CN112763713A - 基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒 - Google Patents

基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒 Download PDF

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CN112763713A
CN112763713A CN202011574376.5A CN202011574376A CN112763713A CN 112763713 A CN112763713 A CN 112763713A CN 202011574376 A CN202011574376 A CN 202011574376A CN 112763713 A CN112763713 A CN 112763713A
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逄越
李庆伟
滕洪明
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Liaoning Normal University
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Abstract

本发明公开一种基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒，有捕获试剂、检测缓冲液及试纸，捕获试剂为生物素化偶联的七鳃鳗免疫蛋白LIP；检测缓冲液为50 mM Tris‑base，0.15 M NaCl，0.01% Tween‑20及7.5% BSA；试纸有底板，在底板上从左至右依次呈阶梯状地固接有加样垫、荧光微球偶联释放垫、硝酸纤维素膜和吸水垫，荧光微球偶联释放垫由铕荧光纳米颗粒与链酶亲和素SA和兔IgG的偶联液喷射在聚酯纤维上制备而成；硝酸纤维素膜上由左到右依次设置有由UMOD单抗构成的检测带和由羊抗兔IgG构成的参照带。

Description

基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试 剂盒
技术领域
本发明属于生物医学检测技术领域，尤其涉及一种基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒。
背景技术
尿路上皮癌是严重威胁人类生命健康的重大疾病之一。“早发现，早诊断，早治疗”是目前尿路上皮癌诊断和治疗的重要发展方向和最有效手段，可显著提高患者生存率。尿路上皮癌包括肾盂癌、膀胱癌、输尿管癌等，现有临床上常用的检查方法有尿液脱落细胞学检查，影像学检查等。尿液脱落细胞学检查的主要标记物是Neu5Gc（N-羟乙酰神经氨酸），检测原理是基于糖链抗体之间的特异性反应，但是由于Neu5Gc存在于除了人和鸡以外的所有哺乳动物体内，对小鼠来说无法引起强烈的免疫应答，因此成功制备单抗的难度较大；虽然Neu5Gc可以诱导鸡产生IgY抗体，但属于多抗，故以Neu5Gc作为癌症标记物特异性较差。
研究表明唾液酸是细胞膜糖蛋白修饰的重要组成部分，已有关于唾液酸修饰UMOD（尿调素）的相关报道，如《尿调素的结构解析及其在人尿路感染时的功能研究》（Cited as:Weiss Gregor L, Stanisich Jessica J, Sauer Maximilian M et al. Architectureand function of human uromodulin filaments in urinary tract infections. [J].Science, 2020, 369: 1005-1010.），该文章的补充材料的实验方法中，公开人尿液中唾液酸修饰UMOD的制备方法，即以硅藻土纯化膀胱癌患者尿液制备Neu5Gc修饰的UMOD糖蛋白。同时文中表明唾液酸修饰UMOD具有促进大肠杆菌凝集，抑制尿道细菌感染的功能。
中国专利号为201310501366.2，名称为“七鳃鳗李蛋白、制备方法及在制备预防和治疗肿瘤疾病药物中的应用”公开了七鳃鳗免疫蛋白LIP的制备方法。
已证明LIP对肿瘤细胞具有独特地识别和选择性杀伤的功能（Cited as: PangYue, Li Changzhi, Wang Shiyue et al. A novel protein derived from lampreysupraneural body tissue with efficient cytocidal actions against tumor cells.[J]. Cell Commun Signal, 2017, 15: 42.）。同时亦有LIP可特异性识别肿瘤细胞膜表面脂筏结构上以Neu5Gc末端修饰的糖型结构的报道（Cited as: Pang Yue, Gou Meng, YangKai et al. Crystal structure of a cytocidal protein from lamprey and itsmechanism of action in the selective killing of cancer cells. [J]. CellCommun Signal, 2019, 17: 54.）。
但是，迄今为止并没有以唾液酸修饰UMOD为标记物而发明的基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的相关报道。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒。
本发明的技术解决方案是：一种基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒，有捕获试剂、检测缓冲液及试纸，其特征在于：所述捕获试剂为生物素化偶联的七鳃鳗免疫蛋白LIP，所述检测缓冲液为50 mM Tris-base，0.15 M NaCl，0.01%Tween-20及7.5% BSA，所述试纸有底板，在底板上从左至右依次呈阶梯状地固接有加样垫、荧光微球偶联释放垫、硝酸纤维素膜和吸水垫，所述荧光微球偶联释放垫由铕荧光纳米颗粒与链酶亲和素SA和兔IgG的偶联液喷射在聚酯纤维上制备而成，所述硝酸纤维素膜上由左到右依次设置有由UMOD单抗构成的检测带和由羊抗兔IgG构成的参照带。
所述生物素化偶联的七鳃鳗免疫蛋白LIP按照如下方法制备：将浓度为0.5 mg/mL的七鳃鳗免疫蛋白LIP水溶液置于试管后加入生物素，室温条件下，充分混匀得到混合液，所述七鳃鳗免疫蛋白LIP的水溶液与生物素的用量比为1 mL：2 mg；将混合液经脱盐柱纯化，得到生物素化偶联的七鳃鳗免疫蛋白LIP。
所述链酶亲和素SA和兔IgG包被的荧光微球偶联液依次按照如下步骤制备：
a. 制备活化羧基化铕荧光纳米微球；
b. 取活化羧基化铕荧光纳米微球、链酶亲和素SA及兔IgG混合，再加入偶联缓冲液将活化羧基化铕荧光纳米微球稀释至2 mg/mL，在37℃条件下反应2 h，所述活化羧基化铕荧光纳米微球、链酶亲和素SA及兔IgG的体积比为2：3：0.5，所述链酶亲和素SA的浓度为1.41 mg/mL，所述兔IgG浓度为5 mg/mL；
c. 将反应液在4 ℃，15000×g条件下离心15 min，取沉淀A；
d. 向沉淀A中加入洗涤液，超声重悬，在4 ℃，15000×g条件下离心15 min，取沉淀B；
e. 向沉淀B中加入封闭液，超声重悬后混匀，得微球悬浮溶液；
f. 将微球悬浮溶液在4 ℃，15000×g条件下离心15 min，取沉淀C；
g. 向沉淀C中加入保存液，超声重悬，在4 ℃，15000×g条件下离心15 min，取沉淀D；
h. 将沉淀D用保存液稀释至2 mg/mL即可；所述保存液中的组分为25 mmol/L的Tris-base、0.15 mol/L的氯化钠、质量浓度为1%的胎牛白蛋白，质量浓度0.05％的吐温-20和质量浓度5%的海藻糖，溶剂为双蒸水，pH 7.8。
本发明所用兔IgG可由鼠IgG替代，当采用鼠IgG时，对应采用羊抗鼠IgG构成参照带。
本发明是以唾液酸修饰UMOD为标记物、基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒，即通过LIP与尿液中修饰UMOD糖蛋白的Neu5Gc结合，结合物中的Neu5Gc修饰UMOD与被预先包被在检测带上的UMOD单抗结合，可准确检测尿液中修饰UMOD的Neu5Gc含量，进而区分正常人、良性疾病（炎症，囊肿等良性肿瘤）和尿路上皮癌。具有操作简单、高灵敏度和特异性等优点，可快速进行大量样本的筛查，具有良好的临床标本检测能力。
附图说明
图1是应用本发明实施例检测不同人尿液的结果示意图。
图2是应用本发明实施例检测各分期膀胱癌患者尿液的结果示意图。
具体实施方式
本发明的基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒，有捕获试剂、检测缓冲液及试纸，所述捕获试剂为生物素化偶联的七鳃鳗免疫蛋白LIP；所述检测缓冲液为50 mM Tris-base，0.15 M NaCl，0.01%Tween-20及7.5%BSA；所述试纸有底板，在底板上从左至右依次呈阶梯状地固接有加样垫、荧光微球偶联释放垫、硝酸纤维素膜和吸水垫，所述荧光微球偶联释放垫由铕荧光纳米颗粒与链酶亲和素SA和兔IgG的偶联液喷射在聚酯纤维上制备而成；所述硝酸纤维素膜上由左到右依次设置有由UMOD单抗构成的检测带和由羊抗兔IgG构成的参照带。UMOD单抗购自ABclonal公司，羊抗兔IgG购自上海生物生工有限生物公司。
所述生物素化偶联的七鳃鳗免疫蛋白LIP按照如下方法制备：取1 mL质量浓度为0.5 mg/mL的七鳃鳗免疫蛋白LIP的水溶液置于试管后加入2 mg生物素，室温条件下，用涡旋混合器充分混匀 2 h，得到混合液；将混合液经离心式脱盐柱纯化，即在4 ℃，5000×g条件下离心5 min，用微量移液器小心吸取流出液，即得到生物素化偶联的七鳃鳗免疫蛋白LIP。七鳃鳗免疫蛋白LIP和生物素的来源分别为：七鳃鳗免疫蛋白LIP按照中国专利号为201310501366.2，名称为“七鳃鳗李蛋白、制备方法及在制备预防和治疗肿瘤疾病药物中的应用”所公开的制备方法制备，生物素购自Thermo生物公司。
所述链酶亲和素SA和兔IgG包被的荧光微球偶联液依次按照如下步骤制备：
a. 按照现有技术的方法制备活化羧基化铕荧光纳米微球：
加入1 mL羧基化铕荧光纳米微球活化缓冲液于试管中，用涡旋混合器充分混匀悬浮羧基化铕荧光纳米微球，在4 ℃，15000×g条件下离心15 min，用微量移液器小心吸走上清液，重复该步骤1次；羧基化铕荧光纳米微球购自Thermo生物公司。
加入14 μL羧基化铕荧光纳米微球活化试剂EDC和130 μL试剂NHS，用涡旋混合器充分混匀悬浮羧基化铕荧光纳米微球，转移至2 mL离心管中，补加854 μL活化缓冲液，室温下用微球混合器旋转30 min，在4 ℃，15000×g条件下离心30 min，用微量移液器小心吸走上清液；
加入1 mL偶联缓冲液，重悬羧基化铕荧光纳米微球，超声重悬微球均匀分散（50W，工作 1s，间隔1s，持续3 min；后续超声条件形同），在4 ℃，15000×g条件下离心30 min，用微量移液器小心吸走上清液，重复该步骤1次，得到活化羧基化铕荧光纳米微球；
b. 取活化羧基化铕荧光纳米微球、链酶亲和素SA及兔IgG混合，再加入偶联缓冲液将活化羧基化铕荧光纳米微球稀释至2 mg/mL，在37℃条件下，用涡旋混合器充分混合，偶联反应2 h；所述活化羧基化铕荧光纳米微球、链酶亲和素SA及兔IgG的体积分别为200mL、300 mL和50 mL，所述链酶亲和素SA的浓度为1.41 mg/mL，所述兔IgG浓度为5 mg/mL；链酶亲和素SA购买自invitrogen公司，兔IgG购自上海生物生工有限生物公司。
. 将偶联反应液在4 ℃，15000×g条件下离心15 min，取沉淀A；
d. 向沉淀A中加入洗涤液，超声重悬，在4 ℃，15000×g条件下离心15 min，取沉淀B，重复该步骤1次；
e. 向沉淀B中加入封闭液，超声重悬后再置于另一离心管中，再用涡旋混合器充分混合悬浮羧基化铕荧光纳米微球，旋转标记1小时，得微球悬浮溶液；
f. 将微球悬浮溶液在4 ℃，15000×g条件下离心15 min，取沉淀C；
g. 向沉淀C中加入保存液，超声重悬羧基化铕荧光纳米微球，在4 ℃，15000×g条件下离心15 min，取沉淀D，重复该步骤1次；
h. 将沉淀D用保存液稀释至2 mg/mL即可；所述保存液中的组分为25 mmol/L的Tris-base、0.15 mol/L的氯化钠、质量浓度为1%的胎牛白蛋白，质量浓度0.05％的吐温-20和质量浓度 5%的海藻糖，溶剂为双蒸水，pH 7.8。
本发明的基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒，使用方法依次按照如下步骤进行：
C1.准备检测样品：取患者尿液1 mL，3000×g条件下离心30 min，去除尿残渣，取上清液即为检测样品；
C2.将C1中检测样品100 μL和100 μL稀释的捕获试剂（生物素化LIP蛋白）混合得混合液，稀释的捕获试剂是将生物素化LIP蛋白与检测试剂按照体积比为1:50稀释；
C3.吸取100 μL步骤C2所得混合液滴加在加样垫上，静置2 min，将试纸条放置于干式荧光免疫分析仪AFS 1000进行检测，观察并记录所述检测带和所述参照带荧光情况，即HT和HC及其比值R（HT/HC）。若检测带荧光强度与参照带荧光强度相比无区别，则检测尿液样本中无修饰UMOD糖蛋白的Neu5Gc；若检测带荧光强度与参照带荧光强度有区别，则检测尿液样本中含有修饰UMOD糖蛋白的Neu5Gc，对照标准曲线即可分析出待测样本中修饰UMOD糖蛋白的Neu5Gc含量；若参照带没有荧光强度，结果无效。
本发明在4 ℃下、三个月内性能稳定，检测指标不发生偏差。
标准品曲线是用磷酸盐缓冲液（PBS：0.01mol/L，pH 7.4）分别配置浓度为0 ng/mL、1 ng/mL、5 ng/mL、10 ng/mL、20 ng/mL、30 ng/mL、50 ng/mL、100 ng/mL的Neu5Gc修饰UMOD标准品溶液，分别用标准品溶液代替检测样品按照上述检测方法进行检测，2 min后观察检测的荧光情况。重复10次平行实验。最终确定本发明检测Neu5Gc修饰UMOD标准品最低浓度为30 ng/mL。Neu5Gc修饰UMOD标准品来源：采用现有技术报道的方法，以硅藻土纯化膀胱癌患者尿液制备Neu5Gc修饰UMOD糖蛋白。
本发明的检测原理：将受检者尿液蛋白与LIP-生物素化溶液混合后，如果尿液样本中有Neu5Gc修饰的UMOD糖蛋白，则修饰UMOD糖蛋白的Neu5Gc可以和LIP-生物素结合形成复合物；复合物中的LIP-生物素可以和荧光微球偶联释放垫中的链霉亲和素SA免疫荧光微球结合，在层析至检测带时，复合物中的Neu5Gc修饰UMOD与被预先包被在检测带上的UMOD单抗结合。检测带上的荧光信号强度与尿液样本中Neu5Gc修饰的UMOD糖蛋白浓度成正比，用仪器对样本检测结果进行判读定量分析。在层析至参照带时，兔IgG偶联的荧光微球可与被预先包被在参照带上的山羊抗兔IgG结合。根据参照带上有无荧光信号，判读样本加样和层析过程是否存在问题。
实验例1：
采用本发明实施639例正常人、25例良性疾病（膀胱炎症8例，膀胱囊肿7例，膀胱息肉6例，肾炎4例）、99例尿路上皮癌患者（52例膀胱癌、20例肾癌和27例前列腺癌）的尿液检测样品进行了检测，结果如图1所示。图1显示正常人尿液样本LIP特异性识别修饰UMOD糖蛋白的Neu5Gc含量在67.63±9.31 ng/mg；25例良性疾病尿液样本中LIP特异性识别修饰UMOD糖蛋白的Neu5Gc含量相对于正常人没有显著性差异，范围在70.71±8.20 ng/mg。结果表明：筛查尿路系统癌症，当尿路存在炎症等良性问题时，LIP特异性识别修饰UMOD糖蛋白的Neu5Gc与正常人识别含量没有差异，不会造成假阳性；52例膀胱癌患者尿液样本LIP特异性识别修饰UMOD糖蛋白的Neu5Gc含量范围在260.25±29.38 ng/mg，20例肾癌患者范围在280.54±11.81 ng/mg，27例前列腺癌范围在264.34±30.63 ng/mg。该试剂盒诊断泌尿上皮癌特异性良好。针对样本数较多的52例膀胱癌患者进一步分析，结果如图2所示。结果显示：不同分期的膀胱癌患者尿液中LIP特异性识别修饰UMOD糖蛋白的Neu5Gc存在差异，膀胱癌处在早期Ta期时，LIP特异性识别尿液中修饰UMOD糖蛋白的Neu5Gc含量显著升高。通过对比检测样本中，LIP可特异性识别尿液中修饰UMOD糖蛋白的Neu5Gc含量范围，从而达到筛查检测样本是否为尿路上皮癌的目的，准确率高达80％以上。

Claims (3)

1. 一种基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒，有捕获试剂、检测缓冲液及试纸，其特征在于：所述捕获试剂为生物素化偶联的七鳃鳗免疫蛋白LIP，所述检测缓冲液为50 mM Tris-base，0.15 M NaCl，0.01% Tween-20及7.5% BSA，所述试纸有底板，在底板上从左至右依次呈阶梯状地固接有加样垫、荧光微球偶联释放垫、硝酸纤维素膜和吸水垫，所述荧光微球偶联释放垫由铕荧光纳米颗粒与链酶亲和素SA和兔IgG的偶联液喷射在聚酯纤维上制备而成，所述硝酸纤维素膜上由左到右依次设置有由UMOD单抗构成的检测带和由羊抗兔IgG构成的参照带。
2. 根据权利要求1所述基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒，其特征在于所述生物素化偶联的七鳃鳗免疫蛋白LIP按照如下方法制备：将浓度为0.5mg/mL的七鳃鳗免疫蛋白LIP水溶液置于试管后加入生物素，室温条件下，充分混匀得到混合液，所述七鳃鳗免疫蛋白LIP的水溶液与生物素的用量比为1 mL：2 mg；将混合液经脱盐柱纯化，得到生物素化偶联的七鳃鳗免疫蛋白LIP。
3.根据权利要求1或2所述基于LIP识别尿液中修饰UMOD的Neu5Gc检验尿路上皮癌的试剂盒，其特征在于所述链酶亲和素SA和兔IgG包被的荧光微球偶联液依次按照如下步骤制备：
a. 制备活化羧基化铕荧光纳米微球；
b. 取活化羧基化铕荧光纳米微球、链酶亲和素SA及兔IgG混合，再加入偶联缓冲液将活化羧基化铕荧光纳米微球稀释至2 mg/mL，在37℃条件下反应2 h，所述活化羧基化铕荧光纳米微球、链酶亲和素SA及兔IgG的体积比为2：3：0.5，所述链酶亲和素SA的浓度为1.41mg/mL，所述兔IgG浓度为5 mg/mL；
c. 将反应液在4 ℃，15000×g条件下离心15 min，取沉淀A；
d. 向沉淀A中加入洗涤液，超声重悬，在4 ℃，15000×g条件下离心15 min，取沉淀B；
e. 向沉淀B中加入封闭液，超声重悬后混匀，得微球悬浮溶液；
f. 将微球悬浮溶液在4 ℃，15000×g条件下离心15 min，取沉淀C；
g. 向沉淀C中加入保存液，超声重悬，在4 ℃，15000×g条件下离心15 min，取沉淀D；
h. 将沉淀D用保存液稀释至2 mg/mL即可；所述保存液中的组分为25 mmol/L的Tris-base、0.15 mol/L的氯化钠、质量浓度为1%的胎牛白蛋白，质量浓度0.05％的吐温-20和质量浓度5%的海藻糖，溶剂为双蒸水，pH 7.8。
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