CN103675280B - 一种检测丙肝核心抗原的标记物及试剂 - Google Patents
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Abstract
本发明涉及丙肝检测技术领域，特别涉及一种检测丙肝核心抗原的标记物，含有摩尔比为20∶1的辣根过氧化物酶与L-赖氨酸羧甲基半胱氨酸聚合物。一种检测丙肝核心抗原的试剂，含有上述的检测丙肝核心抗原的标记物。还含有缓冲体系和显色剂。以L-赖氨酸羧甲基半胱氨酸聚合物为载体使抗体与HRP连接在一起，反应信号可通过连接的HRP催化底物系统而体现，因此，检测的灵敏度大幅提高。利用该方法将HCV核心抗原单克隆抗体与HRP偶联，进而增加抗体与HRP 的比率，增加检测的灵敏度，有效缩短检测的窗口期。
Description
技术领域
本发明涉及丙肝检测技术领域，特别涉及一种检测丙肝核心抗原的标记物，还涉及一种检测丙肝核心抗原的试剂。
背景技术
丙型肝炎是由丙型肝炎病毒( HCV) 感染引起的一种重要的传染病，主要通过血液传播。多数HCV 感染并无症状，感染丙肝病毒后形成慢性感染机率高，多数患者会发展成肝硬化、肝癌。为此，感病后早检出丙肝病毒感染，及时有效的阻断HCV 的传播是非常重要的。
检测丙型肝炎病毒（Hepatitis C virus，HCV）核心抗原可以直观地反映出HCV 的感染状况，并有效缩短HCV 感染的检测窗口期。但由于人在感染后，核心抗原在血液中存在的时间短且含量低，采用高灵敏的方法检测来提高准确性。
在免疫分析方法中，抗原或抗体标记物不仅决定免疫分析方法的分类，而且也决定免疫分析的灵敏度及特异性。传统的标记物为辣根过氧化物酶（HRP）标记抗原或抗体，应用酶上的氨基、巯基或糖蛋白上的羟基作为交联基团，通过过碘酸钠将抗原或抗体与酶直接连接在一起。抗原抗体反应信号通过HRP 催化底物来显现。一般1个抗原或抗体分子上仅能连接1～2 个HRP分子，检测灵敏度低。
发明内容
为了解决以上现有技术中存在的抗原或抗体标记物灵敏度及特异性差的问题，本发明提供了一种增加检测的灵敏度、有效缩短检测的窗口期的检测丙肝核心抗原的标记物。
本发明还提供了含有上述标记物的检测丙肝核心抗原的试剂。
本发明是通过以下措施实现的：
一种检测丙肝核心抗原的标记物，含有摩尔比为20∶1的辣根过氧化物酶与L-赖氨酸羧甲基半胱氨酸聚合物。
所述的标记物，通过以下步骤得到的：
（1）经活化的辣根过氧化物酶，与L-赖氨酸羧甲基半胱氨酸聚合物混合，室温条件下反应2-2.5h，经封闭液处理后分离未连接的辣根过氧化物酶，收集第1 洗脱峰，为L-赖氨酸羧甲基半胱氨酸与HRP的结合物，加入10％体积的10mg／mL EMCS（3-（N-吗啡啉）丙磺酸钠盐），室温反应2.5h洗脱得结合物；
（2）取纯化的单抗，调整浓度至5.0mg/mL，每毫升中加入6.0mgβ-巯基乙胺，37℃保温90min，并洗脱还原，将还原的抗体与结合物按照质量比1∶1的比例混合，4℃避光反应24h，洗脱，收集第1峰，即得。
所述的标记物，优选步骤（2）中洗脱为过Superdex 200 分子筛层析柱。
一种检测丙肝核心抗原的试剂，含有上述的检测丙肝核心抗原的标记物。
所述的试剂，还含有缓冲体系和显色剂。
目前，HCV 核心抗原检测是诊断HCV 感染的新方法，与HCV 抗体检测比较，能缩短HCV 检测的窗口期，但由于HCV 出现在血清中有一定的时限，且含量低，因此需要灵敏度较高的检测方法。标记物为免疫检测中的重要成分，本研究在HCV 检测中抗原、抗体不变的情况下，通过改进标记物进而提高了检测的灵敏度。本研究的标记物也可应用于其他免疫检测中，如果与灵敏度更高的化学发光或免疫荧光法结合，则可以检测更微量的物质。
本研究所制备的聚合物标记物的每个标记物上可连接多个抗体分子，同时在HRP的外围或多聚多肽上连接数个抗体分子。采用双抗体夹心酶联免疫法检测丙型肝炎病毒核心抗原，配以阴阳对照以及显色剂等试剂，定性检测人血清样品中丙型肝炎病毒核心抗原。该系统验证结论的同时，具有耗时短、试剂稳定性好、无污染等优点。
本发明的有益效果：
以L-赖氨酸羧甲基半胱氨酸聚合物为载体使抗体与HRP 连接在一起，反应信号可通过连接的HRP 催化底物系统而体现，因此，检测的灵敏度大幅提高。利用该方法将HCV 核心抗原单克隆抗体与HRP偶联，进而增加抗体与HRP 的比率，增加检测的灵敏度，有效缩短检测的窗口期。
具体实施方式
为了更好的理解本发明，下面结合具体实施例来进一步说明。
实施例1：
一种检测丙肝核心抗原的标记物，是通过以下步骤得到的：
（1）活化的辣根过氧化物酶HRP，与L-赖氨酸羧甲基半胱氨酸聚合物按照20∶ 1的摩尔比混合，室温条件下反应2h，经封闭液处理后分离未连接的HRP，收集第1 洗脱峰，为L-赖氨酸羧甲基半胱氨酸与HRP的结合物，加入10％体积的10mg／mLEMCS，室温反应2.5 h洗脱得到结合物；
（2）纯化的单抗，调整浓度至5.0mg/mL，每毫升中加入6.0mgβ-巯基乙胺，37℃保温90 min洗脱还原，将还原的抗体与结合物按照质量比1∶1的比例混合，4℃避光反应24h，过Superdex 200 分子筛层析柱洗脱，收集第一峰，即得。
对比实施例1：过碘酸钠法得到的标记物
应用传统的配比配方及浓度，HRP：IgG按照摩尔比5:1加入到标记抗体中，经 pH 值调整、封闭等步骤后分离纯化酶标抗体。
灵敏度检测试验
1、血清的处理及保存
为保证实验的准确性，降低干扰，血清的处理及保存严格按以下操作执行 （1）血清在操作过程中避免任何细胞刺激，使用不含热原和内毒素的试管，收集血液后，1000×g离心10分钟将血清和红细胞迅速小心地分离； （2） 细胞上清液---1000×g离心10分钟去除颗粒和聚合物； （3）如果样品不立即使用，应将其分成小部分-70℃保存，避免反复冷冻；如果血清中大量颗粒，检测前先离心或过滤，不要在37℃或更高的温度加热解冻，应在室温下解冻并确保样品均匀地充分解冻。
2、采用两种标记物以ELISA 法检测HCV核心抗原
2.1 实验方法
将两种标记物稀释成ELISA 工作液（含HRP 500ng/mL），用相同的酶标板及检测系统分别进行检测。检测之前，首先对人血清样本进行预处理，将预处理好的血清样本和HCV核心抗原加入含有抗HCV 核心抗原抗体的微孔中，每个小孔加入样本液100μL，37℃反应1小时；用稀释过的清洗液洗板5次后，分别加入100μL两种标记物，37℃条件下反应半小时；清洗一次后加入底物，显色10 min ，测定450 nm下各孔的吸光值。
结果判定：Cutoff 值＝阴性样本平均值×2.1。
2.2 两种标记物检测灵敏度的比较
将HCV 核心抗原系列稀释，每次检测10 孔，分别检测3次，计算A450值的平均值及标准差（s），按公式x+2s得到的A1值与系列稀释抗原的A 值进行比较，其高于A1 值的HCV 核心抗原最低稀释度为检测的灵敏度。
过碘酸钠法标记物的最低检测限为10.0pg/mL，本发明标记物的最低检测限为2.0pg/mL，见表1。
表1 两种标记物灵敏度测定的平均值
2.3 两种标记物检测临床血清标本的比较
分别用两种标记物检测正常人血清标本450份、HCV 抗体阳性血清标本200份和HCV核心抗原阳性血清标本200份，比较检测结果之间的差异。
其中，检测的450 份正常人血清标本，结果均为阴性，符合率100%，表明两者的特异性良好。
本发明标记物与过碘酸钠标记物相比，在使用ELISA针对HCV抗体阳性血清、HCV核心抗原阳性血清检测中均可降低假阴性比例，从而提高检测的灵敏度。见表2。
表2 两种标记物检测临床血清结果比较
本研究所制备的聚合物标记物的每个标记物上可连接多个抗体分子，同时在HRP的外围或多聚多肽上连接数个抗体分子。采用双抗体夹心酶联免疫法检测丙型肝炎病毒核心抗原，配以阴阳对照以及显色剂等试剂，定性检测人血清样品中丙型肝炎病毒核心抗原。该系统验证结论的同时，具有耗时短、试剂稳定性好、无污染等优点。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受实施例的限制，其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式，都包含在本发明的保护范围之内。
Claims (4)
1.一种检测丙肝核心抗原的标记物，其特征在于含有摩尔比为20∶1的辣根过氧化物酶与L-赖氨酸羧甲基半胱氨酸；
是通过以下步骤得到的：
（1）经活化的辣根过氧化物酶，与L-赖氨酸羧甲基半胱氨酸混合，室温条件下反应2-2.5h，经封闭液处理后分离未连接的辣根过氧化物酶，收集第1 洗脱峰，为L-赖氨酸羧甲基半胱氨酸与辣根过氧化物酶的结合物，加入10％体积的10mg／mL 3-（N-吗啡啉）丙磺酸钠盐，室温反应2.5h洗脱得结合物；
（2）取纯化的单抗，调整浓度至5.0mg/mL，每毫升中加入6.0mgβ-巯基乙胺，37℃保温90min，并洗脱还原，将还原的抗体与结合物按照质量比1∶1的比例混合，4℃避光反应24h，洗脱，收集第1峰，即得。
2.根据权利要求1所述的标记物，其特征在于步骤（2）中4℃避光反应24h后的洗脱为过分子筛层析柱洗脱。
3.一种检测丙肝核心抗原的试剂，其特征在于含有权利要求1或2所述的检测丙肝核心抗原的标记物。
4.根据权利要求3所述的试剂，其特征在于还含有缓冲体系和显色剂。
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