Antigen-Antibody Binding s and/ the (the ston of a th single tiger -antibody ed by X bond)for the antigen in the case of hapten-antibody t appears that water molecules are almost totally excluded from saltb ing,a non gens contnb van de for specific recognition. antibody binding originates from an increase in the entropy of inte the buried the same molecules displaced from the interface )and creating shape complement rity be complexation (.e.it is ntropy-driven). the other h make only a little contribution to the overall bindins protein-protein interactions,and considered to be a energy and act mainly to determine the specificity to the critical specificity-determining factor.Saltbridge forma nteraction. However, thermodynamic analyses have Ame)i suggested that a cons rable nun r ant h n bind has n oh ed.local induced nthalny cha ion from the e neg fitting (see above)has been observed. entropy contribution to association. antibody fragmer is used fo As mentioned above,it has been suggested from crystal structures or ant e Antibody Antigen Complex ns)or close contact with small anti where eptide and others)are important.In particular.amo n K =[Complex]/[Antibody][Antigen]. he ent molecules tobe excluded from ciation and dissociation rate constants are defined as follows: Vass=kass [Antibodyl[Antigen]Vaiss=kdiss [Complex] and make hydrogen bonds with both 00 e water mo nd antibody and makes a where v ent the rates of a binding (about )In addition to the direct the rate constants of association and dissociation,respec antiger n chanism.However.for further discussion.a structural study n an nd ki Recent advances in genetic engineering have enabled △Go=-RTIn K antibody fragments to be obtained more easily and the where the gas constant and Tis tem erature. mutants can be conveniently.Some as defined by the equation: binding has been discussed.Thus,biological specificity and affinity often depend on very subtle structural parameters. △G0=△Ho-T△S0 and extensive research is in progress solution have to ntibody bonds hav tight binding.There isa loss of the entropy of free rotation high ecificity. Although K is extremely high and translation of the separate molecules as well as a loss of 5Lmol- )in some protein-ligand interactions ntropy is gained not excee ENCYCLOPEDIA OF LIFE SCIENCES/e 2001 Nature Publishing Group /www.els.net In principle, the increase in van der Waals contacts and/ or varied surfaces upon complexation correlates well with the affinity (the strength of a single antigen–antibody bond) for the antigen in the case of hapten–antibody binding. However, hydrogen bond formation and/or a saltbridge link (also called 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 specific recognition. In protein antigen–antibody interactions, the buried surface is almost the same (
750 A˚ 3 ), and creating shape complementarity between proteins is probably needed. Hydrogen bond formation is more frequently observed in comparison with other protein–protein interactions, and considered to be a critical specificity-determining factor. 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 (see above) has been observed. If a monovalent antibody fragment is used for analysis, the equilibrium of antigen–antibody binding is defined as: CDEFGHIJ CDEFKLD 
M NHOPQLR S where Ka = [Complex] / [Antibody][Antigen]. Association and dissociation rate constants are defined as follows: Vass 5 kass [Antibody][Antigen]Vdiss 5 kdiss [Complex] [2] where Vass and Vdiss represent the rates of association and dissociation, respectively, and kass and kdiss represent the rate constants of association and dissociation, respec￾tively. At equilibrium Vass is equal to Vdiss and from eqns [1] and [2], the following equation is obtained: Ka = kass/kdiss [3] The Gibbs’ energy of formation (DG0) of an antigen– antibody complex is given by: DG0 = 2 RT ln Ka [4] where R is the gas constant and T is temperature. The free energy of complex formation represents a balance between enthalpic (DH0) and entropic (DS0) forces as defined by the equation: DG0 = DH0 2 T DS0 [5] 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 effect is quite significant and, 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–antibody binding 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 specificity to the interaction. However, thermodynamic analyses have suggested that a considerable number of antigen–antibody interactions 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–antibody complexes that shape complementarity of binding surfaces (in the 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 significant contribution 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 significant contribution to the binding (about 1–2 kcal mol 2 1 ). In addition to the direct antigen–antibody hydrogen bonds, solvent-mediated hy￾drogen bond formation should drive the interaction. The structural basis of antigen–antibody binding 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–antibody binding systems have been investigated using mutants, and the role of contact residues in the binding has been discussed. Thus, biological specificity and affinity often depend on very subtle structural parameters, and extensive research is in progress. Kinetic analyses on several antigen–antibody bonds have been performed to investigate the mechanism of creating high specificity. Although Ka is extremely high (
1015L mol 2 1 ), in some protein–ligand interactions (avidin–biotin reaction), the intrinsic affinities of antibodies do not exceed 1010L mol 2 1 (an affinity ceiling). From eqn Antigen–Antibody Binding ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 5