CN111879946A - 一种多指标快速检测荧光免疫层析试剂盒 - Google Patents
一种多指标快速检测荧光免疫层析试剂盒 Download PDFInfo
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Abstract
本发明涉及一种多指标快速检测荧光免疫层析试剂盒，该试剂盒包含检测卡中的荧光垫上涂覆有荧光微球标记抗体的涂层，所述的抗体为C‑反应蛋白、髓过氧化物酶和心脏脂肪酸结合蛋白的单克隆抗体；本发明优化了活化剂配比等参数，实现上述三标志物同时更灵敏、更快捷、更准确检测的需求。
Description
技术领域
本发明涉及生物技术检测领域，具体涉及一种同时检测3个标志物的检测更快捷、更方便的荧光免疫层析试剂盒。
背景技术
荧光免疫层析法检测试剂属于POCT类(point-of-care testing，即时检测或床旁检测) 检测产品，因具有检测方便、快捷、综合成本低等特点，在临床上得到广泛应用。然而，人体的复杂性决定了单一生物标记物难以及时、准确地全面反映受试者身体状况，往往需要同时进行多项检测，再综合判断。门诊量大是我国医疗卫生系统的一大特点，也是产生医患矛盾的一个诱因，无论是医生还是辅助部门都面临很大的工作压力。将多个标记物联合到一个试剂盒中检测，无疑会减少很多重复劳动，大大提高检验效率，从而加快临床医生的诊治效率。
多指标联合检测不但有助于缓解医患矛盾，更有利于保护患者的生命健康。尤其是在心血管疾病领域，心梗、脑梗这类急重症死亡率及致残率均超过40％，严重威胁患者生命安全，给其家庭乃至社会都带来巨大的经济负担和精神痛苦。如何在急症发生前尽快诊断出患者的发病风险，从而采取有效救治措施是减少、甚至避免悲剧发生的关键。多指标联合检测试剂盒是提高临床诊疗效率及患者预后水平的得力助手，但同时检测多个标记物在技术上存在一定难度，不同标记物的性质特点都不同，适用于不同的体系，相互之间会形成干扰，尤其是灵敏度更高的荧光法检测试剂盒。
发明内容
本发明所要解决的技术问题是，目前心血管疾病领域缺乏具有更高灵敏度及准确性且具有早期预警方面功能的多指标联合检测产品。
为解决以上技术问题，本发明提供一种可以在保证灵敏度及准确性的情况下，同时检测出血液中的C-反应蛋白、髓过氧化物酶和心脏脂肪酸结合蛋白三个标记物的荧光免疫层析检测试剂盒，具体技术方案如下：
本发明所述荧光免疫层析试剂盒包含检测卡，该检测卡包括样品垫、荧光垫、具有检测线和质控线的检测垫、吸样垫及底板。其中，所述荧光垫上涂覆有荧光微球标记抗体的涂层，所述的抗体为C-反应蛋白、髓过氧化物酶和心脏脂肪酸结合蛋白的单克隆抗体；该抗体标记过程中所用的活化剂为EDC和NHS，荧光微球与两活化剂的用量比为1: (14-16):(14-16)；所述的检测线有3条，分别为能够与所述涂层中荧光微球标记抗体特异性结合的配对抗体或抗原形成的涂层。
所述的检测卡还包括疏水性外壳，该外壳上设有加样孔及检视窗。所述的样品垫、荧光垫、检测垫、吸样垫和底板组成的试纸条固定在该外壳内。所述的加样孔位于所述样品垫上方，所述检视窗位于所述检测垫上方。
上述的C-反应蛋白(CRP)和髓过氧化物酶(MPO)均是心脑血管疾病的预警标志物，可以通过其含量变化来评估发生心脑血管急重症的风险。所述的心脏脂肪酸结合蛋白(hFABP)是特异性强且灵敏度很高的心肌损伤标记物，可以在无症状或轻微心绞痛症状时检测出心肌损伤状况。该三标记物联合检测，可以更为快捷、更为准确的评价受试者发生心梗等恶性事件的风险，为治疗争取了宝贵时间。
荧光免疫层析检测产品的原理及其结构为公知技术，实现对不同检测对象的检测主要是通过调整工艺部分实现，其中以试剂种类、浓度、pH值、配比等参数的选择尤为重要，由于工艺步骤和涉及试剂繁多，所以工艺筛选优化的工作很难通过一一实践的方法得到，而是需要对实验方案及其结果进行深入分析并在此基础上进行尝试摸索。尤其是多指标同时检测的产品，标记物抗体的性质差异使其具有各自事宜的缓冲系统、用量配比。实现三者间互不干扰且准确检测出其含量，是无法依据常用单个指标检测产品的技术简单调整，或有限次尝试完成的。化学物质间的相互作用还有很多是我们目前科技所不了解的，试验过程中经常会出现理论可行，但结果不理想的状况。
比如本发明活化剂用量的选择。通常荧光微球用量明显多于活化剂用量，而该常用配比范围内，无法得到满足本发明产品中三标志物检测需求的的荧光信号强度。经过大量试验，发现当荧光微球与两活化剂的用量比为1:(14-16):(14-16)时荧光信号很强。活化剂EDC和NHS分别是指碳二亚胺和N-羟基琥珀酰亚胺，其用量比优选为1:1。
在一优选实施例中，本发明所述的荧光微球与两活化剂的用量为1:15:15。
因本发明的活化剂用量较大，因此所述活化剂的加入方式是：先向清洗后荧光微球中加一定量的活化缓冲液，然后分别加入特定用量的EDC和NHS，而不是加活化剂溶液。
本发明所述标记过程的活化时间为25-45分钟，即加入两活化剂后的反应时间为25- 45分钟，低于该时长时偶联率低，荧光信号弱，超过该时长时，微球会出现不同程度的聚集。上述活化时间进一步优选为30分钟，兼顾检测效果、检测效率及检测成本。
在另一优选实施例中，本发明所述标记过程中荧光微球与C-反应蛋白、髓过氧化物酶和心脏脂肪酸结合蛋白三抗体的加入量比为100:13:8:10，即，100μL(1mg/mL)微球需要加入对应C-反应蛋白、髓过氧化物酶和心脏脂肪酸结合蛋白的抗体用量分别为13μg、 8μg和10μg。该配比荧光微球不会聚集且荧光信号最强。
本发明所述荧光垫上的荧光微球标记抗体，还包括鸡IgY抗体。
优选地，质控线通过包被包括但不限于羊抗鼠IgG、羊抗鸡IgY或羊抗兔IgG等抗体形成；所述检测垫为硝酸纤维素膜，进一步优选为孔径5～12um的多孔样结构膜；上样垫、荧光垫的材质为玻璃纤维素膜或无纺布，吸样垫的材质为吸水滤纸，底板的材质为塑料。
所述的荧光微球选自修饰过的聚苯乙烯微球，内部填充镧系元素的螯合物，选自括铕(Eu)、铽(Tb)、钐(Sm)、钕(Nd)或镝(Dy)中的一种。荧光微球检测技术和免疫层析检测技术是已知的，本发明所述的荧光免疫层析试剂盒可在本发明技术方案的基础上采用本领域公知方法或公知方法微调后制备。
本发明提供了一种更为方便、快捷、准确的检测试剂产品，辅助临床及时高效地对心脑血管疾病的高危人群进行诊治，降低非死即残的悲剧发生，同时也有利于减少医保支出，对心梗等急重症的早发现、早治疗具有重要临床的意义和巨大的社会效益。
发明人进一步通过实验来验证本发明的技术效果。
再次重申：以下实验只是本发明研制过程中众多实验中的举例性实验，并未涵盖和穷尽发明人为本发明所做的所有实验，目的仅仅在于阐述不同参数对微球释放效果、荧光信号的影响。
活化剂用量的选择实验
实验方法：按照实施例1中记录的荧光微球标记抗体部分步骤及表1微球及活化剂用量，制做试剂卡。将该试剂卡放入干式荧光免疫分析仪检测，观察信号强度。
表1活化剂用量考察
结果显示，当活化剂用量低于微球用量时，荧光信号非常弱；活化剂用量增加后有所改善，荧光微球与EDC和NHS用量为1:15:15的方5信号非常强，而且与同样EDC和 NHS1:1的方4和方7以及EDC略减量的方6比，方5信号最强。
具体实施方式
实施例1
荧光微球标记抗体部分步骤(1000份)
1)量取荧光微球溶液500μL(1mg/mL)于离心管中，12000r/min，离心15min，弃上清；
2)加入5ml活化缓冲液(0.03mol/L、pH6.0的MES溶液)，12000r/min，离心15min，弃上清；重复清洗一次；再加入5ml活化缓冲液；
3)分别加入特定量的EDC和NHS，常温避光振荡反应一定时间；
4)活化后的溶液，12000r/min，离心15min，弃上清；
5)加入5ml偶联缓冲液(0.01mol/L、pH 7.0的PB缓冲液)，12000r/min，离心15min，弃上清；重复清洗一次；再加入4.0ml偶联缓冲液；
6)加入特定量的抗体，补充适量偶联缓冲液，终体积至5ml，常温避光振荡反应15min。
一种多指标快速检测荧光免疫层析试剂盒，该试剂盒包含检测卡，所述检测卡包括样品垫、荧光垫、具有检测线和质控线的检测垫、吸样垫及底板；其中，所述荧光垫上涂覆有荧光微球标记抗体的涂层，所述的抗体为C-反应蛋白、髓过氧化物酶和心脏脂肪酸结合蛋白的单克隆抗体；该抗体标记过程中所用的活化剂为EDC和NHS用量均为 7.5mg，活化时间为30分钟；所述的检测线有3条，分别为能够与所述涂层中荧光微球标记的三抗体特异性结合的配对抗体形成的涂层。所述质控线上包被羊抗鸡IgY多克隆抗体。按照上述荧光微球标记抗体部分步骤内容及荧光免疫层析试剂卡的其他常规步骤及参数制成试剂卡。
实施例2
与实施例1不同之处在于：所述的活化剂EDC和NHS用量分别为7.0mg和8.0mg，活化时间为25分钟。
实施例3
与实施例1不同之处在于：所述的活化剂EDC和NHS用量分别为8.0mg和7.0mg，活化时间为45分钟。
实施例4
与实施例1不同之处在于：所述的活化剂EDC和NHS用量均为7.0mg，活化时间为 35分钟。
实施例5
与实施例1不同之处在于：C-反应蛋白、髓过氧化物酶和心脏脂肪酸结合蛋白抗体加入量分别为65μg、40μg和50μg。
以上对本发明做了详尽的描述，其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明的精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围内。
Claims (8)
1.一种多指标快速检测荧光免疫层析试剂盒，该试剂盒包含检测卡，所述检测卡包括样品垫、荧光垫、具有检测线和质控线的检测垫、吸样垫及底板，其特征在于：所述荧光垫上涂覆有荧光微球标记抗体的涂层，所述的抗体为C-反应蛋白、髓过氧化物酶和心脏脂肪酸结合蛋白的单克隆抗体；该抗体标记过程中所用的活化剂为EDC和NHS，荧光微球与两活化剂的用量比为：1:(14-16):(14-16)；所述的检测线有3条，分别为能够与所述涂层中荧光微球标记抗体特异性结合的配对抗体或抗原形成的涂层。
2.根据权利要求1所述的试剂盒，其特征在于：所述的两活化剂的用量比为1:1。
3.根据权利要求1所述的试剂盒，其特征在于：所述的荧光微球与两活化剂的用量比为1:15:15。
4.根据权利要求1所述的试剂盒，其特征在于：所述标记过程的活化时间为25-45分钟。
5.根据权利要求4所述的试剂盒，其特征在于：所述标记过程的活化时间为30分钟。
6.根据权利要求1所述的试剂盒，其特征在于：所述标记过程中荧光微球与C-反应蛋白、髓过氧化物酶和心脏脂肪酸结合蛋白三抗体的加入量比为100:13:8:10。
7.根据权利要求1-5任一项所述的试剂盒，其特征在于：所述荧光微球标记抗体，该抗体还包括鸡IgY抗体。
8.根据权利要求7所述的试剂盒，其特征在于：所述的质控线通过包被羊抗鼠IgG、羊抗鸡IgY或羊抗兔IgG抗体形成。
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