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抗体简介

抗体：是高等动物在抗原物质的刺激下由浆细胞产生的一类能与相应抗原发生特异性结合的免疫球蛋白。抗体通常存在于血清合?馨停ㄗ橹海┲小?

单克隆抗体及多克隆抗体：由单一克隆的细胞（通常是单一克隆的杂交瘤细胞）所产生的识别某一抗原表位的单一抗体；多克隆抗体是由多种细胞分别接触相应抗原而产生的多种抗体的总和，多克隆抗体抗原识别抗原的多个表位。

抗体结构：抗体是由四条肽链构成的“Y”形对称的分子，包括两条重链和两条轻链。在“Y”的部位的氨基酸顺序在不同的抗体分子中差别很大，是负责与抗原进行特异性结合的区域，称为可变区，余下的区域则称为恒定区。

抗体分类

抗体分类：根据重链恒定区的血清学类型，可将抗体分为IgM，IgG，IgA，IgD，IgE五类，它们的重链分别为mu, gamma, alpha, delta, epsilon链。在上述每一类别中，按重链构造上的变异又可分为几个亚类，例如人的IgG可分为IgG1，IgG2，IgG3，IgG4四个亚类。

轻链分为两种类型，kappa链和lambda链，但每种抗体中只存在一种类型的轻链。

二抗：二抗是在其它宿主体内制备的能与一抗或一抗片段结合的抗体，上面通常连有酶或荧光素等标签。由于二抗所具备的优点使得其在免疫学实验中得以应用广泛，如western blot（通过与特异性抗体结合来鉴定蛋白质），ELISA（以耦联有酶的抗体或抗原为标记来检测特异性的蛋白质，尤其是相应的抗原或抗体），免疫组织化学（检测组织中的特异性抗原），免疫细胞化学（通过免疫学方法检测细胞的抗原组成），流式细胞术（通过检测激光所激发荧光来鉴定分离不同类型的细胞）及免疫沉淀（通过抗原与抗体的特异性结合作用来分离相应抗原）。二抗针对某一特定物种（如小鼠）的所有抗体均具有特异性，因而使用标记的二抗可以免去对每一个一抗进行标记，大大节省了时间和费用；此外，一个一抗分子可以同时结合几个二抗分子，从而使信号大大增强，提高了实验灵敏度。

二抗可以耦联有几种不同的标记，可以是酶，荧光素，或生物素。

Table 1. 典型的整个IgG分子的二抗

如何选择二抗

通常情况下，某一特定的实验中可能同时有几种二抗可供选择，如何能选择到该实验的二抗，需要综合以下几个方面进行考虑：

一抗的物种来源

一抗是在什么物种来源的，相应的二抗也要是抗该物种的抗体。例如，如果一抗是在小鼠体内制备的，那么你就应当选择抗小鼠的二抗，如果一抗是在兔体内制备的，那么二抗就应当是抗兔的抗体。至于二抗本身是在什么动物中制备的对二抗的质量并无明显的影响。

一抗是属于哪个类或亚类

二抗需与一抗的类别或亚类相匹配。这通常是针对单克隆抗体而言。多克隆抗体主要是IgG类免疫球蛋白，因此相应的二抗就是抗IgG抗体.

单克隆抗体的类别及亚类通常会在产品列表中列出，如果你的一抗是小鼠IgM，那么相应的二抗就应当是抗小鼠IgM，或是抗小鼠IgG抗体。

如果单克隆一抗是小鼠IgG的某一亚类（IgG1，IgG2a，IgG2b，IgG3），那么几乎所有的抗小鼠IgG都可以与之结合，或者你也可以选择专门针对这一亚类的二抗，例如，如果你的一抗是小鼠IgG1，那么?憧梢匝≡窨笽gG1的二抗，此种抗体在双标记实验中尤其适合。

如果你不知道一抗是哪一类别或亚类，那么抗小鼠IgG是一个不错的选择，因为此种抗体可以识别大多数类型的IgG免疫球蛋白。

Table 2. 典型的F（ab’）2片段的二抗

下面总结了几种具有不同特异性的二抗：针对整个抗体分子（H＋L）具有特异性：如抗IgG（H+L），此类抗体既可以与抗体的重链结合也可以与轻链结合，即与抗体分子的Fc，F（ab’）2/Fab部分（见图5）均可反应，抗IgG（H+L）也可以与其他免疫球蛋白家族反应（如IgM和IgA），因为所有的免疫球蛋白都具有相同的轻链（kappa链或lambda链）。

针对Fab片段具有特异性：这类抗体与重链轻链均可以结合，由于它们可以与轻链反应，它们同时也已和具有相同轻链的其他种类的免疫球蛋白反应。

这对Fc片段或重链具有特异性：这类抗体和重链的Fc部分反应，一次它们是类别特异性的（即gamma链特异性抗体只与IgG反应，mu链特异性抗体只识别IgM，依此类推）。

轻链（kappa，labmda）特异性：与所有类别的抗体反应，因为所有类别的抗体均具有相同的lambda链或kappa链。

经过吸附处理的二抗：不同来源的免疫球蛋白含有相似的结构，抗一个物种的抗体可能与其他物种发生交叉反应，一次有些二抗经过了动物或人类IgG吸附处理，以减少非特异性背景。例如，如果是小鼠的组织或细胞，那么选择经小鼠血清或IgG吸附处理过的二抗可以减少非特异性结合。但是此种抗体识别表位的数量被大大减少。

选择哪种形式的二抗，是整个IgG分子，F（ab’）2片段，还是Fab片段？

整个IgG分子：此种抗体适用于多数情况。

F（ab’）2片段：该种抗体是由两个靠二硫键连接的用于结合抗原的Fab片段组成，这些抗体用在特定的情况中，如需要避免抗体与具有Fc受体的细胞结合的情况。

Fab片段：它们只有一个结合位点，一般用来封闭内源性免疫球蛋白。

选用耦联有哪种探针的二抗？

一般来讲，耦联到二抗上的探针主要有酶（辣根过氧化酶HRP和碱性磷酸酶AP或其衍生物APAAP，PAP），荧光基团（FITC, RRX, TR, PE）和生物素。选用哪种探针的二抗主要取决于具体的实验。对于western blot和ELISA，zui常用的二抗是酶标二抗，而细胞或组织标记实验（细胞免疫化学，组织免疫化学，流式细胞术）中通常使用荧光基团标记的二抗，免疫组化中也可以使用辣根过氧化酶或碱性磷酸酶标记的二抗。如果想要更大程度的放大检测信号，可以使用biotin/avidin检测系统。

Table 3. 典型的Fab片段的二抗

下面简单介绍几种二抗探针的优缺点及其适用的实验：

酶：主要有两种不同的酶耦联物，HRP和AP。比较而言，前者更经济、快速、稳定，而后者较前者更为灵敏，通常情况下，HRP广泛应用于免疫组化，western blot和ELISA中，而碱性磷酸酶（AP）更适合于固相的免疫检测实验，如ELISA和western blot.

荧光基团：

1. FITC：FITC是广泛使用的荧光基团，但其zui大的缺点是淬灭快，使用抗淬灭剂可以减轻。
2. AMCA：常用在多标记实验中，如免疫荧光、流式细胞术。其缺点是，淬灭快，且由于肉眼不能很好的检测其所激发的蓝色荧光，因而AMCA耦联的二抗更适用于检测大量存在的抗原。
3. 花青染料（Cy2,Cy3,Cy5）：Cy2比FITC更稳定，且在表面荧光显微镜下，Cy2的荧光FITC更强。Cy3和Cy5比大多数荧光基团更亮，更稳定，背景更弱。二者常在共聚焦显微实验中作多标记。但Cy5的缺点是不能用表面荧光显微镜观察，因此通常用具有远红外检测器的共聚焦显微镜观察。
4. TRITC,RRX,TR:TRITC经常和FITC一起用于双标记实验中，也可以使用RRX或TR，但TR会产生较高的背景。在使用装有氪氢灯的激光共聚焦扫描显微镜作三标记实验时，RRX尤其有用，可以和Cy2（或FITC）及Cy5一起使用，因为RRX的发射波长在Cy2和Cy5中间，且和这两者的重叠很少。
5. PE：常和FITC一起用于流式细胞术双标记实验。
6. 生物素：生物素（biotin）可以和抗生素蛋白（avidin）发生不可逆反应而紧密结合在一起。首先加入生物素标记的二抗，在加入耦联有酶或荧光集团的avidin、ExtrAvidin或streptavidin，后者能够结合于二抗表面的多个位点上，从而极大的放大检测信号。

抗体的纯度：

亲和层析纯化的抗体一般更受人们欢迎，因为这些产品的纯度zui高，产生的非特异性背景zui少。但是有些情况下也要考虑到纯化过程中损失的IgG部分，因为其中含有亲和力很高的抗体，尤其是当目的抗原存在的量很小的时候，高亲和性的IgG部分就显得尤为重要。

常用抗体综述

二抗应用实例分析

为分析二抗的使用情况，我们随机抽取了100篇2007年或2008年发表于Science 或 Journal of Biological Chemistry杂志上的 文章（英文），这些文章涵盖了来自于16个国家的约80个研究机构。收集的数据显示二抗的使用非常广泛，可以应用到几乎所有类型的免疫检测中，尤其是Western blot、免疫组织化学和免疫细胞化学。事实上，二抗在ELISA及流式细胞术中应用也很广泛，不仅用于科学研究，而且广泛的应用于临床分析和疾病诊断。比较而言，二抗在免疫沉淀中的应用相对较少。以下内容是基于对所收集的二抗信息进行统计分析后得出的一些结论，对于您在具体的实验中如何选择的抗体具有借鉴作用。

选择二抗时，您必须考虑到以下因素，如一抗的物种、产生二抗的宿主、一抗的类别或亚类、二抗所耦联的探针类型及二抗的形式（如整个IgG分子，F （ab’） 2片段，或Fab片段）等等。因此选择哪种类型的二抗很大程度上决定于您所使用的一抗是什么，尤其针对种属和类别而言。

通常情况下，一抗可以在山羊、小鼠、兔子、大鼠及绵羊体内制备，其中属小鼠及兔子zui为常见，相应的，二抗的种属也应当与一抗的相一致。而对于制备二抗的宿主，则应选择另外一种不同的动物，一般选用驴、山羊、兔子、绵羊或其他较大的动物，可以实现二抗的大规模制备，其中又属山羊使用zui多。

对于二抗类别或亚类的选择，应遵循与一抗类别或亚类相一致的原则，其中抗IgG使用zui多。这可能是因为除了五种类别的单克隆抗体外，很多一抗是多克隆抗体，属于IgG类免疫球蛋白。此外anti-IgG可以识别几乎所有类型的IgG亚类，因此，在不清楚一抗是属于那个类别或亚类的时候，anti-IgG抗体是个不错的选择。从我们的数据来看，值得注意的是， IgG亚类特异性的二抗（如抗IgG1, 抗IgG2, 抗IgG2a, 抗IgG2b）通常用于双标记实验中，包括免疫细胞化学、免疫组织化学及流式细胞术。

此外，整个IgG分子是zui常用的一种二抗分子形式，而F （ab’） 2片段或Fab片段一般仅用于特殊实验中，如需要避免抗体与具有Fc受体的细胞结合的情况，或需要封闭内源性免疫球蛋白的时候。

对于二抗所耦联的探针的选择，主要取决于具体的实验。对于Western blot，zui常用的探针是辣根过氧化酶（HRP），碱性磷酸酶（AP）的使用也很多，但不如HRP使用广泛。此外，IRDye （如 IRDye 680、IRDye 800） and Alexa Fluor dye （如Alexa 680 ） 等系列染料也偶尔应用于 western blot当中。通过统计分析发现，来自于Amersham Biosciences、Bio-Rad、Cell Signaling、GE Healthcare Bio-Science、Jackson ImmunoResearch Laboratories、LI-COR Biotechnology、Pierce Biotechnology、Santa Cruz Biotechnology、Sigma 及 Zymed Laboratories公司的HRP标记的二抗在Western blot中均有广泛使用；而对于AP标记的二抗，您可以选择Jackson ImmunoResearch Laboratories、Sigma、Bio-Rad 及DAKO公司。

对于免疫荧光检测实验（包括免疫细胞化学及免疫组织化学），应用zui广泛的是耦联有Alexa Fluor系列染料的抗体（如Alexa Fluor 488, 546, 568, 594, 596, 647, 633）、花青染料（Cyanine）系列标记的抗体（如Cy2, Cy3, and Cy5）、FITC及罗丹明（Rhodamine）。其中对于Alexa Fluor标记的二抗，推荐选择Invitrogen 公司或Molecular Probes公司（现属于Invitrogen公司），是的生产Alexa Fluor染料的公司。从我们收集的数据来看，Jackson Immunoresearch Laboratories公司的花青染料（Cyanine）系列标记的二抗使用较多，而对于FITC，Jackson Immunoresearch Laboratories、Santa Cruz Biotechnology、DAKO 及Sigma公司均可选择。Bio-Rad、Chemicon、Invitrogen或Sigma公司的罗丹明标记的二抗使用较好。另外，如果你想放大检测信号，你可以选择生物素标记的二抗，Jackson Immunoresearch Laboratories、Vector Laboratories 或R&D Systems均是不错的选择。

对于流式细胞术，FITC是选用zui多的标签，PE标签也可以使用。

对于ELISA实验，HRP 和 AP标签使用zui多。

在免疫沉淀中，二抗的使用并不是很多，但偶尔也有使用，如用于蛋白的分离，一般选择无耦联标记的二抗。

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