CN112730855A - 一种基于抗人β-HCG双特异性抗体胶体金层析试纸条 - Google Patents
一种基于抗人β-HCG双特异性抗体胶体金层析试纸条 Download PDFInfo
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- CN112730855A CN112730855A CN202011576408.5A CN202011576408A CN112730855A CN 112730855 A CN112730855 A CN 112730855A CN 202011576408 A CN202011576408 A CN 202011576408A CN 112730855 A CN112730855 A CN 112730855A
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Abstract
本发明公开了一种基于抗人β‑HCG双特异性抗体胶体金层析试纸条，所述基于抗人β‑HCG双特异性抗体胶体金层析试纸条包括PVC底板、样本垫、硝酸纤维膜、吸水垫及金标垫，所述样本垫、硝酸纤维膜、吸水垫及金标垫均固定于PVC底板上，所述硝酸纤维膜的两端分别与吸水垫的内端、金标垫的内端搭接，所述样本垫的内端与金标垫的外端搭接，所述硝酸纤维素膜上设有检测线以及质控线；所述金标垫上固化有胶体金‑抗人β‑HCG双特异性抗体结合物及胶体金‑鸡IgY抗体结合物；所述检测线上包被抗人β‑HCG双特异性抗体，所述质控线上包被羊抗鸡IgY多抗。本发明灵敏度高、稳定性好，适合现场快速检测。
Description
技术领域
本发明涉及一种早孕检测试纸条，尤其是涉及一种基于抗人β-HCG双特异性抗体胶体金层析试纸条。
背景技术
成熟女性因受精的卵子移动到子宫腔内着床后，形成胚胎，在发育成长为胎儿过程中，胎盘合体滋养层细胞产生大量的人绒毛膜促性腺激素（human chorionicgonadotropin，HCG），可通过孕妇尿液循环而排泄到尿中。当妊娠1-2.5周时，血清和尿中的HCG水平即可迅速升高，第8孕周达到高峰，至孕期第4个月始降至中等水平，并一直维持到妊娠末期。
HCG是由两个非共价键相连的肽链组成的糖蛋白激素。其单个亚基不具有生物活性，当连接成完整化合物时始具活性，分子量约为4.7万，其主要功能就是刺激黄体，有利于雌激素和黄体酮持续分泌，以促进了宫蜕膜的形成，使胎盘生长成熟。HCG-α亚单位的结构与LH-α，FSH-α及TSH-α亚单位相似，故用完整的抗HCG分子的抗体测定HCG时与LH，FSH及TSH间有免疫交叉反应。但它们的β亚单位各不相同。因此为避免交叉反应，目前均采用抗β-HCG抗体进行特异的HCG检查，近年来还有报道采用抗β-HCG羧基末端肽抗体以进一步提高检测的敏感性和特异性。
免疫胶体金法是将鼠抗人HCG抗体、羊抗鸡IgY多抗分别固定在特制的纤维素膜上并呈两条线上下排列，羊抗鸡IgY多抗线在试纸条上方为阴性对照，鼠抗人HCG多抗在下方为测定线。试纸条中含均匀分布的胶体金标记鼠抗人β-HCG抗体和胶体金标记鸡IgY。检测时将试纸条浸入被检尿液中（液面低于固定的两条抗体线）后迅速取出。尿液沿试带上行，尿中的HCG在上行过程中与胶体金标记抗体结合，待行至鼠抗人β-HCG抗体线时，形成金标记的鼠抗人β-HCG与人HCG夹心复合物而在试带上呈紫红色区带，为HCG阳性反应，试纸上无关的金标记鸡IgY随尿液继续上行至羊抗鸡IgY多抗处时与之形成紫红色的金标记的抗原体复合物是为阴性对照。判断结果时，含HCG的尿液试带可显示上、下两条紫红色线条，而阴性标本则只显出上边一条紫红色线。
现有胶体金法检测试纸条其准确度和灵敏度很大程度上依赖于标记抗体和包被抗体的性能，由于HCG与LH、TSH、FSH的β亚单位虽不相同，但整体β链仅有部分序列不同，且差异化片段长度较小。这样使得使用传统的抗原免疫法制备的抗体特异性不佳，且过小的抗原片段带来免疫及后期纯化的困难，进而不利于最终用于胶体金标记的抗体的灵敏度。
发明内容
本发明是为了解决现有技术的胶体金法检测试纸条所存在的上述技术问题，提供了一种灵敏度高、稳定性好，适合现场快速检测的基于抗人β-HCG双特异性抗体胶体金层析试纸条。
为了实现上述目的，本发明采用以下技术方案：一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，所述基于抗人β-HCG双特异性抗体胶体金层析试纸条包括PVC底板、样本垫、硝酸纤维膜、吸水垫及金标垫，所述样本垫、硝酸纤维膜、吸水垫及金标垫均固定于PVC底板上，所述硝酸纤维膜的两端分别与吸水垫的内端、金标垫的内端搭接，所述样本垫的内端与金标垫的外端搭接，所述硝酸纤维素膜上设有检测线以及质控线；所述金标垫上固化有胶体金-抗人β-HCG双特异性抗体结合物及胶体金-鸡IgY抗体结合物；所述检测线上包被抗人β-HCG双特异性抗体，所述质控线上包被羊抗鸡IgY多抗。
作为优选，所述吸水垫、样本垫及金标垫上设有保护膜。
作为优选，所述样本垫通过以下方法制得：将玻璃纤维裁剪成所需规格后，用样本垫处理液喷涂均匀，烘干。烘干为37℃烘12-16h。
作为优选，所述样本垫处理液通过以下方法制得：80ml超纯水中加入3.58gNa2HPO4·12H2O，0.5g Casein Na，0.9g NaCl，1g 表面活性剂S9，1g PVP，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节pH至8.0，加超纯水定容至100ml。
作为优选，所述硝酸纤维膜通过以下方法制得：将硝酸纤维膜贴在PVC底板上，用包被缓冲液分别将抗人β-HCG双特异性抗体稀释至浓度为1.0mg/ml，将羊抗鸡IgY多抗稀释至浓度为0.5mg/ml，将稀释好的抗人β-HCG双特异性抗体溶液以喷量1μl/cm均匀包被在硝酸纤维膜的检测线上，将稀释好的羊抗鸡IgY多抗溶液以喷量1μl/cm均匀包被在硝酸纤维膜的质控线上；将硝酸纤维膜烘干。烘干为37℃烘干12-16h；检测线与质控线间的间距控制在0.5cm。
作为优选，所述包被缓冲液通过以下方法制得：80ml超纯水中加入1.79gNa2HPO4.12H2O，0.9g Nacl，2g 蔗糖，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节PH至8.0，加超纯水定容至100ml。
作为优选，所述金标垫通过以下方法制得：
（1）用量筒量取需要标记量的粒径40-50nm胶体金溶液，按体积百分含量为1%加入0.1M、pH为7.6的Na2HPO4·12H2O溶液，在磁力搅拌器上边搅拌边用1M NaOH调pH值至7.5，取抗人β-HCG双特异性抗体，按5ug/ml比例标记胶体金，一次性快速加入至胶体金溶液中，继续快速搅拌30min后，按体积百分含量为1%加入100g/L的稳定剂BSA，搅拌20min后10000r/min、10℃离心30min，去除上清，用金标保存液重悬至原体积的1/100，涡旋混匀，得胶体金-抗人β-HCG双特异性抗体结合物，备用。
（2）用量筒量取需要标记量的粒径40-50nm胶体金溶液，按体积百分含量为1%加入0.1M、pH为7.6的Na2HPO4·12H2O溶液，在磁力搅拌器上边搅拌边用1M NaOH调pH值至8.0，取鸡IgY抗体，按10ug/ml比例标记胶体金，一次性快速加入至胶体金溶液中，继续快速搅拌30min后，按体积百分含量为1%加入浓度为100g/L的稳定剂BSA，搅拌20min后10000r/min、10℃离心30min，去除上清，用金标保存液重悬至原体积的1/100，涡旋混匀，即得胶体金-鸡IgY抗体结合物，备用。
（3）将上述胶体金-抗人β-HCG双特异性抗体结合物和胶体金-鸡IgY抗体结合物按体积比5：1比例混合，用金标稀释液对该混合物按体积百分含量50%进行稀释，涡旋混匀，用三维点膜喷金仪以喷量6ul/cm均匀喷涂在金标垫上，烘干。烘干为37℃烘干2-4h。
作为优选，所述金标保存液通过以下方法制得：80ml超纯水中加入1.79g Na2HPO4·12H2O，1g BSA，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节PH至8.0，加超纯水定容至100ml。
作为优选，所述金标稀释液通过以下方法制得：80ml超纯水中加入1.79g Na2HPO4·12H2O，0.5g Casein Na，1g Tween-20，20g 蔗糖，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节PH至8.0，加超纯水定容至100ml。
作为优选，所述抗人β-HCG双特异性抗体通过以下方法制得：
（a）抗人β-HCG双特异性抗体序列设计
通过计算机分子结构模拟及序列分析，优化分泌信号肽及载体表达元件，设计抗人β-HCG双特异性抗体的氨基酸序列，所述抗人β-HCG双特异性抗体的氨基酸序列包含有抗β-HCG表位a和β-HCG表位b的重组重链部分以及β-HCG表位a的轻链部分，所述抗人β-HCG双特异性抗体的重组重链氨基酸序列如SEQ ID No：1所示，所述抗人β-HCG双特异性抗体的轻链氨基酸序列如SEQ ID No：2所示。
（b）抗人β-HCG双特异性抗体基因克隆
在抗体序列不变的前提下，根据CHO细胞偏爱密码子分别将抗人β-HCG双特异性抗体的重组重链氨基酸序列、抗人β-HCG双特异性抗体的轻链氨基酸序列分别转化为相应的核苷酸序列，分别得到抗人β-HCG双特异性抗体的重组重链核苷酸序列和抗人β-HCG双特异性抗体的轻链核苷酸序列；在抗人β-HCG双特异性抗体的重组重链核苷酸序列上下游添加Hind III与BstE II的酶切位点，在抗人β-HCG双特异性抗体的核苷酸序列上下游添加SalI与BsiWI的酶切位点；通过Hind III与BstE II双酶切将抗人β-HCG双特异性抗体的重组重链核苷酸序列连接到表达载体pG4HK上，获得抗人β-HCG双特异性抗体重组重链的表达载体1，质粒命名为pG4HK-β-HCG-1；通过SalI与BsiWI双酶切将抗人β-HCG双特异性抗体的轻链核苷酸序列的融合片段连接到载体pG4HK上，获得抗人β-HCG双特异性抗体轻链表达载体2，质粒命名为pG4HK-β-HCG-2；利用无内毒素大提试剂盒进行质粒大提。
（c）重组表达载体转染真核动物细胞及纯化
将构建好重组表达载体pG4HK-β-HCG-1、pG4HK-β-HCG-2转染CHO-K1细胞，转染前一天将CHO-K1细胞按照1×106/ml的密度传代以保证转染时细胞活力，转染当天将细胞密度调整为2×106/ml进行转染，转染体系每毫升中加入3.2ug重组表达载体，其中pG4HK-β-HCG-1、pG4HK-β-HCG-2的比例为1：1；再转染体系每毫升中加入4.8ug转染试剂PEI；边加边摇匀；37℃，6%二氧化碳摇床转速120rpm培养4小时后，加入1% 500mM VPA以及1% 30g/L L-盐酸半胱氨酸，32℃，6%二氧化碳摇床转速120rpm培养6天后离心收集上清，通过镍琼脂糖亲和层析柱，20mM咪唑溶液去除杂蛋白，300mM咪唑溶液洗脱目的蛋白，收液后，4℃静置30分钟，转至截留分子量为10kD-12kD的透析袋中，于10mmol/L 、pH7.4的PBS中过夜透析；透析后立即取出并分装，于-20℃保存，备用。20mM咪唑配制：咪唑1.36g，加10mmol/L，pH7.4PBS溶液溶解定容至1000mL；300mM咪唑配制：咪唑10.2g，加10mmol/L，pH7.4 PBS溶液溶解定容至500mL。
因此，本发明具有如下有益效果：
（1）通过构建抗人β-HCG双特异性抗体研制了一种利用双抗体夹心法原理、用于早孕检测的胶体金层析试纸条，两种抗原结合区域结合不同的抗原位点，有效的增强了抗体对HCG的结合特异性和靶向性，能极大的提高检测的灵敏度与特异性；
（2）添加的抗原结合区域结合不同的抗原位点，使得同一抗体分子可以结合两倍量的HCG抗原分子，即同样抗体原料用量的制作工艺下可以结合比普通单克隆抗体更多的被测抗原，延缓HOOK效应，避免临床假阴结果的出现。
附图说明
图1是本发明的一种结构示意图。
图中：PVC底板1，样本垫2，硝酸纤维膜3，吸水垫4，金标垫5，检测线6，质控线7，保护膜8。
具体实施方式
下面结合附图和具体实施方式对本发明做进一步的描述。
如图1所示的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，包括PVC底板1、样本垫2、硝酸纤维膜3，吸水垫4，金标垫5，样本垫、硝酸纤维膜、吸水垫及金标垫均固定于PVC底板上，硝酸纤维膜的两端分别与吸水垫的内端、金标垫的内端搭接，样本垫的内端与金标垫的外端搭接，硝酸纤维素膜上设有检测线6以及质控线7；金标垫上固化有胶体金-抗人β-HCG双特异性抗体结合物及胶体金-鸡IgY抗体结合物；检测线上包被抗人β-HCG双特异性抗体，质控线上包被羊抗鸡IgY多抗，吸水垫、样本垫及金标垫上设有保护膜8。
样本垫通过以下方法制得：将玻璃纤维裁剪成宽16mm，长30cm后置于网架上，每条玻纤用3.5ml样本垫处理液喷涂均匀，置于37℃烘箱烘12h备用，样本垫处理液通过以下方法制得：80ml超纯水中加入3.58g Na2HPO4·12H2O，0.5g Casein Na，0.9g NaCl，1g 表面活性剂S9，1g PVP，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节pH至8.0，加超纯水定容至100ml。
硝酸纤维膜通过以下方法制得：将硝酸纤维膜平整贴在PVC底板上，用包被缓冲液分别将抗人β-HCG双特异性抗体稀释至浓度为1.0mg/ml，将羊抗鸡IgY多抗稀释至浓度为0.5mg/ml，采用三维点膜喷金仪将稀释好的抗人β-HCG双特异性抗体溶液以喷量1μl/cm均匀包被在硝酸纤维膜的检测线上，将稀释好的羊抗鸡IgY多抗溶液以喷量1μl/cm均匀包被在硝酸纤维膜的质控线上；将硝酸纤维膜于37℃烘12h，备用；检测线与质控线间的间距控制在0.5cm；其中包被缓冲液通过以下方法制得：80ml超纯水中加入1.79g Na2HPO4.12H2O，0.9g Nacl，2g 蔗糖，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节PH至8.0，加超纯水定容至100ml。
金标垫通过以下方法制得：
（1）用量筒量取需要标记量的粒径40-50nm胶体金溶液，按体积百分含量为1%加入0.1M、pH为7.6的Na2HPO4·12H2O溶液，在磁力搅拌器上边搅拌边用1M NaOH调pH值至7.5，取抗人β-HCG双特异性抗体，按5ug/ml比例标记胶体金，一次性快速加入至胶体金溶液中，继续快速搅拌30min后，按体积百分含量为1%加入100g/L的稳定剂BSA，搅拌20min后10000r/min、10℃离心30min，去除上清，用金标保存液重悬至原体积的1/100，涡旋混匀，得胶体金-抗人β-HCG双特异性抗体结合物，备用；
（2）用量筒量取需要标记量的粒径40-50nm胶体金溶液，按体积百分含量为1%加入0.1M、pH为7.6的Na2HPO4·12H2O溶液，在磁力搅拌器上边搅拌边用1M NaOH调pH值至8.0，取鸡IgY抗体，按10ug/ml比例标记胶体金，一次性快速加入至胶体金溶液中，继续快速搅拌30min后，按体积百分含量为1%加入浓度为100g/L的稳定剂BSA，搅拌20min后10000r/min、10℃离心30min，去除上清，用金标保存液重悬至原体积的1/100，涡旋混匀，即得胶体金-鸡IgY抗体结合物，备用；金标保存液通过以下方法制得：80ml超纯水中加入1.79g Na2HPO4·12H2O，1g BSA，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节PH至8.0，加超纯水定容至100ml；
（3）将上述胶体金-抗人β-HCG双特异性抗体结合物和胶体金-鸡IgY抗体结合物按5：1比例混合，用金标稀释液对该混合物按体积百分含量50%进行稀释，涡旋混匀，用三维点膜喷金仪以喷量6ul/cm均匀喷涂在在宽6mm、长30cm的金标垫上，37℃烘4h；金标稀释液通过以下方法制得：80ml超纯水中加入1.79g Na2HPO4·12H2O，0.5g Casein Na，1g Tween-20，20g 蔗糖，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节PH至8.0，加超纯水定容至100ml。
抗人β-HCG双特异性抗体通过以下方法制得：
（a）抗人β-HCG双特异性抗体序列设计
通过计算机分子结构模拟及序列分析，优化分泌信号肽及载体表达元件，设计抗人β-HCG双特异性抗体的氨基酸序列，所述抗人β-HCG双特异性抗体的氨基酸序列包含有抗β-HCG表位a和β-HCG表位b的重组重链部分以及β-HCG表位a的轻链部分，所述抗人β-HCG双特异性抗体的重组重链氨基酸序列如SEQ ID No：1所示，所述抗人β-HCG双特异性抗体的轻链氨基酸序列如SEQ ID No：2所示；
（b）抗人β-HCG双特异性抗体基因克隆
在抗体序列不变的前提下，根据CHO细胞偏爱密码子分别将抗人β-HCG双特异性抗体的重组重链氨基酸序列、抗人β-HCG双特异性抗体的轻链氨基酸序列分别转化为相应的核苷酸序列，分别得到抗人β-HCG双特异性抗体的重组重链核苷酸序列和抗人β-HCG双特异性抗体的轻链核苷酸序列；在抗人β-HCG双特异性抗体的重组重链核苷酸序列上下游添加Hind III与BstE II的酶切位点，在抗人β-HCG双特异性抗体的轻链核苷酸序列上下游添加SalI与BsiWI的酶切位点；通过Hind III与BstE II双酶切将抗人β-HCG双特异性抗体的重组重链核苷酸序列连接到表达载体pG4HK上，获得抗人β-HCG双特异性抗体重组重链的表达载体1，质粒命名为pG4HK-β-HCG-1；通过SalI与BsiWI双酶切将抗人β-HCG双特异性抗体的轻链核苷酸序列的融合片段连接到载体pG4HK上，获得抗人β-HCG双特异性抗体轻链表达载体2，质粒命名为pG4HK-β-HCG-2；利用无内毒素大提试剂盒进行质粒大提；
（c）重组表达载体转染真核动物细胞及纯化
将构建好重组表达载体pG4HK-β-HCG-1、pG4HK-β-HCG-2转染CHO-K1细胞，转染前一天将CHO-K1细胞按照1×106/ml的密度传代以保证转染时细胞活力，转染当天将细胞密度调整为2×106/ml进行转染，转染体系每毫升中加入3.2ug重组表达载体，其中pG4HK-β-HCG-1、pG4HK-β-HCG-2的比例为1：1；再转染体系每毫升中加入4.8ug转染试剂PEI；边加边摇匀；37℃，6%二氧化碳摇床转速120rpm培养4小时后，加入1% 500mM VPA以及1% 30g/L L-盐酸半胱氨酸，32℃，6%二氧化碳摇床转速120rpm培养6天后离心收集上清，通过镍琼脂糖亲和层析柱，20mM咪唑溶液去除杂蛋白，300mM咪唑溶液洗脱目的蛋白，收液后，4℃静置30分钟，转至截留分子量为10kD-12kD的透析袋中，于10mmol/L 、pH7.4的PBS中过夜透析；透析后立即取出并分装，于-20℃保存，备用；20mM咪唑配制：咪唑1.36g，加10mmol/L，pH7.4PBS溶液溶解定容至1000mL；300mM咪唑配制：咪唑10.2g，加10mmol/L，pH7.4 PBS溶液溶解定容至500mL。
使用方法
检测方法：将待测样本、检测试纸条及其他检测用材料放置室温平衡；将试纸条的样本浸入端（样本垫）插入尿液标本容器中，待液体移行至膜上时取出平放，10min内观察显示结果；结果判读：当检测线显色强度等于或深于G 0.5，为阳性，提示为早孕；当检测线T不显色为阴性；质控线不显色表明试纸条无效或试验失败。
性能验证
a.物理性状：本检测试纸条整洁完整、无毛刺、无破损、无污染，测试宽度为2.5mm。
b.检测灵敏度：用含BSA的PBS （PH7.4）将β-HCG标准品分别稀释至25mIu/ml，50mIu/ml，做好标识，随机取同一批次的试纸条6人份，每个浓度测试3次重复。25mIu/ml的3个测试T线显色为G0.5~G1，为弱阳性，合格；50mIu/ml的3个测试T线显色均强于G4，为阳性，合格。临床诊断灵敏度为25mIu/ml。
c.检测特异性：用含BSA的PBS （PH7.4）分别将LH标准品稀释1000mIU/ml（国标500mIU/ml），FSH标准品稀释至2000mIU/ml（国标1000mIU/ml），TSH标准品稀释至2500μIU/ml（国标1000μIU/ml），做好标识，随机取同一批次的试纸9人份，LH，FSH和TSH分别测试3次，检测线T线均无显色为阴性，合格，特异性高。
d.检测HOOK效应：用含BSA的PBS （PH7.4）将HCG标准品分别稀释至100Iu/ml，200Iu/ml，400Iu/ml，600Iu/ml，800Iu/ml，随机取同一批号试纸条15人份，每个浓度测试3次重复，T线显色强度在200Iu/ml达到顶峰后至800Iu/ml略下降，合格。
e.检测重复性：用含BSA的PBS （PH7.4）将HCG标准品分别稀释至25mIu/ml，300mIu/ml，1000mIu/ml，随机取同一批号试纸条30人份，每个浓度测试10次重复，T线显色强度一致，合格。
f.试纸条稳定性：将同批次试纸条置于37℃条件下，按第0天，第3天，第7天，第14天，第21天，第28天，第35天，第42天，第49天分别取出试纸条，按照b，c，d，e项检测，结果均符合要求。
f.批间差：用含BSA的PBS （PH7.4）将β-HCG标准品稀释至25mIu/ml，取3个批号试纸条，每个批号10人份，共30人份。每个批号试纸条重复检测10次，3个批号的检测线T线显色强度一致，均符合要求。
以上所述的实施例只是本发明的一种较佳的方案，并非对本发明作任何形式上的限制，在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
SEQUENCE LISTING
<110> 杭州新脉生物科技有限公司
<120> 一种基于抗人β-HCG双特异性抗体胶体金层析试纸条
<130> 20201212
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 721
<212> PRT
<213> 人工合成
<220>
<223> 抗人β-HCG双特异性抗体的重组重链氨基酸序列
<400> 1
Met Asn Phe Gly Phe Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly
1 5 10 15
Phe Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys
20 25 30
Pro Gly Gly Ser Leu Lys Val Ser Cys Val Ala Ser Gly Phe Thr Phe
35 40 45
Ser Ser Tyr Ala Met Thr Trp Gly Arg Gln Thr Pro Glu Lys Arg Leu
50 55 60
Glu Trp Val Ala Ser Ile Ser Val Ser Gly Asn Thr Tyr Tyr Pro Asp
65 70 75 80
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile
85 90 95
Val Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr
100 105 110
Tyr Cys Ala Arg Val Asn Asp Tyr Ala Asn Gln Ala Trp Phe Pro Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro
130 135 140
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
145 150 155 160
Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr
195 200 205
Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
210 215 220
His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp
225 230 235 240
Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val
245 250 255
Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr
260 265 270
Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu
275 280 285
Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln
290 295 300
Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser
305 310 315 320
Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys
325 330 335
Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
340 345 350
Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro
355 360 365
Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met
370 375 380
Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn
385 390 395 400
Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr
405 410 415
Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn
420 425 430
Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu
435 440 445
His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys Gly
450 455 460
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
465 470 475 480
Gln Leu Gln Gln Ser Gly Ser Glu Leu Val Arg Pro Gly Ala Ser Val
485 490 495
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Trp Met
500 505 510
His Trp Val Lys Gln Arg His Gly Gln Gly Leu Glu Trp Ile Gly Asn
515 520 525
Ile Tyr Pro Gly Ser Gly Ile Thr Asn Tyr Asp Glu Lys Phe Lys Asn
530 535 540
Lys Gly Ile Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr Met His
545 550 555 560
Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
565 570 575
Gly Gly Arg Gly Leu Asp Val Trp Gly Ala Gly Thr Thr Leu Thr Val
580 585 590
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
595 600 605
Ser Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu
610 615 620
Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn
625 630 635 640
Arg Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu
645 650 655
Ile Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
660 665 670
Gly Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln
675 680 685
Thr Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Arg
690 695 700
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys His His His His His
705 710 715 720
His
<210> 2
<211> 107
<212> PRT
<213> 人工合成
<220>
<223> 抗人β-HCG双特异性抗体的轻链氨基酸序列
<400> 2
Asp Val Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Pro Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Thr Asn Ser Trp Pro Thr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 3
<211> 2163
<212> DNA
<213> 人工合成
<220>
<223> 抗人β-HCG双特异性抗体的重组重链核苷酸序列
<400> 3
atgaactttg gcttttctct gattttcctg gtgctcgtcc tcaagggatt tcagtgcgaa 60
gtgaagctgg tggaatccgg gggaggtctc gttaaaccag gcggtagtct taaggtctct 120
tgcgtagcca gtggtttcac attctcatct tacgcaatga catggggtcg ccagacccca 180
gaaaagagat tggagtgggt ggcctccatc agtgtgtccg gaaatacata ctatcccgat 240
tcagttaaag gcaggttcac aatcagccga gacaacgcaa gaaacattgt gtacttgcaa 300
atgtccagtc tgcggagtga agatacagct atgtattatt gtgccagggt gaacgactat 360
gcaaatcagg cctggttccc ctactgggga cagggcacat tggtgactgt gagcgctgcc 420
aagaccaccc cccccagtgt ctatcctctg gcccccggct ctgcagctca gactaattct 480
atggtgaccc ttggatgctt ggtgaaaggg tatttccctg aaccagtgac agtgacttgg 540
aactctggaa gtctgagtag cggggttcat acattccctg cagtcctcca atctgacttg 600
tacacactgt cctcttctgt cacagtccca tcctctactt ggccatctga aaccgttaca 660
tgcaacgtcg cccatcctgc aagtagtacc aaggttgaca agaagatcgt tcctcgagac 720
tgcggttgca aaccatgcat ctgcactgtc cctgaggtat ccagcgtgtt tatctttccc 780
cctaaaccca aggacgtgct taccattacc ctgacaccta aagtgacctg cgtagttgtc 840
gacatttcaa aagacgaccc tgaagtgcag ttcagttggt ttgttgacga tgtcgaagtg 900
catacagccc agacccagcc tcgagaagag caatttaaca gcacattccg gagtgtgagc 960
gagttgccta tcatgcatca ggattggctt aacggcaagg agttcaagtg tcgggtcaac 1020
tccgccgcat ttccagcacc aatcgaaaag accatttcta agactaaagg acgcccaaaa 1080
gcccctcagg tctacaccat tcccccccca aaggagcaga tggctaagga caaagttagt 1140
ctgacctgca tgatcaccga tttctttcca gaagatatca cagtggagtg gcagtggaat 1200
ggacagccag cagagaacta caagaataca cagcctatta tggacactga tggcagttac 1260
tttgtctact ctaagcttaa cgtgcaaaag tctaattggg aagctggcaa cacatttact 1320
tgttccgtgt tgcacgaagg gctccataat caccacacag aaaagagcct ttcccattct 1380
ccaggcaagg gcggtggggg tagcggcggt ggcgggtccg ggggtggcgg tagccaagtg 1440
cagctccagc agagcggaag tgaactcgtg cggcccgggg cttcagtaaa actgtcatgc 1500
aaggccagcg gctacacttt tacaacctac tggatgcatt gggtcaaaca gcggcacggg 1560
cagggcctgg agtggatcgg caacatctat cctgggtctg gtatcactaa ctatgatgag 1620
aagtttaaga ataagggcat acttaccgtc gataccagtt catcaacagc ctacatgcat 1680
ctgtcaagtc tcgcctctga ggattcagct gtttactatt gtgccagagg tggcagaggt 1740
ctggacgttt ggggtgccgg gacaaccctg accgtaagca gcggcggcgg aggcagcgga 1800
ggtggaggca gtggcggggg aggaagtgac atacagatga cccaaagttc ttccagcttc 1860
tctgtcagct tgggggatag ggtcactatc acctgcaaag ctagcgagga tatctacaac 1920
cggctcgcat ggtaccagca gaagcctgga aatgcccctc gacttcttat cagcggggca 1980
acctcattgg agactggcgt tcccagtaga ttctctggca gtggatctgg caaagactac 2040
accctgagta ttacctcact gcagaccgag gatgtggcta cttattattg tcagcagtac 2100
tggtcaaccc gtactttcgg agggggaacc aagcttgaga tcaagcatca ccaccaccat 2160
cac 2163
<210> 4
<211> 321
<212> DNA
<213> 人工合成
<220>
<223> 抗人β-HCG双特异性抗体的轻链核苷酸序列
<400> 4
gatgtgctcc ttactcagtc acctgctatt ctgtctgtca gccctggaga gcgggtgtcc 60
ttttcctgca gagcaagtca atccatcggg acatcaattc actggtatca gcaacggacc 120
aatggacctc cccgcctgct cattaaatac gccagcgaat ctatctctgg aatcccatcc 180
aggttcagcg gttccggttc agggacagat ttcactctca gcatatcctc cgtggagagt 240
gaggacatcg ctgattatta ctgccagcag acaaattctt ggcctacaac ctttgggggg 300
ggaactaaac tcgagatcaa g 321
Claims (10)
1.一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述基于抗人β-HCG双特异性抗体胶体金层析试纸条包括PVC底板（1）、样本垫（2）、硝酸纤维膜（3）、吸水垫（4）及金标垫（5），所述样本垫、硝酸纤维膜、吸水垫及金标垫均固定于PVC底板上，所述硝酸纤维膜的两端分别与吸水垫的内端、金标垫的内端搭接，所述样本垫的内端与金标垫的外端搭接，所述硝酸纤维素膜上设有检测线（6）以及质控线（7）；所述金标垫上固化有胶体金-抗人β-HCG双特异性抗体结合物及胶体金-鸡IgY抗体结合物；所述检测线上包被抗人β-HCG双特异性抗体，所述质控线上包被羊抗鸡IgY多抗。
2.根据权利要求1所述的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述吸水垫、样本垫及金标垫上设有保护膜（8）。
3.根据权利要求1所述的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述样本垫通过以下方法制得：将玻璃纤维裁剪成所需规格后，用样本垫处理液喷涂均匀，烘干。
4. 根据权利要求3所述的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述样本垫处理液通过以下方法制得：80ml超纯水中加入3.58g Na2HPO4·12H2O，0.5g Casein Na，0.9g NaCl，1g 表面活性剂S9，1g PVP，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节pH至8.0，加超纯水定容至100ml。
5.根据权利要求1所述的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述硝酸纤维膜通过以下方法制得：将硝酸纤维膜贴在PVC底板上，用包被缓冲液分别将抗人β-HCG双特异性抗体稀释至浓度为1.0mg/ml，将羊抗鸡IgY多抗稀释至浓度为0.5mg/ml，将稀释好的抗人β-HCG双特异性抗体溶液以喷量1μl/cm均匀包被在硝酸纤维膜的检测线上，将稀释好的羊抗鸡IgY多抗溶液以喷量1μl/cm均匀包被在硝酸纤维膜的质控线上；将硝酸纤维膜烘干。
6. 根据权利要求5所述的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述包被缓冲液通过以下方法制得：80ml超纯水中加入1.79g Na2HPO4.12H2O，0.9gNacl，2g 蔗糖，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节PH至8.0，加超纯水定容至100ml。
7.根据权利要求1所述的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述金标垫通过以下方法制得：
（1）用量筒量取需要标记量的粒径40-50nm胶体金溶液，按体积百分含量为1%加入0.1M、pH为7.6的Na2HPO4·12H2O溶液，在磁力搅拌器上边搅拌边用1M NaOH调pH值至7.5，取抗人β-HCG双特异性抗体，按5ug/ml比例标记胶体金，一次性快速加入至胶体金溶液中，继续快速搅拌30min后，按体积百分含量为1%加入100g/L的稳定剂BSA，搅拌20min后10000r/min、10℃离心30min，去除上清，用金标保存液重悬至原体积的1/100，涡旋混匀，得胶体金-抗人β-HCG双特异性抗体结合物，备用；
（2）用量筒量取需要标记量的粒径40-50nm胶体金溶液，按体积百分含量为1%加入0.1M、pH为7.6的Na2HPO4·12H2O溶液，在磁力搅拌器上边搅拌边用1M NaOH调pH值至8.0，取鸡IgY抗体，按10ug/ml比例标记胶体金，一次性快速加入至胶体金溶液中，继续快速搅拌30min后，按体积百分含量为1%加入浓度为100g/L的稳定剂BSA，搅拌20min后10000r/min、10℃离心30min，去除上清，用金标保存液重悬至原体积的1/100，涡旋混匀，即得胶体金-鸡IgY抗体结合物，备用；
（3）将上述胶体金-抗人β-HCG双特异性抗体结合物和胶体金-鸡IgY抗体结合物按体积比5：1比例混合，用金标稀释液对该混合物按体积百分含量50%进行稀释，涡旋混匀，用三维点膜喷金仪以喷量6ul/cm均匀喷涂在金标垫上，烘干。
8. 根据权利要求7所述的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述金标保存液通过以下方法制得：80ml超纯水中加入1.79g Na2HPO4·12H2O，1gBSA，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节PH至8.0，加超纯水定容至100ml。
9. 根据权利要求7所述的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述金标稀释液通过以下方法制得：80ml超纯水中加入1.79g Na2HPO4·12H2O，0.5gCasein Na，1g Tween-20，20g 蔗糖，0.05g 防腐剂Proclin300，搅拌至完全溶解，用1M 盐酸调节PH至8.0，加超纯水定容至100ml。
10. 根据权利要求1或5或7所述的一种基于抗人β-HCG双特异性抗体胶体金层析试纸条，其特征在于，所述抗人β-HCG双特异性抗体通过以下方法制得：
（a）抗人β-HCG双特异性抗体序列设计
通过计算机分子结构模拟及序列分析，优化分泌信号肽及载体表达元件，设计抗人β-HCG双特异性抗体的氨基酸序列，所述抗人β-HCG双特异性抗体的氨基酸序列包含有抗β-HCG表位a和β-HCG表位b的重组重链部分以及β-HCG表位a的轻链部分，所述抗人β-HCG双特异性抗体的重组重链氨基酸序列如SEQ ID No：1所示，所述抗人β-HCG双特异性抗体的轻链氨基酸序列如SEQ ID No：2所示；
（b）抗人β-HCG双特异性抗体基因克隆
在抗体序列不变的前提下，根据CHO细胞偏爱密码子分别将抗人β-HCG双特异性抗体的重组重链氨基酸序列、抗人β-HCG双特异性抗体的轻链氨基酸序列分别转化为相应的核苷酸序列，分别得到抗人β-HCG双特异性抗体的重组重链核苷酸序列和抗人β-HCG双特异性抗体的轻链核苷酸序列，抗人β-HCG双特异性抗体的重组重链核苷酸序列如SEQ ID No：3所示，抗人β-HCG双特异性抗体的轻链核苷酸序列如SEQ ID No：4所示；在抗人β-HCG双特异性抗体的重组重链核苷酸序列上下游添加Hind III与BstE II的酶切位点，在抗人β-HCG双特异性抗体的轻链核苷酸序列上下游添加SalI与BsiWI的酶切位点；通过Hind III与BstE II双酶切将抗人β-HCG双特异性抗体的重组重链核苷酸序列连接到表达载体pG4HK上，获得抗人β-HCG双特异性抗体重组重链的表达载体1，质粒命名为pG4HK-β-HCG-1；通过SalI与BsiWI双酶切将抗人β-HCG双特异性抗体的轻链核苷酸序列的融合片段连接到载体pG4HK上，获得抗人β-HCG双特异性抗体轻链表达载体2，质粒命名为pG4HK-β-HCG-2；利用无内毒素大提试剂盒进行质粒大提；
（c）重组表达载体转染真核动物细胞及纯化
将构建好重组表达载体pG4HK-β-HCG-1、pG4HK-β-HCG-2转染CHO-K1细胞，转染前一天将CHO-K1细胞按照1×106/ml的密度传代以保证转染时细胞活力，转染当天将细胞密度调整为2×106/ml进行转染，转染体系每毫升中加入3.2ug重组表达载体，其中pG4HK-β-HCG-1、pG4HK-β-HCG-2的比例为1：1；再转染体系每毫升中加入4.8ug转染试剂PEI；边加边摇匀；37℃，6%二氧化碳摇床转速120rpm培养4小时后，加入1% 500mM VPA以及1% 30g/L L-盐酸半胱氨酸，32℃，6%二氧化碳摇床转速120rpm培养6天后离心收集上清，通过镍琼脂糖亲和层析柱，20mM咪唑溶液去除杂蛋白，300mM咪唑溶液洗脱目的蛋白，收液后，4℃静置30分钟，转至截留分子量为10kD-12kD的透析袋中，于10mmol/L 、pH7.4的PBS中过夜透析；透析后立即取出并分装，于-20℃保存，备用。
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