抗体的亲和力与亲合力.pdf

Affinity and Avidity of AntibodiesAntibody AffinityAffinity measures the strength of interaction between an epitope and an antibody’s antigen binding site. It is defined by the same basic thermodynamic principles that govern any reversible biomolecular interaction:o K A= affinity constanto[Ab]= molar concentration of unoccupied binding sites on the antibodyo[Ag]= molar concentration of unoccupied binding sites on the antigeno[Ab-Ag]= molar concentration of the antibody-antigen complexIn other words, K A describes how much antibody-antigen complex exists at the point when equilibrium is reached. The time taken for this to occur depends on rate of diffusion and is similar for every antibody. However, high-affinity antibodies will bind a greater amount of antigen in a shorter period of time than low-affinity antibodies. K A can therefore vary widely for antibodies from below 105mol-1to above 1012mol-1, and can be influenced by factors including pH, temperature and buffer composition.The affinity of monoclonal antibodies can be measured accurately because they are homogeneous and selective for a single epitope. Polyclonal antibodies are heterogeneous and will contain a mixture of antibodies of different affinities recognizing several epitopes – therefore only an average affinity can be determined.Antibody AvidityAvidity gives a measure of the overall strength of an antibody-antigen complex. It is dependent on three major parameters:o Affinity of the antibody for the epitope (see above)o Valency of both the antibody and antigeno Structural arrangement of the parts that interactAll antibodies are multivalent e.g.IgGs are bivalent and and IgMs are decavalent. The greater an immunoglobulin’s valency (number of antigen binding sites), the greater the amount of antigen it can bind. Similarly, antigens can demonstrate multivalency because they can bind to more than one antibody. Multimeric interactions between an antibody and an antigen help their stabilization.A favorable structural arrangement of antibody and antigen can also lead to a more stable antibody-antigen complex as illustrated in Figures 1 and 2.Figure 1.An immobilized antigen (a high local concentration of available epitopes) provides more opportunity for the antibody-antigen complex to form than free antigen in solution over the same time period. Once the first antigen binding arm of an antibody attaches to an antigen on a solid support, the chances of a bivalent interaction are greatly improved. Many immunoassays like Western blotting and ELISA exploit this principle.Figure 2.When an antigen is mixed with a polyclonal antibody, multivalent interactions may lead to large, stable (high avidity) structures being formed. This is because the antigen may be bound by several antibodies, each recognizing a different epitope. Polyclonal antibodies are therefore ideal for immunoprecipitation experiments.Further Useful ReadingHow we improve the affinity of our recombinant monoclonal antibodies generated using HuCAL technology through affinity maturation。

转载某理工大学讲义，如果有更新的研究文章更好，呵呵第一节抗原抗体反应的原理抗原与抗体能够特异性结合是基于抗原决定簇(表位 )与抗体超变区的沟槽分子表面的结构互性与亲合性而结合的。

一、抗原抗体的结合力抗原抗体间结合为非共价键结合，有四种分子间引力参与。

(1)静电引力：是抗原抗体分子带有相反电荷的氨基和羧基基团之间相互吸引的力。

又称为库伦引力。

这种引力的大小与两电荷间的距离的平方成反比。

两个电荷距离越近，静电引力越强。

(2)范登华引力：是抗原与抗体两个大分子外层轨道上电子之间相互作用时，因两者电子云中的偶极摆动而产生吸引力。

能促使抗原抗体相互结合，这种引力的能量小于静电引力。

(3)氢键结合力：是抗体上亲水基团与相应抗原彼此接近时，相互间可形成氢键，使抗原抗体相互结合。

氢键结合力较范登华引力强。

(4)疏水作用力：是抗原表位与抗体超变区靠近时，相互间正、负极性消失，亲水层也立即失去，排斥了两者之间的水分子，使抗原抗体进一步相互吸引，促进其结合。

疏水作用力是这些力中最强的，对维系抗原抗体结合作用最大。

二、抗原抗体的亲合性和亲合力亲合性是指抗体分子上一个抗原结合点与对应的抗原表位之间相互适应而存在的引力，它是抗原抗体之间固有的结合力，可用平衡常数 K 来表示： K=K1/K2 ，K 值越大，亲合性越高；亲合性越高，与抗原结合越牢。

抗体的亲合力是指抗体结合部位与抗原表位之间结合的强度，与抗体结合价直接相关，即所谓多价优势，如 IgG 为两价，亲合力为单价的103倍，IgM为5〜10价，亲合力为单价的 107倍。

由于抗原抗体的结合反应是可逆的，若抗体的亲合力高，与抗原分子结合牢固，不易解离；反之即容易解离。

三、亲水胶体转化为疏水胶体大多数抗原为蛋白质，抗体是球蛋白，它们溶解在水中皆为胶体溶液，不会发生自然沉淀。

这种亲水胶体的形成机制是因蛋白质含有大量的氨基和羧基残基，在溶液中这些残基带有电荷，由于静电作用，在蛋白质分子周围出现了带相反电荷的电子云并形成了水化层，由于电荷的相斥，就避免了蛋白质分子间靠拢、凝集和沉淀。

Affinity and Avidity of AntibodiesAntibody Affinityantigen Affinity measures the strength of interaction between an epitope and an antibody’s binding site. It is defined by the same basic thermodynamic principles that govern any reversible biomolecular interaction:o K A = affinity constanto[Ab] = molar concentration of unoccupied binding sites on the antibodyo[Ag] = molar concentration of unoccupied binding sites on the antigeno[Ab-Ag] = molar concentration of the antibody-antigen complexIn other words, K A describes how much antibody-antigen complex exists at the point when equilibrium is reached. The time taken for this to occur depends on rate of diffusion and is similarfor every antibody. However, high-affinity antibodies will bind a greater amount of antigen in a shorter period of time than low-affinity antibodies. K A can therefore vary widely for antibodies from below 105 mol-1 to above 1012 mol-1, and can be influenced by factors including pH, temperature and buffer composition.The affinity of monoclonal antibodies can be measured accurately because they are homogeneous and selective for a single epitope. Polyclonal antibodies are heterogeneous and will contain a mixture of antibodies of different affinities recognizing several epitopes –therefore only an average affinity can be determined.Antibody AvidityAvidity gives a measure of the overall strength of an antibody-antigen complex. It is dependenton three major parameters:o Affinity of the antibody for the epitope (see above)o Valency of both the antibody and antigeno Structural arrangement of the parts that interactAll antibodies are multivalent e.g. IgGs are bivalent and and IgMs are decavalent. The greater an immunoglobulin’s valency (number of antigen binding sites), the greater the amount of antigen it can bind. Similarly, antigens can demonstrate multivalency because they can bind to more thanone antibody. Multimeric interactions between an antibody and an antigen help their stabilization.A favorable structural arrangement of antibody and antigen can also lead to a more stable antibody-antigen complex as illustrated in Figures 1 and 2.Figure 1. An immobilized antigen (a high local concentration of available epitopes) provides more opportunity for the antibody-antigen complex to form than free antigen in solution over the same time period. Once the first antigen binding arm of an antibody attaches to an antigen on a solid support, the chances of a bivalent interaction are greatly improved. Many immunoassays like Western blotting and ELISA exploit this principle.Figure 2. When an antigen is mixed with a polyclonal antibody, multivalent interactions may leadto large, stable (high avidity) structures being formed. This is because the antigen may be boundby several antibodies, each recognizing a different epitope. Polyclonal antibodies are therefore ideal for immunoprecipitation experiments.Further Useful Readingo How we improve the affinity of our recombinant monoclonal antibodies generated using HuCAL? technology through affinity maturation。

题目：噬菌体文库系列——抗体亲和力成熟摘要：多策略组合应用，搭配噬菌体展示技术，效果棒棒哒近年来，随着抗体药物的广泛上市，抗体已经取代基因治疗成为生物制药领域的主要生力军。

而抗体在疾病诊断、治疗和预防方面的作用很大程度上取决于其亲和力的高低，随着抗体工程技术的不断发展，如何提高抗体亲和力已成为抗体工程的难题之一。

围绕这一问题人们已经从不同的角度展开了研究，例如，提高抗体库的质量、增加抗体库的多样性、进行抗体重链或轻链的替换以及点突变有目的进行氨基酸替换等都能不同程度地提高抗体亲和力。

抗体亲和力表示抗体与抗原结合能力的大小。

抗体亲和力成熟是指机体正常存在的一种免疫功能状态。

在体液免疫中，再次应答所产生抗体的平均亲和力高于初次免疫应答，这种现象称为抗体亲和力成熟。

噬菌体展示技术作为一种先进的抗体库构建技术能结合多种技术手段，于体外实现抗体的亲和成熟，并配合亲和筛选方法获得具有高亲和力的抗体。

在噬菌体抗体展示技术中，VH和VL基因的随机重组，在一定程度上模拟了体内抗体亲和力成熟的过程。

如果结合其它技术则可以使抗体的亲和力提高到一个更高的层次，下面介绍一些抗体亲和力成熟的方法。

体外抗体亲和力成熟的几种策略要想实现抗体的亲和力体外成熟，就必须充分地了解天然抗体的亲和力体内成熟原理，设计模拟体内可能出现和存在的变化，从而促进抗体的体外进化。

天然抗体的亲和力成熟可以分为体细胞高频突变和克隆选择两个过程。

在天然抗体亲和力成熟的过程中，抗原刺激下的体细胞高频突变有着举足轻重的作用，因此亲和力体外成熟的策略也多在抗体基因突变水平上，即采用各种突变方法来模拟体内的高频突变。

1.随机突变（1）错配PCR通过改变PCR反应条件，提高核酸错配率将随机突变引入基因序列。

该技术可以通过提高镁离子浓度、加入锰离子、失衡4种脱氧核苷三磷酸（dNTPs）浓度、使用低保真DNA 聚合酶等方法，来提高抗体基因的突变率。

除此之外，突变率的高低也可以采用改变模板DNA 的复制次数进行控制。

抗原抗体反应：是指抗原与相应抗体在体内或体外发生的特异性结合反应。

抗原抗体间的结合力涉及静电引力、范德华力、氢键和疏水作用力，其中疏水作用力最强，它是在水溶液中两个疏水基团相互接触，由于对水分子的排斥而趋向聚集的力。

亲和性（affinity）：是指抗体分子上一个抗原结合点与一个相应抗原表位（AD）之间的结合强度，取决于两者空间结构的互补程度。

亲合力（avidity）：是指一个完整抗体分子的抗原结合部位与若干相应抗原表位之间的结合强度，它与亲和性、抗体的结合价、抗原的有效AD数目有关。

抗原抗体反应的特点：特异性、可逆性、比例性、阶段性。

带现象（zone phenomenon）：一种抗原-抗体反应的现象。

在凝集反应或沉淀反应中，由于抗体过剩或抗原过剩，抗原与抗体结合但不能形成大的复合物，从而不出现肉眼可见的反应现象。

抗体过量称为前带，抗原过量称为后带。

免疫原（immunogen）：是指能诱导机体免疫系统产生特异性抗体或致敏淋巴细胞的抗原。

免疫佐剂（immuno adjustvant）：简称佐剂，是指某些预先或与抗原同时注入体内，可增强机体对该抗原的免疫应答或改变免疫应答类型的物质。

半抗原（hapten）：又称不完全抗原，是指仅具有与抗体结合的能力(抗原性)，而单独不能诱导抗体产生（无免疫原性）的物质。

当半抗原与蛋白质载体结合后即可成为完全抗原。

载体（carrier）：结合后能给予半抗原以免疫原性的物质。

载体效应：初次免疫与再次免疫时，只有使半抗原结合在同一载体上，才能使机体产生对半抗原的免疫应答，该现象称为~。

单克隆抗体（McAB）：将单个B细胞分离出来，加以增殖形成一个克隆群落，该B细胞克隆产生的针对单一表位、结构相同、功能均一的抗体，即~。

多克隆抗体（PcAb）：天然抗原分子中常含多种不同抗原特异性的抗原表位，以该抗原物质刺激机体免疫系统，体内多个B细胞克隆被激活，产生含有针对不同抗原表位的免疫球蛋白，即~基因工程抗体（GEAb）：是利用DNA重组及蛋白工程技术，从基因水平对编码抗体的基因进行改造和装配，经导入适当的受体细胞后重新表达的抗体。

Affinity and Avidity of AntibodiesAntibody Affinityantigen Affinity measures the strength of interaction between an epitope and an antibody’s binding site. It is defined by the same basic thermodynamic principles that govern any reversible biomolecular interaction:o K A = affinity constanto[Ab] = molar concentration of unoccupied binding sites on the antibodyo[Ag] = molar concentration of unoccupied binding sites on the antigeno[Ab-Ag] = molar concentration of the antibody-antigen complexIn other words, K A describes how much antibody-antigen complex exists at the point when equilibrium is reached. The time taken for this to occur depends on rate of diffusion and is similarfor every antibody. However, high-affinity antibodies will bind a greater amount of antigen in a shorter period of time than low-affinity antibodies. K A can therefore vary widely for antibodies from below 105 mol-1 to above 1012 mol-1, and can be influenced by factors including pH, temperature and buffer composition.The affinity of monoclonal antibodies can be measured accurately because they are homogeneous and selective for a single epitope. Polyclonal antibodies are heterogeneous and will contain a mixture of antibodies of different affinities recognizing several epitopes –therefore only an average affinity can be determined.Antibody AvidityAvidity gives a measure of the overall strength of an antibody-antigen complex. It is dependenton three major parameters:o Affinity of the antibody for the epitope (see above)o Valency of both the antibody and antigeno Structural arrangement of the parts that interactAll antibodies are multivalent e.g. IgGs are bivalent and and IgMs are decavalent. The greater valency (number of antigen binding sites), the greater the amoun t of an immunoglobulin’santigen it can bind. Similarly, antigens can demonstrate multivalency because they can bind tomore than one antibody. Multimeric interactions between an antibody and an antigen help their stabilization.A favorable structural arrangement of antibody and antigen can also lead to a more stable antibody-antigen complex as illustrated in Figures 1 and 2.Figure 1. An immobilized antigen (a high local concentration of available epitopes) provides more opportunity for the antibody-antigen complex to form than free antigen in solution over the sametime period. Once the first antigen binding arm of an antibody attaches to an antigen on a solid support, the chances of a bivalent interaction are greatly improved. Many immunoassays like Western blotting and ELISA exploit this principle.Figure 2. When an antigen is mixed with a polyclonal antibody, multivalent interactions may leadto large, stable (high avidity) structures being formed. This is because the antigen may be boundby several antibodies, each recognizing a different epitope. Polyclonal antibodies are therefore ideal for immunoprecipitation experiments.进一步阅读Further Useful ReadingHow we improve the affinity of our recombinant monoclonal antibodies generated using HuCAL technology through affinity maturation。

Affinity and Avidity of AntibodiesAntibody AffinityAffinity measures the strength of interaction between an epitope and an antibody’s antigen binding site. It is defined by the same basic thermodynamic principles that govern any reversible biomolecular interaction:o K A= affinity constanto[Ab]= molar concentration of unoccupied binding sites on the antibodyo[Ag]= molar concentration of unoccupied binding sites on the antigeno[Ab-Ag]= molar concentration of the antibody-antigen complexIn other words, K A describes how much antibody-antigen complex exists at the point when equilibrium is reached. The time taken for this to occur depends on rate of diffusion and is similar for every antibody. However, high-affinity antibodies will bind a greater amount of antigen in a shorter period of time than low-affinity antibodies. K A can therefore vary widely for antibodies from below 105mol-1to above 1012mol-1, and can be influenced by factors including pH, temperature and buffer composition.The affinity of monoclonal antibodies can be measured accurately because they are homogeneous and selective for a single epitope. Polyclonal antibodies are heterogeneous and will contain a mixture of antibodies of different affinities recognizing several epitopes – therefore only an average affinity can be determined.Antibody AvidityAvidity gives a measure of the overall strength of an antibody-antigen complex. It is dependent on three major parameters:o Affinity of the antibody for the epitope (see above)o Valency of both the antibody and antigeno Structural arrangement of the parts that interactAll antibodies are multivalent e.g.IgGs are bivalent and and IgMs are decavalent. The greater an immunoglobulin’s valency (number of antigen binding sites), the greater the amount of antigen it can bind. Similarly, antigens can demonstrate multivalency because they can bind to more than one antibody. Multimeric interactions between an antibody and an antigen help their stabilization.A favorable structural arrangement of antibody and antigen can also lead to a more stable antibody-antigen complex as illustrated in Figures 1 and 2.Figure 1.An immobilized antigen (a high local concentration of available epitopes) provides more opportunity for the antibody-antigen complex to form than free antigen in solution over the same time period. Once the first antigen binding arm of an antibody attaches to an antigen on a solid support, the chances of a bivalent interaction are greatly improved. Many immunoassays like Western blotting and ELISA exploit this principle.Figure 2.When an antigen is mixed with a polyclonal antibody, multivalent interactions may lead to large, stable (high avidity) structures being formed. This is because the antigen may be bound by several antibodies, each recognizing a different epitope. Polyclonal antibodies are therefore ideal for immunoprecipitation experiments.Further Useful Readingo How we improve the affinity of our recombinant monoclonal antibodies generated using HuCAL® technology through affinity maturation。

抗体亲和⼒测定-电泳条带迁移法电泳条带迁移分析是⼀种简单⽽背景较低的亲和常数测定⽅法,适合于溶液中抗原-抗体反应的抗体亲和常数测定。

此法可以在不同的实验条件下测定抗体与抗原的结合能⼒。

但由于抗体的浓度低,因此需要⾼灵敏度的⽅法进⾏检测。

在该实验中,放射性核素或荧光素标记的抗体与不同浓度的抗原⼀起孵育,抗原抗体结合达到平衡后,进⾏⾮变性凝胶电泳,使游离的和已结合抗原的抗体得以分离,然后⽤⾃显影或荧光成像系统测定胶上含抗体的条带。

该⽅法要求抗原-抗体复合物⽐较稳定,因此不太适合亲和⼒低、解离快的抗体的亲和⼒常数的测定。

该实验也可⽤于测定较低的动⼒学解离常数(K off<10-3s-1)的抗体。

抗原抗体结合作为双分⼦反应,解离常数K dis依赖于动态的解离和结合:K dis=K off/K on。

K off可以简单理解为抗原抗体复合物趋于发⽣不可逆解离的强度,如抗体的浓度稀释到⼤⼤低于k dis时,K与抗原-抗体复合物半衰期(t1/2)的关系式浓度稀释到⼤⼤低于K时,k off与抗原-抗体复合物半衰期(t1/2)的关系式k off=0.692/（t1/2）⽤电泳条带迁移分析实验测定K off时,在其中⼀个被标记的抗原-抗体复合物⽔溶液中加⼊过量的未标记的竞争分⼦,并孵育不同的时间。

最后的反应混合物进⾏⾮变性凝胶电泳,并对电泳条带的迁移情况进⾏分析。

观察发⽣动⼒学竞争时标记复合物条带密度的衰减指数,标记条带强度随着竞争结合时间的增加⽽衰减。

因此可以得到⼀个衰减曲线,当条带强度衰减到开始时的⼀半时所⽤的时间即可视为t1/2,根据上述公式即可算出K off。

⼀、材料1) 缓冲液A:50mmol/L Tris(pH7.4),200mmo/L Nacl,12 mmol /L MgCl2。

2) PBS:8 g NaCl,0.2g KCl,1.44g Na2HPO4和0.24 g KH2PO4溶于1L双蒸⽔中,调pH为7.43) 上样缓冲液:0.4g蔗糖和0.5mg溴酚蓝溶于10 mL PBS中。