CN112782407A - γ干扰素诱导单核细胞蛋白在血清中表达量的检测方法及应用 - Google Patents
γ干扰素诱导单核细胞蛋白在血清中表达量的检测方法及应用 Download PDFInfo
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Abstract
一种γ干扰素诱导单核细胞蛋白在血清中表达量的检测方法及应用，一种γ干扰素诱导单核细胞蛋白在血清中表达量的检测方法包括如下步骤：采集血清样本、设置零孔、在标准品孔及样本孔中加入标准品及血清、每孔加入生物素标记抗体工作液、将板内液体甩干后加液、将板内液体甩干后在其余每孔中加入TMB显色液、在其余每孔中加入终止液、用酶标仪测量板内各孔OD值、计算出三种蛋白浓度。MIG对于喉癌的发生有着重要意义，可为喉癌诊断提供重要依据，可弥补内镜和影像学检查的不足，减少有创检查，提高喉癌诊断的灵敏度与特异度。
Description
技术领域
本发明涉及一种检测方法及应用，特别是一种γ干扰素诱导单核细胞蛋白在血清中表达量的检测方法及应用。
背景技术
喉癌是头颈部的常见恶性肿瘤之一，影响喉癌的发生因素很多，主要的影响因素为吸烟和饮酒，它的早期没有特异性的临床表现，有患者仅可表现为声音嘶哑，咽喉部异物感等症状，不易引起患者重视。由于喉部在呼吸和发声方面的重要功能，喉癌可严重影响患者的日常生活。
现阶段研究中发现许多与喉癌密切相关的标志物，例如血清肿瘤标志物Cyfra21-1,CA724,CA199,SCCAg,TK1等，但是都缺乏高度特异性及敏感度而无法在临床上进行广泛推广。需要更多的研究来确定新的基于血清的肿瘤标志物，这些肿瘤标志物可能被用于筛查喉癌早期诊断的一般或高危人群，或预测CRC的预后和治疗反应，因此对喉癌肿瘤标志物的寻找有非常大的临床应用价值，是现今喉癌诊断研究的重点和热门。
发明内容
本发明的目的是提供一种γ干扰素诱导单核细胞蛋白在血清中表达量的检测方法。
实现本发明上述目的的技术方案是：一种γ干扰素诱导单核细胞蛋白在血清中表达量的检测方法，包括如下步骤：
一、采集血清样本；
二、按照样本量设置零孔，TMB显色孔，标准品孔及样本孔，并标识；
三、用移液枪分别在标准品孔及样本孔中加入标准品及血清100ul，注意不要碰触到孔壁和孔底,盖上板贴，至37℃恒温箱中孵育2h。
四、标准品孔及样本孔中每孔加入生物素标记抗体工作液100 ul，注意不要碰触到孔壁和孔底，盖上新板贴，放置于37℃恒温箱中，静置1 h；
五、将板内液体甩干，孔内未见明显附壁水滴，洗板3次，每孔加入洗液工作液200ul，浸泡2min。洗板后在滤纸上拍干，可见孔壁和孔底未见明显附壁水滴；
六、用移液枪分别在先前设置的标准品孔及样本孔中加液，每孔加入过氧化物酶标记亲和素工作液100 ul，注意不要碰触到孔壁和孔底。盖上新板贴，置于恒温箱中，温度恒定37℃，静置1 h；
七、将板内液体甩干，孔内未见明显附壁水滴，洗板5次，每孔加入洗液工作液200ul，浸泡2min。洗板后在滤纸上拍干，可见孔壁和孔底未见明显附壁水滴；
八、除空白孔外，用移液枪在其余每孔中加入TMB显色液90ul，注意不要碰触到孔壁和孔底，在37℃恒温箱中避光显色25min；
九、看到标准品孔中有明显颜色反应后。除空白孔外，在其余每孔中加入终止液50ul；
十、加入终止液后可见孔内液体转为黄色，观察到反应后5min内，用酶标仪测量板内各孔OD值，设定测量波长为450nm；
十一、计算出三种蛋白浓度。
本发明实验所用的ELISA法检测MIG检测试剂盒由上海华美生物有限公司提供。
MIG(CXCL9)是γ-干扰素诱导单核细胞产生的小分子蛋白质，属于趋化因子α-亚家族（CXC家族）。有研究报道：MIG在黑色素瘤、乳腺癌、胃癌等多种恶性肿瘤细胞和组织中高表达，并与CXCR3结合，介导癌细胞的定向移动。有研究证实：MIG/CXCR3的相互作用对肿瘤细胞有较强的趋化性，诱导肿瘤穿透微血管壁，对中晚期肿瘤患者其肿瘤细胞的血道转移及新的转移灶形成有着至关重要的作用。MIG利用配体CXCR3吸引Th1细胞，NK细胞，NKT细胞，M1巨噬细胞进入肿瘤部位可以作为一种有效的促进肿瘤策略。其中Mig-7(迁移诱导蛋白)是新近发现的一种特异性表达于肿瘤细胞的蛋白质，在正常组织中没有表达，有研究表示，MIG-7（迁移诱导蛋白7）在肿瘤细胞中胞膜及胞质中高表达，在不同病理分型和不同病理类型的肿瘤细胞中有明显特异性，并且通过实验验证了其作为肿瘤标志物的巨大潜力。
综上，MIG对于喉癌的发生有着重要意义，可为喉癌诊断提供重要依据，可弥补内镜和影像学检查的不足，减少有创检查，提高喉癌诊断的灵敏度与特异度。
以下结合具体实施方式对本发明的详细内容作进一步描述。
具体实施方式
实施例一：
一种γ干扰素诱导单核细胞蛋白在血清中表达量的检测方法，包括如下步骤：
一、采集血清样本；
二、按照样本量设置零孔，TMB显色孔，标准品孔及样本孔，并标识；
三、用移液枪分别在标准品孔及样本孔中加入标准品及血清100ul，注意不要碰触到孔壁和孔底,盖上板贴，至37℃恒温箱中孵育2h。
四、标准品孔及样本孔中每孔加入生物素标记抗体工作液100 ul，注意不要碰触到孔壁和孔底，盖上新板贴，放置于37℃恒温箱中，静置1 h；
五、将板内液体甩干，孔内未见明显附壁水滴，洗板3次，每孔加入洗液工作液200ul，浸泡2min。洗板后在滤纸上拍干，可见孔壁和孔底未见明显附壁水滴；
六、用移液枪分别在先前设置的标准品孔及样本孔中加液，每孔加入过氧化物酶标记亲和素工作液100 ul，注意不要碰触到孔壁和孔底。盖上新板贴，置于恒温箱中，温度恒定37℃，静置1 h；
七、将板内液体甩干，孔内未见明显附壁水滴，洗板5次，每孔加入洗液工作液200ul，浸泡2min。洗板后在滤纸上拍干，可见孔壁和孔底未见明显附壁水滴；
八、除空白孔外，用移液枪在其余每孔中加入TMB显色液90ul，注意不要碰触到孔壁和孔底，在37℃恒温箱中避光显色25min；
九、看到标准品孔中有明显颜色反应后。除空白孔外，在其余每孔中加入终止液50ul；
十、加入终止液后可见孔内液体转为黄色，观察到反应后5min内，用酶标仪测量板内各孔OD值，设定测量波长为450nm；
十一、计算出三种蛋白浓度。
本发明实验所用的ELISA法检测MIG检测试剂盒由上海华美生物有限公司提供。
Claims (2)
1.一种γ干扰素诱导单核细胞蛋白在血清中表达量的检测方法，其特征是包括如下步骤：
一、采集血清样本；
二、按照样本量设置零孔，TMB显色孔，标准品孔及样本孔，并标识；
三、用移液枪分别在标准品孔及样本孔中加入标准品及血清100ul，注意不要碰触到孔壁和孔底,盖上板贴，至37℃恒温箱中孵育2h；
四、标准品孔及样本孔中每孔加入生物素标记抗体工作液100 ul，注意不要碰触到孔壁和孔底，盖上新板贴，放置于37℃恒温箱中，静置1 h；
五、将板内液体甩干，孔内未见明显附壁水滴，洗板3次，每孔加入洗液工作液200ul，浸泡2min；
洗板后在滤纸上拍干，可见孔壁和孔底未见明显附壁水滴；
六、用移液枪分别在先前设置的标准品孔及样本孔中加液，每孔加入过氧化物酶标记亲和素工作液100 ul，注意不要碰触到孔壁和孔底；
盖上新板贴，置于恒温箱中，温度恒定37℃，静置1 h；
七、将板内液体甩干，孔内未见明显附壁水滴，洗板5次，每孔加入洗液工作液200ul，浸泡2min；
洗板后在滤纸上拍干，可见孔壁和孔底未见明显附壁水滴；
八、除空白孔外，用移液枪在其余每孔中加入TMB显色液90ul，注意不要碰触到孔壁和孔底，在37℃恒温箱中避光显色25min；
九、看到标准品孔中有明显颜色反应后；
除空白孔外，在其余每孔中加入终止液50ul；
十、加入终止液后可见孔内液体转为黄色，观察到反应后5min内，用酶标仪测量板内各孔OD值，设定测量波长为450nm；
十一、计算出三种蛋白浓度。
2.一种γ干扰素诱导单核细胞蛋白在血清中表达量作为肿瘤标志物的应用。
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| CN103954754A (zh) * | 2014-04-30 | 2014-07-30 | 广东省结核病控制中心 | 一种活动性肺结核的免疫诊断试剂盒 |
| EP3073268A1 (en) * | 2015-03-27 | 2016-09-28 | Deutsches Krebsforschungszentrum Stiftung des Öffentlichen Rechts | Biomarker panel for diagnosing cancer |
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| CN103954754A (zh) * | 2014-04-30 | 2014-07-30 | 广东省结核病控制中心 | 一种活动性肺结核的免疫诊断试剂盒 |
| EP3073268A1 (en) * | 2015-03-27 | 2016-09-28 | Deutsches Krebsforschungszentrum Stiftung des Öffentlichen Rechts | Biomarker panel for diagnosing cancer |
| CN109748967A (zh) * | 2017-11-02 | 2019-05-14 | 北京蛋白质组研究中心 | 具有抑制肿瘤进程活性的融合蛋白BTN3A3-Ig及其制备方法与应用 |
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