抗原和抗体，犹如敌人和卫士。抗原是一些属于自身正常物质之外的异种或异体物质，如细菌、病毒、寄生虫、毒素等。这些抗原能刺激人体的免疫系统，并由淋巴细胞产生特殊物质--免疫球蛋白，亦称为抗体。抗体能与抗原特异性地结合，拖住抗原，帮助免疫细胞吞噬、清除抗原。根据抗体结构和功能，可分为五类，分别称做 igG、IgA、IgM、IgD、IgE。其中 igG是人体内最主要的抗体。在血中的含量占血清总抗体的80％。人体大多数抗菌性、抗病毒性、抗毒素性的抗体属 igG。婴儿出生后3个月才能合成 igG抗体，2～3岁达到成人水平。出生前由母亲通过胎盘供给。IgG抗体对防止新生儿出生数周内的感染起很大作用。临床上使用的丙种球蛋白（主要是IgG）来治疗缺少或无免疫球蛋白的病人，以暂时提高病人的抗病能力。
igA是人体分泌液中的主要抗体，有抗菌、抗病毒能力。是粘膜防御感染的重要因素。如果 igA缺乏，则易患呼吸道感染。新生儿体内的 igA抗体，需从母乳中获得（所以提倡母乳喂养），4～6个月后的婴儿可产生 igA抗体，到青少年期即达到成人水平。
Antigen –Antibody Binding
Izumi Kumagai, Tohoku University, Sendai, Japan Kouhei Tsumoto, Tohoku University, Sendai, Japan
Antibodies are a family of glycoproteins that bind specifically to foreign molecules
(antigens).The binding between antibodies and antigens has high specificity and affinity resulting from various structural and energetic aspects.
. Antigen –Antibody Binding . Antigenic Epitope
. Forces Involved in Antigen-binding . Affinity and Avidity
. Biological Significance of Antibody Affinity and
Antibodies (immunoglobulins)are produced by the immune system of vertebrates and are essential for the prevention and resolution of infection by foreign invaders such as viruses. Antibodies are a family of variable glycoproteins that bind speciﬁcallyto foreign molecules (antigens).The most striking feature of antigen–antibodyinteractions is their high speciﬁcityand aﬃnity.In this article, the structural and energetic aspects of antigen–antibody binding are described, focusing in particular on howan antibody speciﬁcallyrecognizes its cognate antigen and binds to it tightly.
A binding strength between an antigenic determinant in an antigen (epitope)and an antigen-binding site in an antibody (paratope)is termed aﬃnity.Each antibody unit has at least two antigen-binding sites, and is therefore bivalent, or multivalent, to its antigen. The functional combining strength of an antibody with its antigen, which is related to both the aﬃnityof the reaction and the valencies of the antibody, is termed avidity. The signiﬁ-cance of avidity to antigen–antibodybinding is also described.
Antigen –Antibody Binding
The basic structure of an antibody (immunoglobulin)molecule comprises two identical light chains and two identical heavy chains linked together by disulﬁdebonds. There are ﬁveclasses of immunoglobulins (IgG,IgA, IgM, IgD and IgE), which diﬀerin amino acid sequence and number of domains in the constant regions of the heavy chains. There are two diﬀerentisotypes of light chains (l and k ). Immunoglobulin G (IgG)is the major type of immunoglobulin in normal serum and the most extensively investigated (Figure 1). The remarkable feature of the antibody molecule is revealed by comparison of amino acid sequences from various immunoglobulin molecules. This shows that immunoglobulins are composed of various copies of a folding unit of about 100amino acids, each of which forms an independent similar structure called the
immunoglobulin fold (Figure 2). The N-terminal domain of each polypeptide (heavyand light chains) is highly variable, while the remaining domains have constant sequences. The former domain is called the variable region (Vregion), while the latter is the constant region (Cregion). In addition, a comparison of V region sequences shows that variability is not uniformly distributed but concen-trated into three areas called the hypervariable regions. Investigations into the structures of various antigen–antibody complexes have demonstrated that the domain structure of the antibody molecule is a b barrel consisting of nine antiparallel b strands (Vregions) and seven C regions, and that the hypervariable regions are clustered at the end of the variable domain arms (Figure 3). The antigen-combining site of antibodies is formed almost entirely by six polypeptide segments, three from light variable domains and three from heavy variable domains. These segments showvariability in sequence as w ell as in number of residues, and it is this variability that provides the basis for the diversity found in the binding characteristics of the diﬀerentantibodies. These six hypervariable segments are often referred to as the complementarity-determining regions or CDRs.
It was realized that a more constant sequence of residues outside of the CDRs was required to maintain the essential immunoglobulin fold that results in the CDRs being brought into three-dimensional proximity. These residues are referred to as the framework regions. The framework residues do not usually form bonds with the antigen. However, they are essential for producing the folding of the V domains and maintaining the integrity of the binding site. Thus, the antibody-binding sites are formed by six segments of variable structure (CDRs)supported by a scaﬀoldingof essentially invariant architecture (frame-work regions). This characteristic structure has led to several approaches to the artiﬁcialdesign of novel antibodies by grafting newCDRs onto existing antibodies (Figure 3).
Canonical structure of CDRs
The antigen-binding speciﬁcityof an antibody is deﬁnedby the physical and chemical properties of its CDR surface. These in turn are determined by the conformation of
Figure 1Structure of immunoglobulin G (IgG).(a)Schematic representation of a typical IgG. The L and H chains are shown as solid lines, with the intramolecular disulfide linking Cys residues (S-S),that are characteristic of each immunoglobulin domain as shown in Figure 2. (b)Three-dimensional structure of IgG. The drawing is a composite of the crystal structures of human IgG Fab (PDBentry 21G2), and of human IgG Fc (PDBentry 1FC2). Some of the residues connecting Fab with Fc have not been visualized in the crystal structure; their probable location is indicated by the unconnected circles. Drawn using
Figure 2The immunoglobulin fold. The variable domain of a light chain is shown. Drawn using
individual CDRs, by the relative disposition of the CDRs, and by the nature and disposition of the side-chains of the amino acids in the CDRs. The structure of antibodies shows that antibodies can recognize inﬁnitelyvariable
antigens by varying the amino acid sequences of the CDR loops (i.e.the surface structures of antigen-recognizing regions). While the structure of the CDR loops might vary randomly, there are certain preferred conformations. Such preferred conformations can be deduced from the lengths and sequences of these regions.
CDR loop conformations in the antigen–antibodycomplexes whose three-dimensional structures have been solved by X-ray crystallographic study have been exten-sively analysed. Examination of the sequences of variable domains of unknown structure has shown that many have CDR loops that are similar in size to those of one of the known structures and contain identical residues at the sites responsible for the observed conformation. For ﬁveof the six hypervariable regions of most immunoglobulins (theonly exception is CDR of heavy chain 3), there seems to be only a small repertoire of main-chain conformations, most of which are known from the set of immunoglobulin structures so far determined. These observations have interesting implications for the molecular mechanism involved in the generation of antibody diversity, since the combination of limited numbers of CDR loop structure creates inﬁnitespeciﬁcitytoward foreign molecules. Sequence variations within the hypervariable regions modulate the surface that these canonical structures present to the antigens, altering the speciﬁcityand aﬃnityof the antibodies. Sequence variations within both
residues (Tyr,Trp and Asn) seem to have a propensity for being in the CDRs, and for participating in antigen recognition. It seems that the aromatic side-chains (Tyr,Trp) are more exposed to the solvent than in usual water-soluble proteins, and they are frequently found to be involved in the interaction with the ligand. This is explained by their large size (hydrophobiceﬀect),large polarization (vander Waals interactions), ability to form hydrogen bonds, and rigidity (lessloss of conformational entropy upon complexation). Thus, the concentration of aromatic rings would give a certain ‘stickiness’to the CDRs and give diverse speciﬁcityto antibodies. Speciﬁcityfor a particular antigen would arise from the complemen-tarity of the shapes of the interacting surfaces created by the proper positioning of the aromatic rings and the correct location of polar and/orcharged groups.
Role of structural changes upon complex formation in antigen –antibody binding
On comparison of an antigen structure and the antibody with its complex, local conformational changes (oftencalled induced ﬁtting)of both molecules have often been observed, creating high speciﬁcityand aﬃnity.It can be supposed that induced ﬁttingof an antibody to its antigen is critical for high speciﬁcityand aﬃnity.Induced ﬁttingcan be achieved (1)by small movements of side-chains, (2)by structural modiﬁcationssuch as deformation of CDR loops, or (3)by a change in the relative orientation of variable domains. All of these also seem to play a critical role in antigen–antibodyinteraction. In some antigen–antibody complexes, the signiﬁcanceof (3)has been suggested. As for (1)and (2),although the ﬂexibilityof the binding site leads to entropic loss upon complex formation, a greater interaction due to a more precise ﬁttingmust result in an overall increase in binding energy. Favourable energy change is in part or completely compensated for by unfavourable entropic loss (seebelow). Therefore, the binding characteristics must be analysed structurally by both antigen-free and complex form.
It is known that diversity in the germline antibody population is generated by the association of V, D and J gene segments with additional diversity occurring at the joining regions (Figure 4). In addition, somatic hypermuta-tion, which alters the speciﬁcamino acid by speciﬁcmutations of the gene encoding V regions, provides further diversity and leads to increased aﬃnityand speciﬁcityas the immune response proceeds, generating aﬃnity-ma-tured antibody. A comparison of the structures of germline antibodies and aﬃnity-maturedantibodies shows that the former display signiﬁcantconformational changes upon complex formation, whereas the antigens bind to the mature antibodies by a lock-and-key mechanism. Thus, it can be speculated that germline antibodies may
Figure 3Structure of complementarity-determining regions (CDRs).Variable domains of a murine immunoglobulin G (composedof heavy chain and light chain) are shown (fromHyHEL10structure, Tsumoto and Kumagai, unpublished results). The hypervariable regions, CDRs
comprising the antigen-binding site are shown by thick lines. These are located at the one edge of the b -barrel structure. Drawn using
framework and the hypervariable regions shift the canonical structures relative to each other by small but signiﬁcantamounts.
Contrary to other biomolecular interactions, antibodies can recognize various foreign antigens (e.g.small mole-cules, DNA, soluble proteins, surface proteins on viruses) by varying the hypervariable regions. The six CDR loops from the two chains at the rim of the eight-strand barrel provide an ideal arrangement for generating antigen-binding sites of diﬀerentshapes depending on the size and sequence of the loops. DiﬀerentCDR structures form according to the antigen structure. These CDR structures can create ﬂat,extended binding surfaces for protein antigens, a speciﬁcgroove for a peptide, DNA and carbohydrate, or speciﬁcdeep binding cavities for small molecules called haptens. A hapten is a simple chemical molecule that has the ability to bind antibody and can induce speciﬁcantibody production when it is attached to a carrier molecule such as albumin or Ficoll.
It has been pointed out that the distribution of amino acids in variable domains seems to be biased, and certain
(H chain polypeptide)
come together on the surface when the polypeptide chain folds to form the native protein.
Various researchers have suggested that any macro-molecule can be antigenic, and that all accessible areas of a protein can potentially be bound by antibodies. ‘Discon-tinuous’seems to be a more accurate description of nonlinear epitopes since they are assembled from residues from diﬀerentportions of the polypeptide chain. However, recent research has suggested that not all areas seem to contribute equally to the binding. Small sets of surface residues on antigens make a signiﬁcantcontribution to the interaction (calledenergetic epitope), while the rest of the antibody-binding regions seem to make additional con-tributions to the binding energy, although with some exceptions (antibody–idiotypeantibody binding).
Limitation of antibody specificity –cross-reactivity
As mentioned above, the most striking feature of the antigen–antibodyinteraction is its high speciﬁcityand aﬃnity.However, in some cases, antibodies can recognize other antigens. This is called cross-reactivity, i.e. the ability of a binding site to accommodate antigens other than the original immunogen. The structural basis of this cross-reactivity has been investigated in several antigen–anti-body-binding systems. For example, antisteroid (proges-terone)–antibody(DB3)cross-reacts with several steroids, and is seen, from X-ray crystallography, to bind to alternative binding subsites, i.e. diﬀerentbinding orienta-tions of the steroids can be formed in these binding sites. Anti-hen egg lysozyme antibody (D11.15)cross-reacts with several avian lysozymes, in some cases with a higher aﬃnitythan that for the original immunogen, hen lysozyme (calledheteroclitic binding). In this case residues diﬀerentfrom the original immunogen were found to be located around the edge of the epitope. Thus, it was concluded that a stereochemically permissive environment for the variant antigen residues at the antibody–antigeninterface was required for cross-reactivity.
V H C
Figure 4Molecular basis for antibody diversity. Only the case of heavy chain has been shown. In the human genome, one of the about 80VH genes (inmouse, about 100) recombines with one of 30D segments (inmouse, about ten), and one of six J segments (inmouse, four) producing a functional V-D-J gene in the B cell. The recombined DNA is transcribed, spliced and translated into a polypeptide chain. Half of the VH genes in human B cell seem to be pseudogenes.
multiple conﬁgurationsupon antigen binding, and com-bined with somatic hypermutation, this could stabilize the conﬁgurationwith optimal complementary to antigen. The structural plasticity of CDRs, especially in the case of germline antibodies, may adopt many diﬀerentconforma-tions, enabling them to accommodate many diﬀerentantigenic structures, which might lead to polyreactivity (i.e.reactivity to various antigens). Thus, adequate stickiness and the plasticity of CDRs may create high speciﬁcityand aﬃnityof antibodies toward target anti-gens.
Forces Involved in Antigen-binding
Each antibody binds to a particular part of the antigen called the antigenic determinant (orepitope). The term epitope was ﬁrstproposed by Jerne, to include surface conﬁgurations,haptenic groups, speciﬁcareas and so on. For protein antigens, it was later proposed that epitopes might be subdivided into sequential epitopes (involvinga single continuous length of the polypeptide chain) and conformational epitopes, in which several discrete amino acid sequences, widely separated in the primary structure,
The binding of an antigen to an antibody takes place by the formation of multiple noncovalent bonds between the antigen and the amino acids of the binding site. The strength of a single antigen–antibodybond is the antibody aﬃnity.It is produced by summation of the attractive and repulsive forces (vander Waals interactions, hydrogen bonds, salt bridges and hydrophobic force). The interac-tion of the antibody-combining site with antigen can be investigated thermodynamically and kinetically by using monovalent antibody fragments (fragmentsof variable regions or Fab).
In principle, the increase in van der Waals contacts and/or varied surfaces upon complexation correlates well with the aﬃnity(thestrength of a single antigen–antibodybond) for the antigen in the case of hapten–antibodybinding. However, hydrogen bond formation and/ora saltbridge link (alsocalled ion pairing, a noncovalent bond formed when a charged residue (e.g.aspartate) attracts its oppositely charged group (e.g.lysine)) seem to be required for speciﬁcrecognition. In protein antigen–antibodyinteractions, the buried surface is almost the same
3), and creating shape complementarity between ( 750A
proteins is probably needed. Hydrogen bond formation is more frequently observed in comparison with other protein–proteininteractions, and considered to be a critical speciﬁcity-determiningfactor. Saltbridge forma-tion (e.g.aspartate–lysine)is not always seen, and seems not to be necessary. Although no gross conformational change upon binding has been observed, local induced ﬁtting(seeabove) has been observed.
If a monovalent antibody fragment is used for analysis, the equilibrium of antigen–antibodybinding is deﬁnedas:
Antibody Antigen () Complex
K a =[Complex]/[Antibody][Antigen].
Association and dissociation rate constants are deﬁnedas
V ass 5k ass [Antibody][Antigen]V diss 5k diss [Complex]
where V ass and V diss represent the rates of association and dissociation, respectively, and k ass and k diss represent the rate constants of association and dissociation, respec-tively. At equilibrium V ass is equal to V diss and from eqns and ,the following equation is obtained:
K a =k ass /k diss
The Gibbs’energy of formation (D G 0) of an antigen–antibody complex is given by:
D G 0=2RT ln K a
where R is the gas constant and T is temperature.
The free energy of complex formation represents a balance between enthalpic (D H 0) and entropic (D S 0) forces as deﬁnedby the equation:
D G 0=D H 02T D S 0
In general, antigens and antibodies in solution have to overcome large entropic barriers before they can form a tight binding. There is a loss of the entropy of free rotation and translation of the separate molecules as well as a loss of conformational entropy of mobile segments and of side-chains upon binding. On the other hand, entropy is gained
when water molecules are displaced from the surfaces that become the newinterface. This latter eﬀectis quite signiﬁcantand, in the structures observed by X-ray analysis, it appears that water molecules are almost totally excluded from the interface by the close contact between antibodies and antigens. Enthalpic contributions arise from van der Waals interactions and hydrogen bond formation.
It is believed that the driving force in antigen–antibodybinding originates from an increase in the entropy of solvent molecules displaced from the interface upon complexation (i.e.it is entropy-driven). On the other hand, hydrogen bond formation and van der Waals interactions make only a little contribution to the overall binding energy and act mainly to determine the speciﬁcityto the interaction. However, thermodynamic analyses have suggested that a considerable number of antigen–antibodyinteractions are enthalpy-driven, i.e. they make favourable enthalpy changes with some opposition from the negative entropy contribution to association.
As mentioned above, it has been suggested from crystal structures of antigen–antibodycomplexes that shape complementarity of binding surfaces (inthe case of protein antigens) or close contact with small antigens (hapten,peptide and others) are important. In particular, almost all solvent molecules have been observed to be excluded from the interfaces, and therefore hydrophobic interactions are supposed to make a signiﬁcantcontribution to the interaction. However, a recent high-resolution crystal-lographic study shows that several water molecules remain in the interface and make hydrogen bonds with both antigen and antibody. The water molecule complements the imperfect structural complementarity between antigen and antibody and makes a signiﬁcantcontribution to the binding (about1–2kcal mol 21). In addition to the direct antigen–antibodyhydrogen bonds, solvent-mediated hy-drogen bond formation should drive the interaction.
The structural basis of antigen–antibodybinding is fundamentally important for clarifying the binding me-chanism. However, for further discussion, a structural study using X-ray crystallographic study or nuclear magnetic resonance (NMR)should be combined with an energetic study using thermodynamics and kinetics. Recent advances in genetic engineering have enabled antibody fragments to be obtained more easily, and the mutants can be constructed more conveniently. Some antigen–antibodybinding systems have been investigated using mutants, and the role of contact residues in the binding has been discussed. Thus, biological speciﬁcityand aﬃnityoften depend on very subtle structural parameters, and extensive research is in progress.
Kinetic analyses on several antigen–antibodybonds have been performed to investigate the mechanism of creating high speciﬁcity.Although K a is extremely high ( 1015L mol 21), in some protein–ligandinteractions (avidin–biotinreaction), the intrinsic aﬃnitiesof antibodies do not exceed 1010L mol 21(anaﬃnityceiling). From
,the ceiling originates from the limits for association and dissociation rate constants. The maximum association rate constant for the binding of a monomeric protein antigen by its antibody is approximately 105–106, a value controlled by the diﬀusioncoeﬃcientsof the reactant molecules and veriﬁedexperimentally. In contrast, no limitation for dissociation rate constant seems to exist, and aﬃnitychanges appear mostly as variations of the dissociation rate constant. Nevertheless, the k diss of the naturally prepared antibody molecule is ﬁxedat 1023–1024, and it is considered that dissociation rates which are too slow would not be selected in the immune system. Thus, the aﬃnityceiling of an antibody for any antigen is around 1010L mol 21.
Biological Significance of Antibody Affinity and Multivalency
An antibody with high aﬃnityfor its antigen can function most eﬀectivelyin the immune system (e.g.in biological reactions such as haemagglutination, virus neutralization, enzyme inactivation, haemolysis, immune elimination of foreign antigens, etc.). However, an antibody molecule with high aﬃnityfor target antigen does not usually exist in the primary naive antibody library, and antibody aﬃnityusually increases during an immune response (calledaﬃnitymaturation) in vivo . Nature provides us with numerous examples of molecules with low-intrinsic aﬃnitybinding sites that are capable of high-avidity interactions with their targets due to multivalent binding. For example, the lowintrinsic aﬃnityof IgM produced during the primary immune response is compensated by its penta-meric structure, resulting in a high avidity toward repetitive antigenic determinants present on the surface of bacteria or viruses. Thus, one of the most eﬃcientways to increase the binding activity of an antibody to a surface (e.g.cell surface) is to make use of the multivalency eﬀect.Therefore, even if the intrinsic aﬃnityof an antibody molecule toward various invaders (e.g.viruses, proteins) is relatively low, high avidity can overcome the low intrinsic aﬃnity,leading to the production of antibody molecules with high intrinsic aﬃnityfor the target antigen through aﬃnitymaturation in the immune system.
Affinity and Avidity
The strength of a single antigen–antibodybond is termed the antibody aﬃnity.It is produced by summation of the attractive and repulsive forces mentioned above. However, since each monoclonal antibody produced by hybridoma technology has two antigen-binding sites and antibodies obtained from serum contain polyclonal antibodies which can bind multiple antigenic determinants, antibodies are potentially multivalent in their reaction with antigen. When an antigen carrying multiple copies of the antigenic determinant (macromoleculesor microorganisms) com-bines with a multivalent antibody, the binding strength is greatly increased because all of the antigen–antibodybonds must be broken simultaneously before the antigen and antibody can dissociate. Thus, total binding energy between a multivalent antigen and more than one of the antigen-binding sites of an antibody is greater than the summation of the aﬃnityof each binding site for an antigen.
The strength with which a multivalent antibody binds a multivalent antigen is termed avidity, to diﬀerentiateit from the aﬃnityof the bond between a single antigenic determinant and an individual combining site. The avidity of an antibody for its antigen is determined by the sum of all of the individual interactions taking place between individual antigen-binding sites of antibodies and deter-minants on the antigens. The avidity of an antibody for its antigen strongly depends on the aﬃnitiesof the individual combining sites for the determinants on the antigens. It is greater than the summation of these aﬃnitiesif both antigen-binding sites of an antibody can combine with the antigen. The eﬀectiverange of antibody valence is from 2(IgG)up to 10(IgM),and the advantage of multivalence to the functional aﬃnity(asopposed to the aﬃnityof monovalent interactions, termed intrinsic aﬃnity)is estimated to be 103–7. Thus, even when each antigen-binding site has only a lowaﬃnity(e.g.IgM produced early in immune responses), antibodies can function eﬀectivelyin the immune system.
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