CN101178402B - 聚合酶标记抗体的制备方法 - Google Patents
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Abstract
本发明属于酶免疫技术领域，具体涉及一种聚合酶标记抗体的制备方法。聚合酶标记抗体的制备方法，其特征在于它包括如下步骤：在非离子表面活性剂存在下，使可溶性辣根过氧化物酶(HRP)胶束化，以NaIO4先将HRP表面的糖分子氧化成醛基，然后利用HRP上的氨基与醛基发生缩合反应生成聚合酶(Poly-HRP)，再利用聚合酶上过量的醛基与IgG上的氨基相结合，生成可溶性的Poly-HRP-IgG，用NaBH4将所生成的不饱和键还原后，生成稳定的聚合酶标记抗体(Poly-HRP-IgG)。该方法制备过程简单，制备的聚合酶标记抗体在应用时具有较快的显色速度，较高的灵敏度和更好的贮存稳定性。
Description
技术领域
本发明属于酶免疫技术领域，具体涉及一种聚合酶标记抗体(Poly-HRP-IgG)的制备方法。
技术背景
酶标记物包括酶标记抗原、酶标记抗体和酶标记SPA等。酶标记物质量的好坏直接关系到免疫酶技术的成功与否，因此被称为关键的试剂。酶标记物中最常用的是酶标记抗体，它是将酶与特异性抗体经适当方法连接而成。酶标记抗体的质量主要取决于纯度好、活性强及亲和力高的酶和抗体，其次要有良好的制备方法。目前，高质量的酶(如辣根过氧化物酶，简称HRP)国内已有商品供应。高质量的抗体则可通过提取纯化而获得。Dako公司在美国申请了一项发明专利(US005543332A 1996)，主要内容和关键技术是利用水溶性高分子—右旋糖苷作聚合载体分子，将多个联乙烯砜单体(在单体末端为游离且具有反应活性的乙烯基)以共价键连接其上，自由的乙烯基可以结合有功能基团的抗原、半抗原、抗体、核苷酸等被标记物质；中国专利申请号03150895.2，公开了一种新型的免疫球蛋白与辣根过氧化物酶交联技术，关键技术基于水溶性的聚合载体分子—右旋糖苷，它可共价结合多个联乙烯砜单体(在单体末端为游离且具有反应活性的乙烯基)，自由的乙烯基可以结合有功能基团的抗原、半抗原、抗体、核苷酸等被标记物质，生成的HRP-右旋糖苷-RAM复合物一步免疫分析法比LASB三步分析法操作简易。但无论是Dako的专利(US005543332A 1996)还是中国专利申请号03150895.2，其主要问题来自于试剂制备过程中需要用到聚合物载体分子——右旋糖苷和具有毒性和危险性的乙烯砜，且制备过程步骤多，复杂。
发明内容
本发明的目的在于提供一种聚合酶标记抗体(Poly-HRP-IgG)的制备方法，该方法制备过程简单，制备的聚合酶标记抗体在应用时具有较快的显色速度，较高的显色灵敏度和更好的贮存稳定性。
为了实现上述目的，本发明的技术方案是：在非离子表面活性剂存在下，使可溶性HRP胶束化，以NaIO4先将HRP表面的糖分子氧化成醛基，然后利用HRP上的氨基与醛基发生缩合反应生成聚合酶(Poly-HRP)，再利用Poly-HRP上过量的醛基与IgG上的氨基相结合，生成可溶性的Poly-HRP-IgG，用NaBH4将所生成的不饱和键还原后，生成稳定的聚合酶标记抗体。
聚合酶标记抗体的制备方法，其特征在于它包括如下步骤：
1)氧化反应：取8mg辣根过氧化物酶，加8mmol NaIO4，反应20分钟，得到含醛基的HRP；
2)聚合形成多聚酶：步骤1)得到的含醛基的HRP中加入2ml质量浓度为3％的非离子表面活性剂，再用1M Na2CO3-NaHCO3溶液调pH至8.8-9.2，然后4℃旋转反应过夜，第二天室温反应1小时，得多聚酶；
3)纯化：步骤2)得到的多聚酶用10mM磷酸盐生理缓冲液(PBS)透析换液3次，得透析物；
4)连接IgG：取出步骤3)得到的透析物，加入IgG 0.27mg；
5)用10ul浓度为1M的Na2CO3-NaHCO3缓冲溶液调PH至8.3-8.7，旋转反应4小时；
6)还原稳定化：加入120ul浓度为5mg/ml NaHB4，摇匀，室温静置还原90分钟；
7)纯化：10mM磷酸盐生理缓冲液(PBS)透析，换液3次，取出透析物，得聚合酶标记抗体Poly-HRP-IgG。
本发明所用的HRP(Horseradish Peroxidase，HRP，辣根过氧化物酶)广泛分布于植物界，是能呈现有色化学反应的酶，具有来源广泛，易于纯化，比活性高，性质稳定，酶活性和量能用简单方法测定的特点。HRP由无色的酶蛋白和棕色的铁卟啉结合而成的糖蛋白，糖含量18％。HRP由多个同功酶组成，分子量为40,000，等电点为pH3～9，酶催化的最适pH因供氢体不同而稍有差异，多在pH5左右。HRP的辅基和酶蛋白最大吸收光谱分别为403nm和275nm，一般以OD403nm/OD275nm的比值RZ(德文Reinheit Zahl)表示酶的纯度。高纯度的酶RZ值应在3.0左右(最高可达3.4)。RZ值越小，非酶蛋白就越多。HRP的催化反应需要底物过氧化氢(H2O2)和供氢体(DH2)。供氢体多为无色的还原型染料，通过反应可生成有色的氧化型染料(D)。本发明所用供氢体是DAB(3，3-二氨基联苯胺)，形成的产物为深桔黄色或棕色，可溶性好，在避光处颜色稳定，空白可近于无色。
本发明所用的非离子表面活性剂，能吸附在HRP周围，使HRP胶束化，具有显著调节聚合酶Poly-HRP聚合度的作用。所述非离子表面活性剂包括聚乙二醇类非离子表面活性剂、多元醇类非离子表面活性剂。聚乙二醇类非离子表面活性剂，如烷基聚氧乙烯醚、烷基酚聚氧乙烯醚等；多元醇类非离子表面活性剂，如Span类、Tween类等。
本发明的有益效果：
1、在非离子表面活性剂存在时，胶束化的HRP在氧化剂过碘酸钠作用下自交联生成多聚酶(Poly-HRP)，使制备过程得以简化。
2、本发明的关键技术基于非离子表面活性剂存在时，胶束化的辣根过氧化物酶(HRP)在氧化剂过碘酸钠作用下自交联生成多聚酶(Poly-HRP)，然后将此多聚酶在不进行氧化还原反应的条件下与IgG结合成Poly-HRP-IgG；通过调节非离子表面活性剂的用量，控制反应时的pH值和反应时间，来调节Poly-HRP聚合度的大小，从而使后续生成的聚合酶标记抗体(Poly-HRP-IgG)在应用时具有较快的显色速度，较高的显色灵敏度和更好的贮存稳定性。所获聚合酶标记抗体的产率高，酶与Ig的活性无重大损失，免疫分析时显色灵敏度高，速度快。
3、用非放射性标记法替代同位素标记，可以避免放射性对人的伤害；避免使用有毒或对人具有危害性的试剂。
附图说明
图1是本发明实施例1的Poly-HRP-IgG应用于免疫组织化学实验的阳性结果图。
图2是本发明实施例1的Poly-HRP-IgG应用于免疫组织化学实验的阴性对照图。
具体实施方式
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实施例。
实施例1：
聚合酶标记抗体的制备方法，它包括如下步骤：
1)氧化反应：取8mg辣根过氧化物酶(HRP)，加8mmol NaIO4，反应20分钟；NaIO4(氧化剂)的加入起氧化作用，得到含醛基的HRP；
2)聚合形成多聚酶(Poly-HRP)：步骤1)得到的含醛基的HRP中加入2ml质量浓度为3％的非离子表面活性剂TritonX-100，再用1M Na2CO3-NaHCO3溶液调pH至8.8-9.2(最佳为9.0)，然后4℃旋转反应过夜，第二天室温反应1小时，得多聚酶(Poly-HRP)；非离子表面活性剂加入形成胶束化包覆；
3)纯化：步骤2)得到的多聚酶10mM磷酸盐生理缓冲液(PBS)透析换液3次，得透析物；
4)连接IgG：取出步骤3)得到的透析物(透析物中含HRP 2mg)，加入IgG 0.27mg；
5)用10ul(微升)浓度为1M的Na2CO3-NaHCO3缓冲溶液调PH至8.3-8.7(最佳为8.5)，旋转反应4小时；
6)还原稳定化：加入120ul(微升)浓度为5mg/ml NaHB4(还原剂，用2mM NaOH配制)，摇匀，室温静置还原90分钟；
7)纯化：10mM PBS透析换液3次；取出透析物，得聚合酶标记抗体(Poly-HRP-IgG)。
酶活性的检验：
按实施例1的步骤1)和2)得到的聚合酶Poly-HRP，具有高的催化活性。其催化活性的检验方法和效果如下：
1、方法：HRP溶解成1mg/ml的原液，临用前取0.1ml稀释到250ml；底物I为H2O2，取质量浓度30％H2O2 1ml(1毫升)用蒸馏水稀释到100ml，使用前取1ml用pH6.0的0.01MPB(磷酸盐缓冲液)稀释到100ml；底物II为1％邻联茴香胺甲醇溶液。底物II 0.05ml和底物I 6.0ml混匀后，取2.9ml加入反应用比色皿，其余加入对照比色皿。在反应皿中加入酶稀释液0.1ml，即记录为零时，迅速混匀，以对照皿为空白，每隔15秒记录反应皿的A460nm共1-2分钟。HRP活性(U/mg)＝ΔA460nm/分÷(11.3×HRP mg数/每ml反应物)。
2、结果：按实施例1的步骤1)和2)得到的聚合酶Poly-HRP酶活性为未交联前的80％左右，符合进入下一步交联IgG的要求。
Poly-HRP-IgG应用于免疫组织化学实验结果：
目前最普遍地免疫组织化学方法是三步法，而利用本发明所涉及的Poly-HRP-IgG，可以将三步法缩减为两步法，简化了操作步骤，试验证明，根据实施例1得到的Poly-HRP-IgG可以用于免疫组织化学两步法实验，实验结果见图1(阳性结果)，图中抗原阳性胞核胞浆着色(深棕色)明显，图2(阴性对照)，细胞核、组织间质和胞浆等无非特异着色(深棕色)，两图中蓝色部分为阴性细胞核苏木素复染后的呈色。
本发明所列举的非离子表面活性剂，都能实现本发明；调pH值的上限、下限都能实现本发明，在此不一一列举实施例。
Claims (1)
1.聚合酶标记抗体的制备方法，其特征在于它包括如下步骤：
1)氧化反应：取8mg辣根过氧化物酶，加8mmol NaIO4，反应20分钟，得到含醛基的HRP；
2)聚合形成多聚酶：步骤1)得到的含醛基的HRP中加入2ml质量浓度为3％的非离子表面活性剂，再用1M Na2CO3-NaHCO3溶液调pH至8.8-9.2，然后4℃旋转反应过夜得多聚酶；
所述的非离子表面活性剂为TritonX-100；
3)纯化：步骤2)得到的多聚酶用10mM磷酸盐生理缓冲液透析换液3次，得透析物；
4)连接IgG：取出步骤3)得到的透析物，加入IgG 0.27mg；
5)用10μl浓度为1M的Na2CO3-NaHCO3缓冲溶液调pH至8.3-8.7，旋转反应4小时；
6)还原稳定化：加入120μl浓度为5mg/ml NaHB4，摇匀，室温静置还原90分钟；
7)纯化：10mM磷酸盐生理缓冲液透析，换液3次，取出透析物，得聚合酶标记抗体。
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| CN104237527B (zh) * | 2014-03-18 | 2016-09-28 | 杭州浙大迪迅生物基因工程有限公司 | 高灵敏检测过敏原特异性抗体IgE的试剂盒及方法 |
| CN107037207B (zh) * | 2017-06-20 | 2018-08-24 | 山东莱博生物科技有限公司 | 一种丙肝抗体多聚体酶标记物及其制备与应用 |
| CN109781983A (zh) * | 2019-03-14 | 2019-05-21 | 武汉原谷生物科技有限责任公司 | 一种多聚酶法快速免疫组化试剂盒及其免疫组化方法 |
| CN110865183B (zh) * | 2019-11-25 | 2022-11-18 | 润方(长春)生物科技有限公司 | 一种利用磁珠制备hrp标记抗体的方法 |
| CN113358863A (zh) * | 2021-06-10 | 2021-09-07 | 武汉原谷生物科技有限责任公司 | 一种多聚酶标记的制作方法 |
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