CN112763713B - 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 PDFInfo
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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
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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