CN114113608A - 一种快速检测肺癌肿瘤标志物的免疫层析试纸条及其制备方法 - Google Patents
一种快速检测肺癌肿瘤标志物的免疫层析试纸条及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条及其制备方法，所述试纸条包括底板，所述底板上依次排列样品垫、金标垫、NC膜及吸水纸；所述NC膜粘附于底板上，自吸水纸方向依次设置质控线（C线），细胞角蛋白19片段检测线（T1线）和癌胚抗原检测线(T2线)；所述金标垫和样品垫设置在NC膜下端，吸水纸设置在NC膜上端，所述样品垫、金标垫、NC膜和吸水纸重叠设置。本试纸可以快速实现全血中的细胞角蛋白19片段（CYFRA21‑1）、癌胚抗原（CEA）的同时检测，操作简易，可视化的结果判读，灵敏度高、特异性强，成本低，无需专业人员和大型仪器，适用于肺癌高风险人群的监测及肺癌术后预后，不受地域和时间的限制，适用于家庭和社区。
Description
技术领域
本发明专利涉及一种快速检测肺癌肿瘤标志物的免疫层析试纸条及其制备方法，应用于临床检测领域。
背景技术
肺癌是最常见的恶性肿瘤之一，其发病率及死亡率位居全球及中国恶性肿瘤的首位。肺癌按细胞形态特征与分化程度可分为小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)，其中非小细胞肺癌占所有肺癌的80％～85％。早期肺癌患者症状较轻，临床表现不明显，晚期肺癌患者预后较差，总体5年生存率仅为19.7％，严重影响我国居民的生命健康。早诊断早治疗是提高肺癌患者生存率、减轻经济负担的关键。
目前，肺癌的临床诊断手段包括肺穿刺活检、影像学病理诊断(CT、X射线、MRI)、细胞学检查、血液肿瘤标志物检查、肺支气管镜检查等。早期肺癌患者异常表现不明显，影像学检查特异性较低，常常无法辨别肺部良性结节与早期肺癌。且对于早期患者而言，细胞学检查及穿刺活检等有创检查的阳性率低且反复取材困难，血清肿瘤标志物检查是临床肺癌重要的辅助检测方法，血清肿瘤标志物属于肿瘤细胞增殖中合成、释放或肿瘤与宿主细胞相互作用而形成的一类物质，指示着肿瘤的发生发展。临床上可通过化学、免疫、分子生物学或蛋白组学方法，在血液、尿液、脑脊髓液等体液中检测该类物质，其在肿瘤疾病诊断及病情分期中均具有重要价值。
癌胚抗原(CEA)为广谱性的肿瘤标志物，正常人血清中含量极低，小于5ng/ml，CEA水平升高常见于大肠癌、胃癌、肺癌、乳腺癌、甲状腺髓样癌等。临床报道显示，30％～70％的非小细胞肺癌(NSCLC)患者CEA水平升高，且常见于腺癌与晚期鳞癌。因此，CEA水平升高可作为肺癌诊断的依据。
细胞角蛋白-19片段(CYFRA21-1)属细胞角蛋白家族，当肿瘤细胞被破坏/死亡时，大量可溶性CYFRA21-1片段释放入血，使血液中CYFRA21-1水平升高。目前，CYFRA21-1被认为是一种主要用于检测肺癌的肿瘤标记物，尤其对非小细胞肺癌中的鳞癌，特异性较高。血清CYFRA21-1诊断临界值一般为3.3μg/L，其检测灵敏度为51％～77％，特异性为87％～96％，各类非小细胞肺癌阳性检出率为70％～85％。是监测肺癌患者病情、提示预后的良好指标。
肿瘤标志物对肿瘤的早期筛查、鉴别诊断、预后判断具有极其重要的意义，但单一肿瘤标志物的检测灵敏度和特异性肺癌有着不同的表现，联合检测可以提高检测的灵敏度。研究表明，CEA和CYFRA21-1联合检测是对于非小细胞肺癌的最佳标志物组合，两者均可作为独立的肺癌预后指标，且其水平和预后发展密切相关，多项研究考察了两者组合对于非小细胞肺癌的预后预测效果，认为两者组合是非小细胞肺癌预后的预测因素，对疗效的预测和病程的检测均有重大意义。
因此，我们建立了基于胶体金免疫层析原理的癌胚抗原(CEA)、细胞角蛋白19片段(CYFRA21-1)两联免疫层析试纸条，可同时检测全血或血清中的CEA和CYFRA21-1，具有操作简单、检测快速、成本低廉且无需检测仪器等优点，用于肺癌的预后预测和病程检测。
发明内容
针对现有问题的不足，本发明的目的是提供一种快速检测肺癌肿瘤标志物的免疫层析试纸条及其制备方法。本发明提供的免疫层析试纸条灵敏度高，特异性好，检测快速，成本低廉，实现CYFRA21-1和CEA的快速检测，检测限分别为3.3ng/ml(CYFRA21-1)以及5ng/ml(CEA)，可用于肺癌的辅助诊断和肺癌病人的预后监测。
本发明解决其技术问题采用的技术方案是：
一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，所述试纸条包括底板，所述底板上依次排列样品垫、金标垫、NC膜及吸水纸；所述NC膜粘附于底板上，自吸水纸方向依次设置质控线(C线)，细胞角蛋白19片段检测线(T1线)和癌胚抗原检测线(T2线)；所述金标垫和样品垫设置在NC膜下端，吸水纸设置在NC膜上端，所述样品垫、金标垫、NC膜和吸水纸重叠设置。
作为本申请的优选技术方案，所述质控线(C线)固定有羊抗鼠二抗。
优选的，所述细胞角蛋白19片段检测线(T1线)固定有细胞角蛋白19片段(CYFRA21-1)包被抗体。
更优选的，所述癌胚抗原检测线(T2线)固定有癌胚抗原(CEA)包被抗体。
优选的，包被抗体为肿瘤标志物的单克隆抗体。
作为本申请的优选技术方案，所述金标垫上固化有细胞角蛋白19片段(CYFRA21-1)胶体金标记抗体；癌胚抗原(CEA)胶体金标记抗体。
优选的，标记抗体为肿瘤标志物的单克隆抗体，针对的抗原决定簇不应与包被抗体相同。
作为本申请的优选技术方案，所构建试纸条裸条宽度为4-5mm，各组分相互紧密衔接，重叠约1-2mm。
作为本申请的优选技术方案，所述底板为PVC材质或硬质不吸水的材质。
作为本申请的优选技术方案，所述样品垫为全血分离膜，上包被抗RBC抗体，可以有效地分离血清与血细胞，最大程度抑制溶血，解决了背景颜色深的问题。
作为本申请的优选技术方案，所述金标垫为玻璃纤维素膜，所述胶体金粒径优选为15～40nm。
作为本申请的优选技术方案，所述NC膜为硝酸纤维素膜。
作为本申请的优选技术方案，所述质控线、细胞角蛋白19片段(CYFRA21-1)检测线以及癌胚抗原(CEA)检测线，依次相距0.4-0.8cm。
作为本申请的优选技术方案，所述吸水纸为高质量纯棉绒浆滤纸。
有益效果
本发明的检测原理为双抗体夹心法，其优点：与现有产品相比，该发明检测快速、成本低廉、只需要50μl全血即可实现检测，不需要大型仪器设备和专业操作人员，不受地域和时间的限制，适用于家庭及基层单位使用，且灵敏度高、特异性好、安全可靠。
附图说明
图1为本发明一种快速检测肺癌肿瘤标志物的金标免疫层析试纸条的结构示意图。
图2为免疫层析试纸条原理图。
图3为免疫层析试纸条示意图及结果判定图。
图4为免疫层析试纸条检测特异性实验结果照片。
图5、图6分别为不同浓度的CYFRA21-1标准品以及CEA标准品检测结果照片。
图7为肺癌样本的实际应用效果图。
具体实施方式
以下结合实施例对本发明做进一步详细说明。所用试剂或者仪器设备未注明生产厂商的，均视为可以通过市场购买的常规产品。
本具体实施方式中选用的细胞角蛋白19片段标记抗体来自厦门同仁心，型号为ZL28，癌胚抗原标记抗体来自广东菲鹏，型号为CEA01；选用的细胞角蛋白19片段包被抗体来自厦门同仁心，型号为ZL27，癌胚抗原包被抗体来自广东菲鹏，型号为CEA00。其他企业的同类产品亦可，本申请不一一列出。
实施例1肺癌肿瘤标志物免疫层析试纸条的制备方法
胶体金的制备：玻璃仪器酸缸浸泡过夜，取出后去离子水冲洗干净，干燥备用；配制万分之一的氯金酸溶液；加热煮沸后加入0.8～1.4ml 1％新制柠檬酸三钠溶液，并继续加热至溶液颜色不再变化后，停止加热，冷却至室温，保存备用。
CYFRA21-1及CEA标记抗体标记胶体金：取1～10ml保存备用的胶体金溶液，调整pH至抗体标记范围(pH7.4～8.0)，后加入10～40微升0.2mg/ml的标记抗体混匀，静置30min后加入的200微升封闭剂(封闭剂选用5％BSA溶液或0.5％酪蛋白酸钠溶液都可，本实施例选用5％BSA溶液)封闭，封闭完成后4℃下离心弃去上清，复溶液重悬浓缩至原来体积的10％～20％，即制得金标抗体，于4℃保存备用。
其中，复溶液成分如下：1～5％BSA、3～10％糖类、1％表面活性剂、0.5％惰性蛋白，溶于PBS缓冲液。
金标垫预处理：采用玻璃纤维素膜作为固体材料，将结合垫浸泡于预处理液后取出，37℃烘箱干燥过夜，干燥密封保存备用。
金标垫的制备：预处理过的结合垫裁剪成宽4mm的长条，喷涂1μl/ml的上述胶体金标记复合物，37℃烘箱干燥，即制得金标垫，干燥密封保存备用。
NC膜区域检测条带的固化：以硝酸纤维素膜作为固相材料，使用PBS缓冲液稀释包被抗体，采用金标划膜仪按1μl/cm的浓度将稀释后的抗体包被于相应的预设区域，37℃烘箱干燥过夜，干燥密封保存备用。
其中，检测线1：0.5mg/ml细胞角蛋白19片段包被抗体；检测线2：0.7mg/ml癌胚抗原包被抗体；质控线：0.1mg/ml羊抗鼠二抗。
试纸条的组装：将包被有抗体的NC膜粘贴于PVC底板上，从上到下依次组装吸水纸、NC膜、金标垫、全血样品垫，各组分相互衔接，并各自重叠约1-2mm，装入塑料卡壳即制得免疫层析试纸条，干燥密封保存备用。
实施例2试纸条的检测特异性评价
检测方法：采用PBS缓冲液将CYFRA21-1、CEA、GP73、PSA、CRP、SSA标准品稀释成200ng/ml的溶液，取200μL上述标准品滴加至试纸条加样孔进行检测，15min后观察检测结果。
结果判定：结合图3与图4，仅在滴加CYFRA21-1、CEA标准品后，试纸条检测线出现条带，在PBS及其他标准品下均无假阳性，且CYFRA21-1、CEA检测互不干扰，即检测特异性良好。
实施例3试纸条的灵敏度评价
检测方法：使用PBS缓冲液将CYFRA21-1、CEA标准品稀释至一定浓度：CYFRA21-1标准品(3.3、10、50、100、500ng/mL)；CEA标准品(5、20、100、500ng/mL)各稀释完成的溶液分别取100μL加至试纸条加样孔进行检测，15min后观察检测结果。
结果判定：结合图5与图6，当CYFRA21-1标准品浓度为3.3ng/ml时，CYFRA21-1检测线仅刚刚出线，随其浓度增加，条带逐渐加深；CEA检测线在CEA标准品浓度为5ng/ml时，刚刚出线，条带亦随浓度增加，即该试纸条检测灵敏度满足临床检测要求。
实施例4临床样本检测
检测方法：使用PBS缓冲液50μL稀释50μL样本全血或25μL血清/血浆，加至制备好的试纸条加样孔中进行检测，10min后观察检测结果。
结果判定：结合图7判读肺癌病人样本的检测结果，并与商用Elisa试剂盒的检测结果进行比对，结果显示，试纸条检测结果与Elisa试剂盒结果一致，表明该试纸条具有良好的临床使用价值。
综上所述，本发明构建的快速检测血清肺癌标志物的免疫层析试纸条，检测性能评价良好，具有较高的检测灵敏度及检测特异性，稳定性、重复性良好，适用于家庭和基层使用。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。
Claims (10)
1.一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所述试纸条包括底板，所述底板上依次排列样品垫、金标垫、NC膜及吸水纸；所述NC膜粘附于底板上，自吸水纸方向依次设置质控线，细胞角蛋白19片段检测线和癌胚抗原检测线；所述金标垫和样品垫设置在NC膜下端，吸水纸设置在NC膜上端，所述样品垫、金标垫、NC膜和吸水纸重叠设置。
2.根据权利要求1所述的一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所述质控线固定有羊抗鼠二抗；优选的，所述细胞角蛋白19片段检测线固定有细胞角蛋白19片段包被抗体；更优选的，所述癌胚抗原检测线固定有癌胚抗原包被抗体。
3.根据权利要求1所述的一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所述金标垫上固化有细胞角蛋白19片段胶体金标记抗体；癌胚抗原胶体金标记抗体。
4.根据权利要求1所述的一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所构建试纸条裸条宽度为4-5mm，各组分相互紧密衔接，重叠1-2mm。
5.根据权利要求1所述的一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所述底板为PVC材质或硬质不吸水的材质。
6.根据权利要求1所述的一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所述样品垫为全血分离膜，上包被抗RBC抗体。
7.根据权利要求1所述的一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所述金标垫为玻璃纤维素膜；优选的，胶体金粒径为15~40nm。
8.根据权利要求1所述的一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所述NC膜为硝酸纤维素膜。
9.根据权利要求1所述的一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所述质控线、细胞角蛋白19片段检测线以及癌胚抗原检测线，依次相距0.4-0.8cm。
10.根据权利要求1所述一种快速检测肺癌血清肿瘤标志物的免疫层析试纸条，其特征在于，所述吸水纸为纯棉绒浆滤纸。
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