CN115327110A - 时间分辨荧光微球建立的双酚s侧流层析试纸条及方法 - Google Patents
时间分辨荧光微球建立的双酚s侧流层析试纸条及方法 Download PDFInfo
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Abstract
时间分辨荧光微球建立的双酚S侧流层析试纸条及方法,所述试纸条包含BPS完全抗原、时间分辨荧光微球标记的BPS单克隆抗体。利用5‑溴戊酸甲酯做为中间产物制备了BPS的完全抗原,然后以BPS完全抗原免疫小鼠获得了BPS高亲和力和高特异性的单克隆抗体。随后,使用时间分辨荧光微球作为标记物建立了BPS的侧流层析试纸条,并发现该试纸条的检测范围为1.2~625 ng/mL。本发明可以用于水样中的环境基质,生物样品,食品和药品中的BPS检测工作。同时该方法与目前常用的化学方法相比,更为简便,灵敏,检测时间仅为10 min,不需要昂贵的设备和复杂的前处理过程。
Description
技术领域
本发明属于环境检测领域,具体涉及一种时间分辨荧光微球建立的双酚S侧流层析试纸条及方法。
背景技术
双酚S(Bisphenol S, BPS)是一种双酚A(Bisphenol A, BPA)的替代品,因为与BPA相比具有更高的热稳定性和光稳定性,被广泛用于生产聚碳酸酯塑料和树脂。但是随着BPS在“BPA-free”产品中的使用越来越多,其进入环境的潜力也越来越大,导致BPS在各种环境基质和生物样品中时有检出。如太湖水体中BPS的浓度均值为16 ng/L;中国成人尿液样本(n=160)中BPS的检出浓度为0.24 µg/L。研究表明BPS具有激素活性会干扰生物体正常的内分泌功能。如环境浓度的BPS暴露会对斑马鱼发育和生殖产生毒性效应。另外,母体在低浓度的BPS暴露下会导致子代神经和内分泌系统受损。鉴于BPS在环境中具有较高的浓度和毒性效应,因此亟需建立BPS的检测方法。
目前BPS的检测方法以高效液相色谱和气相色谱-质谱法为主,上述方法虽然具有较高的精度和准确性,但是其复杂的样品前处理过程和昂贵的设备等问题使上述方法难以普及,限制了BPS的检测工作。免疫学检测方法是利用抗体对抗原的识别作用对BPS进行检测,其中侧流层析免疫试纸条因为具有成本低、分析时间短、简单便携和可现场测量等优点,被大量用于快速检测工作。如基于金纳米颗粒建立了检测吡虫啉的侧流免疫层析试纸条,可有效替代最常用的ELISA检测方法。但是目前常规的BPS测流层析试纸条通常是以胶体金作为标记物,因为其本身背景值较高,且是通过离子吸附的方式与抗体结合,存在结合力较弱等缺点,导致检测灵敏度不高。时间分辨荧光微球,是一种利用稀有金属开发的荧光微球,荧光背景值较低,与常规的荧光标记物相比,更加稳定不易淬灭,另外这种荧光微球表面带有羧基基团,可以与抗体通过共价键连接,结合力更稳定。
因此本发明通过制备了BPS高亲和力和高特异性的单克隆抗体,并将该抗体与时间分辨荧光微球偶联建立BPS的侧流层析试纸条,以期为BPS检测工作提供一种性能良好和检测简便的检测方法。
发明内容:
本发明的目的在于克服现有技术存在的缺点,提供一种时间分辨荧光微球建立的双酚S侧流层析试纸条及方法,该试纸条具有灵敏、快捷和准确的特点。
时间分辨荧光微球建立的双酚S侧流层析试纸条,其特征在于该试纸条包含BPS完全抗原、时间分辨荧光微球标记的BPS单克隆抗体;
所述的BPS完全抗原使用5-溴戊酸甲酯作为连接臂,所述BPS完全抗原的载体蛋白为牛血清白蛋白、卵清蛋白、钥孔血蓝蛋白、甲状腺球蛋白、纤维蛋白原或明胶。
所述时间分辨荧光微球标记的BPS单克隆抗体中的BPS单克隆抗体,由权利要求1所述BPS完全抗原制得。
所述BPS完全抗原通过以下方法制得:
首先称取0.25 g BPS加入50 mL乙腈中搅拌至完全溶解,随后加入0.415 g碳酸钾反应1 h后,将0.293 g 5-溴戊酸甲酯加热回流24 h后,移除有机溶剂后将得到的固体过柱(石油醚/乙酸乙酯)纯化,即获得BPS的半抗原;称取10 mg BPS半抗原、8.9 mg碳酰二亚胺(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC)、6.2 mg N-羟基琥珀酰亚胺(1-hydroxy-5-pyrrolidinedione, NHS)溶于4 mL N,N-二甲基甲酰胺(N,N-Dimethylformamide, DMF)中,室温下避光反应2 h;将上述溶液逐滴加入到2 mL 含有20mg卵清蛋白(Ovalbumin, OVA)或者15 mg 牛血清白蛋白(Bovine albumin, BSA)的磷酸缓冲液中,4 ℃反应震荡过夜后,6000 r/min离心5 min取上清,置于0.01 M PBS中透析32 h,每8 h更换一次透析液;所得溶液再次离心获得上清液即为完全抗原其中加入卵清蛋白时制得的完全抗原记为BPS-BSA,作为免疫抗原;加入牛血清白蛋白时制得的完全抗原记为BPS-OVA,作为包被抗原。
所述时间分辨荧光微球标记的BPS单克隆抗体通过以下步骤制备:
取0.5 mg 时间分辨荧光微球,加入浓度为0.5 mg/mL EDC和NHS,室温反应30 min后,12000 r/min离心10 min后弃上清液,加入1 mL PBS重新沉淀后,使用超声波震荡均匀,加入总量为0.3 mg BPS单克隆抗体室温反应2 h后,加入终浓度为0.25% 的OVA封闭30min,离心弃上清后加入抗体复溶液(0.01 M Tris-HCl,5% 蔗糖,1% OVA,pH8.0)重新悬浮沉淀,即获得时间分辨荧光微球标记的BPS单克隆抗体。
所述试纸条通过以下步骤完成组装:
将时间分辨荧光微球标记的BPS单克隆抗喷涂于玻璃纤维膜后作为结合垫,进行干燥,然后将1.25 mg/mL羊抗鼠抗体和0.18 mg/mL BPS-BSA喷涂于硝酸纤维素膜,然后将硝酸纤维素膜、结合垫、样品垫、吸水垫依次组装于PVC板上后,使用斩切机剪切成4 mm的条状,即为时间分辨荧光微球建立的双酚S侧流层析试纸条。
所述时间分辨荧光微球标记的BPS侧流层析试纸条,主要用于环境介质和生物样品中BPS的快速检测。
发明优点
本发明使用5-溴戊酸甲酯作为连接臂制备了BPS半抗原,由于其具有更长的碳链,可以使BPS特征基团充分暴露,从而获得具有更高亲和力和特异性的BPS单克隆抗体。随后,使用时间分辨荧光微球作为标记物,由于时间分辨荧光微球通过镧系金属铕制备,具有较低的背景值的同时,荧光更加稳定,便于开发具有较高的灵敏度的检测方法。另外,本发明制备的测流层析试纸条操作简单、便于携带、检测成本低廉,无需辅助仪器设备便可完成相应的检测,尤其适用于野外快速检测样品中的BPS。
附图说明
图1为BPS半抗原合成图。
图2为BPS完全抗原合成图。
图3为BPS完全抗原红外光谱鉴定结果图。
图4为BPS完全抗原聚丙烯酰胺凝胶电泳结果图。
图5为BPS侧流层析试纸条最佳T线和C线优化结果图。
图6为试纸条对于不同浓度BPS的实际检测图。
图7为BPS侧流层析试纸条标准曲线。
具体实施方式
时间分辨荧光微球建立的双酚S侧流层析试纸条,其主要结构包括PVC胶板,PVC胶板表面设有样品垫、吸水垫、硝酸纤维素膜和结合垫;其特征在于该试纸条包含BPS完全抗原、时间分辨荧光微球标记的BPS单克隆抗体;所述BPS完全抗原包被于硝酸纤维素膜上,所述时间分辨荧光微球标记的BPS单克隆抗体包被在结合垫上。
下面通过具体实施例并结合附图对本发明作进一步阐述。
实施例1、BPS半抗原和单克隆抗体的制备
(1)BPS半抗原的合成
首先,按照图1所示制备BPS半抗原,称取0.25 g BPS加入50 mL乙腈中搅拌至完全溶解,随后加入0.415 g碳酸钾反应1 h后,将0.293 g 5-溴戊酸甲酯加热回流24 h后,移除有机溶剂后,将得到的固体过柱(石油醚/乙酸乙酯)纯化,即可获得BPS的半抗原。
(2)完全抗原的合成
按照图2所示制备BPS完全抗原,称取10 mg BPS半抗原、8.9 mg EDC、6.2 mg NHS溶于4 mL DMF中,室温下避光反应2 h。将上述溶液逐滴加入到2 mL 含有20 mg OVA或者15mg BSA的磷酸缓冲液中,4 ℃反应震荡过夜后,6000 r/min离心5 min取上清,置于0.01 MPBS中透析32 h,每8 h更换一次透析液。所得溶液再次离心获得上清液即为完全抗原(BPS-OVA/BPS-BSA)。其中BPS-BSA为免疫抗原,BPS-OVA为包被抗原。将制备的完全抗原进行红外光谱扫描,结果如图3所示,与BSA、OVA偶联后的完全抗原红外吸收峰出现了明显的特征峰,图4的聚丙烯酰胺凝胶电泳结果也显示,完全抗原分子量明显大于BSA或OVA,以上结果均证实本发明制备了BPS的完全抗原。
(3)BPS特异性抗体的制备
选取3只健康Balb/C小鼠作为实验生物,将100 µg的BPS-BSA和等体积的弗氏完全佐剂乳化后注射小鼠腹腔。每间隔14天,使用相同剂量的BPS-BSA与弗氏不完全佐剂充分乳化之后对小鼠进行免疫。第4次免疫结束后3天,将小鼠处死取脾脏备用。然后复苏小鼠骨髓瘤细胞SP2/0,将小鼠骨髓瘤细胞和小鼠脾脏细胞按照1:5的比例进行混合后,加入1 mL 聚乙二醇1500,静置90 s后,1000 r/min离心10 min,将上清弃去,加入培养基重悬,并转入96孔板进行培养后,使用有限稀释法获得能分泌BPS抗体的细胞株,将细胞株注射入小鼠体内后获得腹水,使用Protein G亲和层析柱对抗体进行纯化,获得BPS特异性抗体。
(4)时间分辨荧光微球标记抗体
首先,取0.5 mg 荧光微球,加入浓度为0.5 mg/mL EDC和NHS,室温反应30 min后,12000 r/min离心10 min后弃上清液,加入1 mL PBS重新沉淀后,使用超声波震荡均匀。加入总量为0.3 mg抗体室温反应2 h后,加入终浓度为0.25% OVA封闭30 min,离心弃上清后加入抗体复溶液(0.01 M Tris-HCl,5% 蔗糖,1% OVA,pH8.0)重新悬浮沉淀,即获得荧光微球标记的BPS单克隆抗体。
(5)结合垫的制备:取一条长取一条长10 cm,宽约0.5 cm的玻璃纤维膜,将上述400 µL时间分辨荧光微球标记的BPS单克隆抗体均匀地涂在该玻璃纤维膜上,于37℃干燥,塑封袋密封后,于4℃保存备用。
(6)试纸条最佳T线和C线浓度的优化:为确定最佳的质控线浓度(Control line,C),将0.16~10 mg/mL的羊抗鼠IgG抗体喷涂于硝酸纤维素膜作为C线,显色后观察结果。随后确定最佳的检测线浓度(Testing line, T),将2.5 mg/mL的羊抗鼠抗体喷涂于硝酸纤维素膜作为C线,将浓度为0.02~1.4 mg/mL的BPS-OVA喷涂于硝酸纤维膜作为T线,显色后观察最佳的T线浓度。结果如图5显示,当C线浓度为1.25 mg/mL,T线浓度为0.18 mg/mL时,侧流层析试纸条的检测效果最佳。
(7)PVC板上依次贴硝酸纤维素膜、胶体金结合垫、样品垫、吸水垫,将组装好的PVC板用斩切机纵向剪切成宽度为4 mm的条状,即为BPS侧流层析试纸条。试纸条对于不同浓度的BPS的实际检测图及标准曲线如图6所示。结果发现在1.2~625 ng/mL的范围下,标准曲线呈现良好的线性关系(图7)。
本发明的一种检测BPS的侧流层析试纸条,可用于环境基质和生物样本的快速检测,使用时将待测样品滴加侧流层析试纸条的样品垫处,10 min后观察显色反应。
Claims (5)
1.时间分辨荧光微球建立的双酚S侧流层析试纸条,其特征在于该试纸条包含BPS完全抗原、时间分辨荧光微球标记的BPS单克隆抗体;
所述的BPS完全抗原使用5-溴戊酸甲酯作为连接臂,所述BPS完全抗原的载体蛋白为牛血清白蛋白、卵清蛋白、钥孔血蓝蛋白、甲状腺球蛋白、纤维蛋白原或明胶。
2.根据权利要求1所述的试纸条,其特征在于所述时间分辨荧光微球标记的BPS单克隆抗体中的BPS单克隆抗体,由权利要求1所述BPS完全抗原制得。
3.根据权利要求1所述的试纸条,其特征在于所述BPS完全抗原通过以下方法制得:
首先称取0.25 g BPS加入50 mL乙腈中搅拌至完全溶解,随后加入0.415 g碳酸钾反应1 h后,将0.293 g 5-溴戊酸甲酯加热回流24 h后,移除有机溶剂后将得到的固体过柱纯化,即获得BPS的半抗原;称取10 mg BPS半抗原、8.9 mg碳酰二亚胺、6.2 mg N-羟基琥珀酰亚胺溶于4 mL N,N-二甲基甲酰胺中,室温下避光反应2 h;将上述溶液逐滴加入到2 mL 含有20 mg卵清蛋白或者15 mg 牛血清白蛋白的磷酸缓冲液中,4℃反应震荡过夜后,6000 r/min离心5 min取上清,置于0.01 M PBS中透析32 h,每8 h更换一次透析液;所得溶液再次离心获得上清液即为BPS完全抗原,其中加入卵清蛋白时制得的完全抗原记为BPS-BSA,作为免疫抗原;加入牛血清白蛋白时制得的完全抗原记为BPS-OVA,作为包被抗原。
4.如权利要求3所述的试纸条,其特征在于所述时间分辨荧光微球标记的BPS单克隆抗体通过以下步骤制备:
取0.5 mg 时间分辨荧光微球,加入浓度为0.5 mg/mL EDC和NHS,室温反应30 min后,12000 r/min离心10 min后弃上清液,加入1 mL PBS重新沉淀后,使用超声波震荡均匀,加入总量为0.3 mg BPS单克隆抗体室温反应2 h后,加入终浓度为0.25% 的OVA封闭30 min,离心弃上清后加入抗体复溶液(0.01 M Tris-HCl,5% 蔗糖,1% OVA,pH8.0)重新悬浮沉淀,即获得荧光微球标记的BPS单克隆抗体。
5.如权利要求4所述的试纸条,其特征在于所述试纸条通过以下步骤完成组装:
将时间分辨荧光微球标记的BPS单克隆抗喷涂于玻璃纤维膜作为结合垫后,进行干燥,然后将1.25 mg/mL羊抗鼠抗体和0.18 mg/mL BPS-BSA喷涂于硝酸纤维素膜,然后将硝酸纤维素膜、结合垫、样品垫、吸水垫依次组装于PVC板上后,使用斩切机剪切成4 mm的条状,即为时间分辨荧光微球建立的双酚S侧流层析试纸条。
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