肺癌：629例，III+IV期肺癌：351例。
2.结果
正常人血清中GASP-2的含量为4.75±1.26ng/ml，肺癌病人血清中GASP-2含量为31.55±5.43ng/ml(p＝0.000)。
肺癌病人中，男性血清中GASP-2含量为33.78±5.43ng/ml，女性血清中GASP-2含量为30.87±4.43ng/ml(p＝0.236)；腺癌病人血清中GASP-2含量为26.96±2.72ng/ml，鳞癌病人血清中GASP-2含量为25.19±1.82ng/ml，小细胞癌病人血清中GASP-2含量为119.94±3.84ng/ml(p＝0.000)；I期肺癌病人血清中GASP-2含量为14.44±2.42ng/ml，II期肺癌病人血清中GASP-2含量为28.16±1.82ng/ml，III期肺癌病人血清中GASP-2含量为42.78±1.84ng/ml，IV期肺癌病人血清中GASP-2含量为57.10±3.50/ml(P＝0.000)。
为了探讨检测肺癌患者血清GASP-2浓度诊断肺癌的临床价值，本研究应用Logistic回归方程计算GASP2浓度的诊断价值，结果发现：检测肺癌患者血清GASP-2浓度诊断肺癌的敏感度为81.59％，特异度为86.41％，阳性似然比为6.0037，阴性似然比为0.2131，准确度为83.58％，阳性预测值为89.5％，阴性预测值为76.77％(表1)。
表1 logistic回归方程估计检测GASP-2浓度的诊断肺癌的临床价值
如果将血清GASP-2浓度的CUT-OFF值定为：12.87ng/ml时，诊断肺癌的敏感度为95.1％，特异度为96.0％，如果将血清GASP-2浓度的CUT-OFF值定为12.75ng/ml时，诊断肺癌的敏感度为90.2％，特异度为96.9％；如果将血清GASP-2浓度的CUT-OFF值定为24.56ng/ml，诊断肺癌的敏感度为95.1％，特异度为99.5％；如果将血清GASP-2浓度的CUT-OFF值定为30.41ng/ml时，诊断肺癌的敏感度为90.2％，特异度为99.5％。
实验例2肺癌高危人群血清GASP-2浓度与确诊情况追踪
1.方法
连续5年持续检测肺癌高发现场肺癌高危人群(1233例)与对照人群(899例)血清GASP-2浓度，并用医学影像学等常规肺癌诊断方法对前述人群进行肺癌诊断。
注：
肺癌高发现场，即肺癌发病率高的地区，本实验例中为云南宣威、个旧；
肺癌高危人群满足以下条件：1.年龄：≥45岁；2.吸烟史：吸烟≥20包-年；3.家族肿瘤病史：长期环境致癌剂暴露。
2.结果
在4年随访研究周期结束时，肺癌高发人群现场高危人群组共有128例人群在研究周期的不同时间段被诊断患肺癌，对照人群组研究期结束时没有一人被诊断为肺癌；在4年研究周期中对照组人群血清GASP-2浓度无明显变化，而高危人群组人群血清GASP-2浓度成线性上升趋势，表明血清GASP-2浓度随着肺癌的发生、进展而逐渐升高，两组间各时间点比较均有显著性差异(P＝0.000)，检测血清GASP-2浓度能过预测肺癌高危人群发生肺癌的风险(图1,2)。
本实验例结果表明：肺癌高危人群血清GASP-2含量较普通人群(对照人群)明显更高，可通过检测血清中GASP-2含量来评价肺癌易感性。
综上，本发明的诊断试剂盒能以较高的准确度辅助诊断肺癌，且不受肺癌类型与阶段的限制；并能提前对肺癌的易感性进行检测，对未患肺癌但血清GASP-2浓度高的人提前做出预警。

Claims (5)

1.检测G蛋白偶联受体相关分选蛋白-2的试剂在制备小细胞肺癌早期诊断和易感性检测试剂盒中的用途。
2.如权利要求1所述的用途，其特征在于：所述检测G蛋白偶联受体相关分选蛋白-2的试剂是ELISA检测试剂。
3.如权利要求1所述的用途，其特征在于：所述检测G蛋白偶联受体相关分选蛋白-2的试剂是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G蛋白偶联受体相关分选蛋白-2的试剂是放射性免疫检测试剂。
5.如权利要求1所述的用途，其特征在于：所述检测G蛋白偶联受体相关分选蛋白-2的试剂是蛋白芯片检测试剂。
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