8536d_ch06_137-160 8/1/02 12: 41 PM Page 137 mac79 Mac 79: 45_BWrpQldsby et al. /Immunology 5e chapter 6 Antigen-Antibod Interactions. Principles and applications HE ANTIGEN-ANTIBODY INTERACTION IS A BIMO- lecular association similar to an enzyme-substrate interaction, with an important distinction: it does not lead to an irreversible chemical alteration in either the antibody or the antigen. The association between an ant Fluorescent Antibody Staining Reveals Intracellular body and an antigen involves various noncovalent interac tions between the antigenic determinant, or epitope, of the antigen and the variable-region(VH/Vi) domain of the an a Strength of Antigen-Antibody Interactions tibody molecule, particularly the hypervariable regions, or complementarity-determining regions( CDRs). The exquis ite specificity of antigen-antibody interactions has led to the m Precipitation Reactions development of a variety of immunologic assays, which can be used to detect the presence of either antibody or antigen. Agglutination Reactions Immunoassays have played vital roles in diagnosing diseases, Radioimmunoassa nonitoring the level of the humoral immune response, and identifying molecules of biological or medical interest. a Enzyme-Linked Immunosorbent Assay These assays differ in their speed and sensitivity, some are Western Blotting strictly qualitative, others are quantitative. This chapter ex amines the nature of the antigen-antibody interaction, and it describes various immunologic assays that measure or ex- Immunofluorescence ploit this interaction Flow Cytometry and Fluorescence a Alternatives to Antigen-Antibody reactions Strength of Antigen-Antibody Immunoelectron Microscopy Interactions antibody (Ag-Ab)binding include hydrogen bonds, ionic Antibody Affinity Is a Quantitative Measure bonds, hydrophobic interactions, and van der Waals interac- of Binding Strength tions (Figure 6-1). Because these interactions are individu- The combined strength of the noncovalent interactions be ally weak(compared with a covalent bond), a large number tween a single antigen-binding site on an antibody and a sin of such interactions are required to form a strong Ag-Ab in- gle epitope is the affinity of the antibody for that epitope teraction.Furthermore, each of these noncovalent interac- Low-affinity antibodies bind antigen weakly and tend to dis- tions operates over a very short distance, generally about 1 mm(I angstrom, A); consequently, a strong Ag- sociate readily, whereas high-affinity antibodies bind antigen Ab interaction depends on a very close fit between the anti. more tightly and remain bound longer. The association be- tween a binding site on an antibody (Ab)with a monovalent gen and antibody. Such fits require a high degree or antigen(Ag)can be described by the equation complementarity between antigen and antibody, a require ment that underlies the exquisite specificity that character- izes antigen-antibody interactions g+ ab= Ag-Ab
chapter 6 Antibody Affinity Is a Quantitative Measure of Binding Strength The combined strength of the noncovalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope. Low-affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen more tightly and remain bound longer. The association between a binding site on an antibody (Ab) with a monovalent antigen (Ag) can be described by the equation k1 Ag Ab 34 Ag-Ab k1 ■ Strength of Antigen-Antibody Interactions ■ Cross-Reactivity ■ Precipitation Reactions ■ Agglutination Reactions ■ Radioimmunoassay ■ Enzyme-Linked Immunosorbent Assay ■ Western Blotting ■ Immunoprecipitation ■ Immunofluorescence ■ Flow Cytometry and Fluorescence ■ Alternatives to Antigen-Antibody Reactions ■ Immunoelectron Microscopy Antigen-Antibody Interactions: Principles and Applications T - - lecular association similar to an enzyme-substrate interaction, with an important distinction: it does not lead to an irreversible chemical alteration in either the antibody or the antigen. The association between an antibody and an antigen involves various noncovalent interactions between the antigenic determinant, or epitope, of the antigen and the variable-region (VH/VL) domain of the antibody molecule, particularly the hypervariable regions, or complementarity-determining regions (CDRs). The exquisite specificity of antigen-antibody interactions has led to the development of a variety of immunologic assays, which can be used to detect the presence of either antibody or antigen. Immunoassays have played vital roles in diagnosing diseases, monitoring the level of the humoral immune response, and identifying molecules of biological or medical interest. These assays differ in their speed and sensitivity; some are strictly qualitative, others are quantitative. This chapter examines the nature of the antigen-antibody interaction, and it describes various immunologic assays that measure or exploit this interaction. Strength of Antigen-Antibody Interactions The noncovalent interactions that form the basis of antigenantibody (Ag-Ab) binding include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions (Figure 6-1). Because these interactions are individually weak (compared with a covalent bond), a large number of such interactions are required to form a strong Ag-Ab interaction. Furthermore, each of these noncovalent interactions operates over a very short distance, generally about 1 107 mm (1 angstrom, Å); consequently, a strong AgAb interaction depends on a very close fit between the antigen and antibody. Such fits require a high degree of complementarity between antigen and antibody, a requirement that underlies the exquisite specificity that characterizes antigen-antibody interactions. Fluorescent Antibody Staining Reveals Intracellular Immunoglobin 8536d_ch06_137-160 8/1/02 12:41 PM Page 137 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch06_137-160 8/1/02 12: 41 PM Page 138 mac79 Mac 79: 45_BWrpQldsby et al. /Immunology 5e 138 RT II Generation of B-Cell and T-Cell Respons VISUALIZING CONCEPTS CH,,-NH,+ o C-CH,-CH,-Ionic bond Hydrophob CHa CH H-CH- CH CH2-C、∠+H3N一CH2- Ionic bond FIGURE 6-1 The interaction between an antibody and an anti- drophobic groups together; and (4) van der Waals interactions gen depends on four types of noncovalent forces: (1)hydrogen between the outer electron clouds of two or more atoms. In an bonds, in which a hydrogen atom is shared between two elec. aqueous environment, noncovalent interactions are extremely tronegative atoms: (2)ionic bonds between oppositely charged weak and depend upon close complementarity of the shapes of residues;(3) hydrophobic interactions, in which water forces hy. antibody and antigen. where k, is the forward (association)rate constant and k-i is the association constant Ka for several Ag-Ab interactions the reverse(dissociation) rate constant. The ratio k,/k-1 is For example, the k, for the DNP-L-lysine system is about the association constant Ka (i.e, k/k-1= Ka), a measure of one fifth that for the fluorescein system, but its k-I is 200 affinity Because Ka is the equilibrium constant for the above times greater; consequently, the affinity of the antifluores reaction, it can be calculated from the ratio of the molar con- cein antibody Ka for the fluorescein system is about 1000 centration of bound Ag-Ab complex to the molar concentra- fold higher than that of anti-DNP antibody. Low-affinity ons of unbound antigen and antibody at equilibrium as Ag-Ab complexes have K values between 10"and 10 follows: L/mol; high-affinity complexes can have Ka values as high K,=lAg-Ab [BiLaG] For some purposes, the dissociation of the antigen-anti dy complex is of interest: The value of Ka varies for different Ag-Ab complexes and depends upon both k,, which is expressed in units of liters/mole/second(L/mol/s), and k-l, which is expressed units of 1/second. For small haptens, the forward rate con- cal of K. The equilibrium constant for that reaction is Kd, the recipro- stant can be extremely high; in some K, can be as as 4 X 10 L/mol/s, approaching the theoretical upper limit Kd=[Ab][Ag/(Ab-Ag=1/Ka of diffusion-limited reactions(10 L/mol/s). For larger pro- tein antigens, however, ki is smaller, with values in the range and is a quantitative indicator of the stability of an Ag-Ab of 10 L/mol/s complex; very stable complexes have very low values of K The rate at which bound antigen leaves an antibodys and less stable ones have higher values. binding site (i.e, the dissociation rate constant, k-1) plays a The affinity constant, Ka, can be determined by equilib major role in determining the antibody's affinity for an rium dialysis or by various newer methods. Because equilib- antigen. Table 6-1 illustrates the role of k-1 in determining rium dialysis remains for many the standard against which
where k1 is the forward (association) rate constant and k1 is the reverse (dissociation) rate constant. The ratio k1/k1 is the association constant Ka (i.e., k1/k1 Ka), a measure of affinity. Because Ka is the equilibrium constant for the above reaction, it can be calculated from the ratio of the molar concentration of bound Ag-Ab complex to the molar concentrations of unbound antigen and antibody at equilibrium as follows: Ka The value of Ka varies for different Ag-Ab complexes and depends upon both k1, which is expressed in units of liters/mole/second (L/mol/s), and k1, which is expressed in units of 1/second. For small haptens, the forward rate constant can be extremely high; in some cases, k1 can be as high as 4 108 L/mol/s, approaching the theoretical upper limit of diffusion-limited reactions (109 L/mol/s). For larger protein antigens, however, k1 is smaller, with values in the range of 105 L/mol/s. The rate at which bound antigen leaves an antibody’s binding site (i.e., the dissociation rate constant, k1) plays a major role in determining the antibody’s affinity for an antigen. Table 6-1 illustrates the role of k1 in determining [Ag-Ab] [Ab][Ag] the association constant Ka for several Ag-Ab interactions. For example, the k1 for the DNP-L-lysine system is about one fifth that for the fluorescein system, but its k1 is 200 times greater; consequently, the affinity of the antifluorescein antibody Ka for the fluorescein system is about 1000- fold higher than that of anti-DNP antibody. Low-affinity Ag-Ab complexes have Ka values between 104 and 105 L/mol; high-affinity complexes can have Ka values as high as 1011 L/mol. For some purposes, the dissociation of the antigen-antibody complex is of interest: Ag-Ab 34 Ab Ag The equilibrium constant for that reaction is Kd, the reciprocal of Ka Kd [Ab][Ag][Ab-Ag] 1Ka and is a quantitative indicator of the stability of an Ag-Ab complex; very stable complexes have very low values of Kd, and less stable ones have higher values. The affinity constant, Ka, can be determined by equilibrium dialysis or by various newer methods. Because equilibrium dialysis remains for many the standard against which 138 PART II Generation of B-Cell and T-Cell Responses VISUALIZING CONCEPTS FIGURE 6-1 The interaction between an antibody and an antigen depends on four types of noncovalent forces: (1) hydrogen bonds, in which a hydrogen atom is shared between two electronegative atoms; (2) ionic bonds between oppositely charged residues; (3) hydrophobic interactions, in which water forces hydrophobic groups together; and (4) van der Waals interactions between the outer electron clouds of two or more atoms. In an aqueous environment, noncovalent interactions are extremely weak and depend upon close complementarity of the shapes of antibody and antigen. ANTIGEN CH2 ANTIBODY OH ••• O C CH2 CH2 NH2 Hydrogen bond CH2 CH2 NH3 + –O C CH2 CH2 Ionic bond O CH2 CH3 CH CH3 CH3 +H3N CH3 CH CH2 van der Waals CH CH3 CH interactions CH CH3 O O– CH2 C CH2 Ionic bond CH3 Hydrophobic interactions 8536d_ch06_137-160 8/1/02 12:41 PM Page 138 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch06_137-160 8/1/02 12: 41 PM Page 139 mac79 Mac 79: 45_BWrpQldsby et al. /Immunology 5e Antigen-Antibody Interactions: Principles and Applications CHAPTER 6 139 TABLE 6.1 Forward and reverse rate constants (k, and k-1) and association and dissociation constants (Ka and Kd) for three ligand-antibody interactions Antibody Ligand k K Anti-DNP 8×103 1×108 1×10-8 4×103 1×1011 1×10-1 Anti-bovine serum albumin(BSA) 3×105 2×10 1.7×1 08 5.9×10 SOURCE: Adapted from H. N. Eisen, 1990, Immunology, 3rd ed, Harper Row Publishers. other methods are evaluated, it is described here. This proce- of the labeled ligand will be bound to the antibody at equi dure uses a dialysis chamber containing two equal compart- librium, trapping the ligand on the antibody side of the ves ments separated by a semipermeable membrane Antibody is sel, whereas unbound ligand will be equally distributed in placed in one compartment, and a radioactively labeled lig- both compartments. Thus the total concentration of ligand and that is small enough to pass through the semipermeable will be greater in the compartment containing antibody(Fig- membrane is placed in the other compartment(Figure 6-2). ure 6-2b). The difference in the ligand concentration in the Suitable ligands include haptens, oligosaccharides, and oligo- two compartments represents the concentration of ligand peptides In the absence of antibody, ligand added to com- bound to the antibody (i. e, the concentration of Ag-Ab com partment B will equilibrate on both sides of the membrane plex). The higher the affinity of the antibody, the more ligand (Figure 6-2a). In the presence of antibody, however, part is bound. rol: No antibody present Control d equilibrates on both sides equally) Initial state Equilibrium Experimental: Antibody in A Experimental (at equilibrium more ligand in A due to Ab binding) ● Radiolabeled Antibody bound Initial state Equilibrium Time. h FICURE6-2 Determination of antibody affinity by equilibrium dial. sured. (b)Plot of concentration of ligand in each compartment with sis.(a) The dialysis chamber contains two compartments(A and B) time. At equilibrium, the difference in the concentration of radioac separated by a semipermeable membrane Antibody is added to one tive ligand in the two compartments represents the amount of ligand compartment and a radiolabeled ligand to another. At equilibrium, bound to antibod ne concentration of radioactivity in both compartments is mea-
other methods are evaluated, it is described here. This procedure uses a dialysis chamber containing two equal compartments separated by a semipermeable membrane. Antibody is placed in one compartment, and a radioactively labeled ligand that is small enough to pass through the semipermeable membrane is placed in the other compartment (Figure 6-2). Suitable ligands include haptens, oligosaccharides, and oligopeptides. In the absence of antibody, ligand added to compartment B will equilibrate on both sides of the membrane (Figure 6-2a). In the presence of antibody, however, part of the labeled ligand will be bound to the antibody at equilibrium, trapping the ligand on the antibody side of the vessel, whereas unbound ligand will be equally distributed in both compartments. Thus the total concentration of ligand will be greater in the compartment containing antibody (Figure 6-2b). The difference in the ligand concentration in the two compartments represents the concentration of ligand bound to the antibody (i.e., the concentration of Ag-Ab complex). The higher the affinity of the antibody, the more ligand is bound. Antigen-Antibody Interactions: Principles and Applications CHAPTER 6 139 TABLE 6-1 Forward and reverse rate constants (k1 and k1) and association and dissociation constants (Ka and Kd) for three ligand-antibody interactions Antibody Ligand k1 k1 Ka Kd Anti-DNP -DNP-L-lysine 8 107 1 1 108 1 108 Anti-fluorescein Fluorescein 4 108 5 103 1 1011 1 1011 Anti-bovine serum albumin (BSA) Dansyl-BSA 3 105 2 103 1.7 108 5.9 109 SOURCE: Adapted from H. N. Eisen, 1990, Immunology, 3rd ed., Harper & Row Publishers. FIGURE 6-2 Determination of antibody affinity by equilibrium dialysis. (a) The dialysis chamber contains two compartments (A and B) separated by a semipermeable membrane. Antibody is added to one compartment and a radiolabeled ligand to another. At equilibrium, the concentration of radioactivity in both compartments is measured. (b) Plot of concentration of ligand in each compartment with time. At equilibrium, the difference in the concentration of radioactive ligand in the two compartments represents the amount of ligand bound to antibody. (a) Radiolabeled ligand AB AB (b)Concentration of ligand, M 100 50 100 50 Control: No antibody present Control (ligand equilibrates on both sides equally) Experimental: Antibody in A Experimental (at equilibrium more ligand in A due to Ab binding) Ligand bound by antibody 2468 Time, h Initial state Equilibrium AB AB Initial state Equilibrium Antibody D A B A B 8536d_ch06_137-160 8/1/02 12:41 PM Page 139 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch06_137-160 8/1/02 9: 01 AM Page 140 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e 140 PART II Generation of B-Cell and T-Cell Re Since the total concentration of antibody in the equilib- slope is constantly changing, reflecting this antibody hetero ium dialysis chamber is known, the equilibrium equation geneity(Figure 6-3b) With this type of Scatchard plot, it is can be rewritten as possible to determine the average affinity constant, Ko, by de- Ka=[Ab-AgM[(Agl termining the value of Ka when half of the antigen-binding (n-r) sites are filled. This is conveniently done by dete where equals the ratio of the concentration of bound ligand slope of the curve at the point where half of the antigen bind- to total antibody concentration, cis the concentration of free ing sites are filled ligand, and n is the number of binding sites per antibody molecule. This expression can be rearranged to give the Antibody Avidity Incorporates Affinity Scatchard equation: of Multiple Binding Sites K,, The affinity at one binding site does not always reflect the Values for rand c can be obtained by repeating the equi- true strength of the antibody-antigen interaction. When librium dialysis with the same concentration of antibody but complex antigens containing multiple, repeating antigenic with different concentrations of ligand. If Ka is a constant, determinants are mixed with antibodies containing multiple that is, if all the antibodies within the dialysis chamber have binding sites, the interaction of an antibody molecule with the same affinity for the ligand, then a Scatchard plot of r/c an antigen molecule at one site will increase the probability versus r will yield a straight line with a slope of -Ka( Figure of reaction between those two molecules at a second site. The 6-3a. As the concentration of unbound ligand increases, r/c strength of such multiple interactions between a multivalent pproaches 0, and r approaches n, the valency, equal to the antibody and antigen is called the avidity. The avidity of an number of binding sites per antibody molecule antibody is a better measure of its binding capacity within bi- Most antibody preparations are polyclonal, and Ka is ological systems(e.g, the reaction of an antibody with anti- therefore not a constant because a heterogeneous mixture of genic determinants on a virus or bacterial cell antibodies with a range of affinities is present. A Scatchard affinity of its individual binding sites. High avidity can com- plot of heterogeneous antibody yields a curved line whose pensate for low affinity. For example, secreted pentameric (a) Homogeneous antibody (b) Heterogeneous antibody 103 工×108 Slope at rof 1/ n=-Ko Intercept=n 1.0 IGURE6-3Scatchard plots are based on repeated equilibrium graph, antibody #1 has a higher affinity than antibody #2(b )If the dialyses with a constant concentration of antibody and varying con- antibody preparation is polyclonal and has a range of affinities, a centration of ligand. In these plots, r equals moles of bound lig. Scatchard plot yields a curved line whose slope is constantly chang. and/ mole antibody and c is the concentration of free ligand. From a ing. The average affinity constant Ko can be calculated by determin Scatchard plot, both the equilibrium constant(Ka)and the number of ing the value of Ka when half of the binding sites are occupied (i binding sites per antibody molecule(n), or its valency, can be ob. when r= 1 in this example). In this graph, antiserum #3 has a higher tained.(a) If all antibodies have the same affinity, then a Scatchard affinity(Ko= 2.4 X 10) than antiserum #4(Ko=1.25 X 100).Note plot yields a straight line with a slope of -K,. The x intercept is n, the that the curves shown in(a)and(b) are for divalent antibodies such valency of the antibody, which is 2 for igG and other divalent lgs. For as lgG IgM, which is pentameric, n= 10, and for dimeric IgA, n= 4. In this
Since the total concentration of antibody in the equilibrium dialysis chamber is known, the equilibrium equation can be rewritten as: Ka [Ab-Ag][Ab][Ag] c(n r r) where r equals the ratio of the concentration of bound ligand to total antibody concentration,c is the concentration of free ligand, and n is the number of binding sites per antibody molecule. This expression can be rearranged to give the Scatchard equation: c r Kan Kar Values for r and c can be obtained by repeating the equilibrium dialysis with the same concentration of antibody but with different concentrations of ligand. If Ka is a constant, that is, if all the antibodies within the dialysis chamber have the same affinity for the ligand, then a Scatchard plot of r/c versus r will yield a straight line with a slope of Ka (Figure 6-3a). As the concentration of unbound ligand cincreases,r/c approaches 0, and r approaches n, the valency, equal to the number of binding sites per antibody molecule. Most antibody preparations are polyclonal, and Ka is therefore not a constant because a heterogeneous mixture of antibodies with a range of affinities is present. A Scatchard plot of heterogeneous antibody yields a curved line whose slope is constantly changing, reflecting this antibody heterogeneity (Figure 6-3b). With this type of Scatchard plot, it is possible to determine the average affinity constant,K0, by determining the value of Ka when half of the antigen-binding sites are filled. This is conveniently done by determining the slope of the curve at the point where half of the antigen binding sites are filled. Antibody Avidity Incorporates Affinity of Multiple Binding Sites The affinity at one binding site does not always reflect the true strength of the antibody-antigen interaction. When complex antigens containing multiple, repeating antigenic determinants are mixed with antibodies containing multiple binding sites, the interaction of an antibody molecule with an antigen molecule at one site will increase the probability of reaction between those two molecules at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. The avidity of an antibody is a better measure of its binding capacity within biological systems (e.g., the reaction of an antibody with antigenic determinants on a virus or bacterial cell) than the affinity of its individual binding sites. High avidity can compensate for low affinity. For example, secreted pentameric 140 PART II Generation of B-Cell and T-Cell Responses FIGURE 6-3 Scatchard plots are based on repeated equilibrium dialyses with a constant concentration of antibody and varying concentration of ligand. In these plots, r equals moles of bound ligand/mole antibody and c is the concentration of free ligand. From a Scatchard plot, both the equilibrium constant (Ka) and the number of binding sites per antibody molecule (n), or its valency, can be obtained. (a) If all antibodies have the same affinity, then a Scatchard plot yields a straight line with a slope of Ka. The x intercept is n, the valency of the antibody, which is 2 for IgG and other divalent Igs. For IgM, which is pentameric, n 10, and for dimeric IgA, n 4. In this graph, antibody #1 has a higher affinity than antibody #2. (b) If the antibody preparation is polyclonal and has a range of affinities, a Scatchard plot yields a curved line whose slope is constantly changing. The average affinity constant K0 can be calculated by determining the value of Ka when half of the binding sites are occupied (i.e., when r 1 in this example). In this graph, antiserum #3 has a higher affinity (K0 2.4 108 ) than antiserum #4 (K0 1.25 108 ). Note that the curves shown in (a) and (b) are for divalent antibodies such as IgG. 1.0 2.0 r (a) Homogeneous antibody — × 108 r c 2.0 3.0 4.0 #1 #2 Slope = –Ka Intercept = n (b) Heterogeneous antibody 1.0 — × 108 r c 2.0 3.0 4.0 2.0 r 1.0 Slope at r of 1/2 n = –K0 Intercept = n #3 #4 8536d_ch06_137-160 8/1/02 9:01 AM Page 140 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch06_137-160 8/1/02 9: 01 AM Page 141 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e Antigen-Antibody Interactions: Principles and Applications CHAPTER 6 141 IgM often has a lower affinity than IgG, but the high avidity The bacterium Streptococcus pyogenes, for example, expresses of IgM, resulting from its higher valence, enables it to bind cell-wall proteins called M antigens. Antibodies produced to antigen effectively. streptococcal M antigens have been shown to cross-react with several myocardial and skeletal muscle proteins and have been implicated in heart and kidney damage following Cross-Reactivity reptococcal infections. The role of other cross-reacting antigens in the development of autoimmune diseases is dis Although Ag-Ab reactions are highly specific, in some cases cussed in Chapter 20 antibody elicited by one antigen can cross-react with an un- Some vaccines also exhibit cross-reactivity. For instance, related antigen. Such cross-reactivity occurs if two different vaccinia virus, which causes cowpox, expresses cross-reacting antigens share an identical or very similar epitope In the lat pitopes with variola virus, the causative agent of smallpox. ter case, the antibody's affinity for the cross-reacting epitope This cross-reactivity was the basis of Jenner's method of us- is usually less than that for the original epitope. Ing virus to induce immunity to smallpox, as men- Cross-reactivity is often observed among polysaccharide tioned in Chapter I antigens that contain similar oligosaccharide residues. The ABo blood-group antigens, for example, are glycoproteins expressed on red blood cells. Subtle differences in the termi- nal residues of the sugars attached to these surface proteins Precipitation Reactions distinguish the A and B blood-group antigens. An individual Antibody and soluble antigen interacting in aqueous solu lacking one or both of these antigens will have serum anti- tion form a lattice that eventually develops into a visible pre- cipitate Antibodies that aggregate soluble antigens are called not by exposure to red blood cell antigens but by exposure to precipitins. Although formation of the soluble Ag-Ab com ns present plex occurs within minutes, formation of the visible precipi- testinal bacteria. These microbial antigens induce the for- tate occurs more slowly and often takes a day or two to reach mation of antibodies in individuals lacking the similar blood-group antigens on their red blood cells. (In individu- Completion. als possessing these antigens, complementary antibodies Formation of an Ag-Ab lattice depends on the valency of would be eliminated during the developmental stage in oth the antibody and antigen which antibodies that recognize self epitopes are weeded The antibody must be bivalent; a precipitate will not out.)The blood-group antibodies, although elicited by mi form with monovalent Fab fragments crobial antigens, will cross-react with similar oligosaccha rides on foreign red blood cells, providing the basis for The antigen must be either bivalent or polyvalent; that is, blood typing tests and accounting for the necessity of com- it must have at least two copies of the same epitope, or have different epitopes that react with different patible blood types during blood transfusions. a type A in- antibodies present in polyclonal antisera dividual has anti-B antibodies; a type B individual has anti-A; and a type O individual thus has anti-A and anti-B Experiments with myoglobin illustrate the requirement (Table 6-2). at protein antigens be bivalent or polyvalent for a precip- A number of viruses and bacteria hav determi- itin reaction to occur. Myoglobin precipitates well with spe- nants identical or similar to normal host onents. In cific polyclonal antisera but fails to precipitate with a specific some cases, these microbial antigens ha shown to monoclonal antibody because it contains multiple, distinct elicit antibody that cross-reacts with the host-cell compo- epitopes but only a single copy of each epitope( Figure 6-4a) ents, resulting in a tissue-damaging autoimmune reaction. Myoglobin thus can form a crosslinked lattice structure with lyclonal antisera but not with monoclonal antisera. The principles that underlie precipitation reactions are presented because they are essential for an understanding of commonly TAL BLE 6-2 ABO blood types used immunological assays. Although various modifications of the precipitation reaction were at one time the major types Blood type Antigens on RBCs Serum antibodies of assay used in immunology, they have been largely replaced by methods that are faster and, because they are far more sen Anti-B sitive,require only very small quantities of antigen or anti Anti-A body. Also, these modern assay methods are not limited to A and B antigen-antibody reactions that produce a precipitate. Table 6-3 presents a comparison of the sensitivity, or minimu Anti-A and anti-B amount of antibody detectable, by a number of immunoas-
IgM often has a lower affinity than IgG, but the high avidity of IgM, resulting from its higher valence, enables it to bind antigen effectively. Cross-Reactivity Although Ag-Ab reactions are highly specific, in some cases antibody elicited by one antigen can cross-react with an unrelated antigen. Such cross-reactivity occurs if two different antigens share an identical or very similar epitope. In the latter case, the antibody’s affinity for the cross-reacting epitope is usually less than that for the original epitope. Cross-reactivity is often observed among polysaccharide antigens that contain similar oligosaccharide residues. The ABO blood-group antigens, for example, are glycoproteins expressed on red blood cells. Subtle differences in the terminal residues of the sugars attached to these surface proteins distinguish the A and B blood-group antigens. An individual lacking one or both of these antigens will have serum antibodies to the missing antigen(s). The antibodies are induced not by exposure to red blood cell antigens but by exposure to cross-reacting microbial antigens present on common intestinal bacteria. These microbial antigens induce the formation of antibodies in individuals lacking the similar blood-group antigens on their red blood cells. (In individuals possessing these antigens, complementary antibodies would be eliminated during the developmental stage in which antibodies that recognize self epitopes are weeded out.) The blood-group antibodies, although elicited by microbial antigens, will cross-react with similar oligosaccharides on foreign red blood cells, providing the basis for blood typing tests and accounting for the necessity of compatible blood types during blood transfusions. A type A individual has anti-B antibodies; a type B individual has anti-A; and a type O individual thus has anti-A and anti-B (Table 6-2). A number of viruses and bacteria have antigenic determinants identical or similar to normal host-cell components. In some cases, these microbial antigens have been shown to elicit antibody that cross-reacts with the host-cell components, resulting in a tissue-damaging autoimmune reaction. The bacterium Streptococcus pyogenes, for example, expresses cell-wall proteins called M antigens. Antibodies produced to streptococcal M antigens have been shown to cross-react with several myocardial and skeletal muscle proteins and have been implicated in heart and kidney damage following streptococcal infections. The role of other cross-reacting antigens in the development of autoimmune diseases is discussed in Chapter 20. Some vaccines also exhibit cross-reactivity. For instance, vaccinia virus, which causes cowpox, expresses cross-reacting epitopes with variola virus, the causative agent of smallpox. This cross-reactivity was the basis of Jenner’s method of using vaccinia virus to induce immunity to smallpox, as mentioned in Chapter 1. Precipitation Reactions Antibody and soluble antigen interacting in aqueous solution form a lattice that eventually develops into a visible precipitate. Antibodies that aggregate soluble antigens are called precipitins. Although formation of the soluble Ag-Ab complex occurs within minutes, formation of the visible precipitate occurs more slowly and often takes a day or two to reach completion. Formation of an Ag-Ab lattice depends on the valency of both the antibody and antigen: ■ The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments. ■ The antigen must be either bivalent or polyvalent; that is, it must have at least two copies of the same epitope, or have different epitopes that react with different antibodies present in polyclonal antisera. Experiments with myoglobin illustrate the requirement that protein antigens be bivalent or polyvalent for a precipitin reaction to occur. Myoglobin precipitates well with specific polyclonal antisera but fails to precipitate with a specific monoclonal antibody because it contains multiple, distinct epitopes but only a single copy of each epitope (Figure 6-4a). Myoglobin thus can form a crosslinked lattice structure with polyclonal antisera but not with monoclonal antisera. The principles that underlie precipitation reactions are presented because they are essential for an understanding of commonly used immunological assays. Although various modifications of the precipitation reaction were at one time the major types of assay used in immunology, they have been largely replaced by methods that are faster and, because they are far more sensitive, require only very small quantities of antigen or antibody. Also, these modern assay methods are not limited to antigen-antibody reactions that produce a precipitate. Table 6-3 presents a comparison of the sensitivity, or minimum amount of antibody detectable, by a number of immunoassays. Antigen-Antibody Interactions: Principles and Applications CHAPTER 6 141 TABLE 6-2 ABO blood types Blood type Antigens on RBCs Serum antibodies A A Anti-B B B Anti-A AB A and B Neither O Neither Anti-A and anti-B 8536d_ch06_137-160 8/1/02 9:01 AM Page 141 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: