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:
8536d_ch06_137-160 8/1/02 9: 01 AM Page 142 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e 142 PART II Generation of B-Cell and T-Cell Responses (a) POLY CLONAL ANTISERUM Antibody-excess Equivalence ntigen-exce y excess xcess Ag MONOCLONAL ANTIBODY Antibody precipitated FIGURE6-4Precipitation reactions (a)Polyclonal antibodies can zone of antibody excess, in which precipitation is inhibited and anti- form lattices, or large aggregates, that precipitate out of solution. body not bound to antigen can be detected in the supernatant; an ognized by a given monoclonal antibody, the antibody can link only antigen form large insoluble complexes and neither antora However, if each antigen molecule contains only a single epitope rec- equivalence zone of maximal precipitation in which antibody two molecules of antigen and no precipitate is formed. (b)A precip. antigen can be detected in the supernatant; and a zone of antigen ex itation curve for a system of one antigen and its antibodies. This plot cess in which precipitation is inhibited and antigen not bound to of the amount of antibody precipitated versus increasing antigen antibody can be detected in the supernatant concentrations(at constant total antibody) reveals three zones: a Precipitation Reactions in Fluids Yield imentally today, the principles of antigen excess, antibody a Precipitin Curve cess,and equivalence apply to many Ag-Ab reactions A quantitative precipitation reaction can be performed by Precipitation Reactions in Gels Yield placing a constant amount of antibody in a series of tubes Visible Precipitin Lines and adding increasing amounts of antigen to the tubes. At one time this method was used to measure the amount of Immune precipitates can form not only in solution but also in antigen or antibody present in a sample of interest. After the an agar matrix. When antigen and antibody diffuse toward one precipitate forms, each tube is centrifuged to pellet the pre- another in agar, or when antibody is incorporated into the agar itate, the supernatant is poured off, and the amount of and antigen diffuses into the antibody-containing matrix,a precipitate is measured. Plotting the amount of precipitate visible line of precipitation will form. As in a precipitation re against increasing antigen concentrations yields a precipitin action in fluid, visible precipitation occurs in the region of curve. As Figure 6-4b shows, excess of either antibody or equivalence, whereas no visible precipitate forms in regions of antigen interferes with maximal precipitation, which occurs antibody or antigen excess. Two types of immunodiffusion re- in the so-called equivalence zone, within which the ratio of actions can be used to determine relative concentrations of an lattice is formed at equivalence, the complex increases in size relative purity of an antigen preparation. They are radial ina antibody to antigen is optimal. As a large multimolecular tibodies or antigens, to compare antigens, or to determine tl and precip itates out of solution. As show sion(the M ini metho der conditions of antibody excess or antigen excess, extensive diffusion(the Ouchterlony method ) both are carried out in lattices do not form and precipitation is inhibited. Although a semisolid medium such as agar In radial immunodiffusion, the quantitative precipitation reaction is seldom used exper- an antigen sample is placed in a well and allowed to diffuse into
Precipitation Reactions in Fluids Yield a Precipitin Curve A quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of tubes and adding increasing amounts of antigen to the tubes. At one time this method was used to measure the amount of antigen or antibody present in a sample of interest. After the precipitate forms, each tube is centrifuged to pellet the precipitate, the supernatant is poured off, and the amount of precipitate is measured. Plotting the amount of precipitate against increasing antigen concentrations yields a precipitin curve. As Figure 6-4b shows, excess of either antibody or antigen interferes with maximal precipitation, which occurs in the so-called equivalence zone, within which the ratio of antibody to antigen is optimal. As a large multimolecular lattice is formed at equivalence, the complex increases in size and precipitates out of solution. As shown in Figure 6-4, under conditions of antibody excess or antigen excess, extensive lattices do not form and precipitation is inhibited. Although the quantitative precipitation reaction is seldom used experimentally today, the principles of antigen excess, antibody excess, and equivalence apply to many Ag-Ab reactions. Precipitation Reactions in Gels Yield Visible Precipitin Lines Immune precipitates can form not only in solution but also in an agar matrix.When antigen and antibody diffuse toward one another in agar, or when antibody is incorporated into the agar and antigen diffuses into the antibody-containing matrix, a visible line of precipitation will form. As in a precipitation reaction in fluid, visible precipitation occurs in the region of equivalence, whereas no visible precipitate forms in regions of antibody or antigen excess. Two types of immunodiffusion reactions can be used to determine relative concentrations of antibodies or antigens, to compare antigens, or to determine the relative purity of an antigen preparation. They are radial immunodiffusion (the Mancini method) and double immunodiffusion (the Ouchterlony method); both are carried out in a semisolid medium such as agar. In radial immunodiffusion, an antigen sample is placed in a well and allowed to diffuse into 142 PART II Generation of B-Cell and T-Cell Responses FIGURE 6-4 Precipitation reactions. (a) Polyclonal antibodies can form lattices, or large aggregates, that precipitate out of solution. However, if each antigen molecule contains only a single epitope recognized by a given monoclonal antibody, the antibody can link only two molecules of antigen and no precipitate is formed. (b) A precipitation curve for a system of one antigen and its antibodies. This plot of the amount of antibody precipitated versus increasing antigen concentrations (at constant total antibody) reveals three zones: a zone of antibody excess, in which precipitation is inhibited and antibody not bound to antigen can be detected in the supernatant; an equivalence zone of maximal precipitation in which antibody and antigen form large insoluble complexes and neither antibody nor antigen can be detected in the supernatant; and a zone of antigen excess in which precipitation is inhibited and antigen not bound to antibody can be detected in the supernatant. ++++ +_ _ _ __ __ _ _ __ _ _ _ ++ ++ Antigen added Equivalence zone Antibody-excess zone POLYCLONAL ANTISERUM MONOCLONAL ANTIBODY Myoglobin Antigen-excess zone Supernatants excess Ab excess Ag Antibody precipitated (a) (b) 8536d_ch06_137-160 8/1/02 9:01 AM Page 142 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch06_137-160 8/1/02 9: 01 AM Page 143 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e Antigen-Antibody Interactions: Principles and Applications CHAPTER 6 143 TABLE 6-3Sensitivity of various immunoassays RADIAL IMMUNODIFFUSION Assay (ug antibody/ml) Precipitation reaction in fluid 20-200 Precipitation reactions in gels Antibody 98° incorporated Mancini radial immunodiffusion Ouchterlony double immunodiffusion 20-200 Immunoelectrophoresis 20-200 Rocket electrophoresis 2 Precipitate forms ring Agglutination reaction Direct DOUBLE IMMUNODIFFUSION Passive agglutination 0.006-006 Antibody Agglutination inhibition 0.006-0.06 Radioimmunoassay 0.0006-0.006 Enzyme- linked immunosorbent ssay(ELISA) <00001-0.01 ● ELISA using chemiluminescence <00001-0011 Immunofluorescence 0.06-0006 Agar matrix Precipitate The sensitivity depends upon the affinity of the antibody as well as the epi- tope density and distributio FIGURE 6-5 Diagrammatic representation of radial immunodiffu- TNote that the sensitivity of chemiluminescence-based ELISA assays can be sion(Mancini method)and double immunodiffusion(Ouchterlony made to match that of ria. ethod)in a gel. In both cases, large insoluble complexes form in SOURCE: Adapted from N R Rose et al, eds, 1997, Manual of Clinical the agar in the zone of equivalence, visible as lines of precipitation Laboratory Immunology, 5th ed, American Society for Microbiology, (purple regions). Only the antigen (red) diffuses in radial immuno- Washington, D.C. diffusion, whereas both the antibody(blue)and antigen(red)diffuse in double immunodiffusion agar containing a suitable dilution of an antiserum. As the the electric field, and antiserum is added to the troughs ntigen diffuses into the agar, the region of equivalence is es- Antibody and antigen then diffuse toward each other and blished and a ring of precipitation, a precipitin ring, forms produce lines of precipitation where they meet in appropr around the well( Figure 6-5, upper panel). The area of the pre- ate proportions(Figure 6-6a. Immunoelectrophoresis is cipitin ring is proportional to the concentration of antigen By used in clinical laboratories to detect the presence or absence comparing the area of the precipitin ring with a standard curve of proteins in the serum. A sample of serum is elec- (obtained by measuring the precipitin areas of known concen- trophoresed, and the individual serum components are trations of the antigen), the concentration of the antigen sam- identified with antisera specific for a given protein or im ple can be determined. In the Ouchterlony method, both munoglobulin class( Figure 6-6b). This technique is useful in antigen and antibody diffuse radially from wells toward each determining whether a patient produces abnormally low other, thereby establishing a concentration gradient. As equiv- amounts of one or more isotypes, characteristic of certain alence is reached, a visible line of precipitation, a precipitin immunodeficiency diseases. It can also show whether a pa line, forms(Figure 6-5, lower panel) ent overproduces some serum protein, such as albumin, mmunoglobulin, or transferrin. The immunoelectrophe Immunoelectrophoresis Combines retic pattern of serum from patients with multiple myeloma, Electrophoresis and Double for example, shows a heavy distorted arc caused by the large Immunodiffusion amount of myeloma protein, which is monoclonal Ig and therefore uniformly charged( Figure 6-6b). Because immu In immunoelectrophoresis, the antigen mixture is first elec- noelectrophoresis is a strictly qualitative technique that only trophoresed to separate its components by charge. Troughs detects relatively high antibody concentrations(greater than re then cut into the agar gel parallel to the direction of several hundred ug/ml), it utility is limited to the detection
agar containing a suitable dilution of an antiserum. As the antigen diffuses into the agar, the region of equivalence is established and a ring of precipitation, a precipitin ring, forms around the well (Figure 6-5, upper panel). The area of the precipitin ring is proportional to the concentration of antigen. By comparing the area of the precipitin ring with a standard curve (obtained by measuring the precipitin areas of known concentrations of the antigen), the concentration of the antigen sample can be determined. In the Ouchterlony method, both antigen and antibody diffuse radially from wells toward each other, thereby establishing a concentration gradient. As equivalence is reached, a visible line of precipitation, a precipitin line, forms (Figure 6-5, lower panel). Immunoelectrophoresis Combines Electrophoresis and Double Immunodiffusion In immunoelectrophoresis, the antigen mixture is first electrophoresed to separate its components by charge. Troughs are then cut into the agar gel parallel to the direction of the electric field, and antiserum is added to the troughs. Antibody and antigen then diffuse toward each other and produce lines of precipitation where they meet in appropriate proportions (Figure 6-6a). Immunoelectrophoresis is used in clinical laboratories to detect the presence or absence of proteins in the serum. A sample of serum is electrophoresed, and the individual serum components are identified with antisera specific for a given protein or immunoglobulin class (Figure 6-6b). This technique is useful in determining whether a patient produces abnormally low amounts of one or more isotypes, characteristic of certain immunodeficiency diseases. It can also show whether a patient overproduces some serum protein, such as albumin, immunoglobulin, or transferrin. The immunoelectrophoretic pattern of serum from patients with multiple myeloma, for example, shows a heavy distorted arc caused by the large amount of myeloma protein, which is monoclonal Ig and therefore uniformly charged (Figure 6-6b). Because immunoelectrophoresis is a strictly qualitative technique that only detects relatively high antibody concentrations (greater than several hundred g/ml), it utility is limited to the detection Antigen-Antibody Interactions: Principles and Applications CHAPTER 6 143 TABLE 6-3 Sensitivity of various immunoassays Sensitivity∗ Assay ( g antibody/ml) Precipitation reaction in fluids 20–200 Precipitation reactions in gels Mancini radial immunodiffusion 10–50 Ouchterlony double immunodiffusion 20–200 Immunoelectrophoresis 20–200 Rocket electrophoresis 2 Agglutination reactions Direct 0.3 Passive agglutination 0.006–0.06 Agglutination inhibition 0.006–0.06 Radioimmunoassay 0.0006–0.006 Enzyme-linked immunosorbent assay (ELISA) 0.0001–0.01 ELISA using chemiluminescence 0.0001–0.01† Immunofluorescence 1.0 Flow cytometry 0.06–0.006 ∗ The sensitivity depends upon the affinity of the antibody as well as the epitope density and distribution. † Note that the sensitivity of chemiluminescence-based ELISA assays can be made to match that of RIA. SOURCE: Adapted from N. R. Rose et al., eds., 1997, Manual of Clinical Laboratory Immunology, 5th ed., American Society for Microbiology, Washington, D.C. RADIAL IMMUNODIFFUSION Antibody incorporated in agar Antigen Antigen diffusion Precipitate forms ring DOUBLE IMMUNODIFFUSION Agar matrix Precipitate Antibody Antigen FIGURE 6-5 Diagrammatic representation of radial immunodiffusion (Mancini method) and double immunodiffusion (Ouchterlony method) in a gel. In both cases, large insoluble complexes form in the agar in the zone of equivalence, visible as lines of precipitation (purple regions). Only the antigen (red) diffuses in radial immunodiffusion, whereas both the antibody (blue) and antigen (red) diffuse in double immunodiffusion. 8536d_ch06_137-160 8/1/02 9:01 AM Page 143 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch06_137-160 8/1/02 9: 01 AM Page 144 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e 144 PART II Generation of B-Cell and T-Cell Responses (a) Ig A Ig G Antibody g M K separates the component antigens on the basis of charge Antiserum serum specific for the indicated antibody class or light chain t l a FIGURE Immunoelectrophoresis of an antigen mixture. (A-light-chain-bearing) antibody. A sample of serum from the patie (a)An antigen preparation (orange) is first electrophoresed, which was placed in the well of the slide and electrophoresed. Then (blue)is then added to troughs on one or both sides of the separated placed in the top trough of each slide. At the concentrations of pa- antigens and allowed to diffuse; in time, lines of precipitation(col- tient's serum used, only anti-IgG and anti-A antibodies produced ored arcs) form where specific antibody and antigen interact. (b) Im- lines of precipitation. [ Part (b ), Robert A. Kyle and Terry A. Katzman, munoelectrophoretic patterns of human serum from a patient with Manual of Clinical Immunology, 1997, N Rose, ed, ASM Press, Wash- myeloma. The patient produces a large amount of a monoclonal lgG ington, D.C., p. 164. of quantitative abnormalities only when the departure from are not sufficiently charged to be quantitatively analyzed normal is striking, as in immunodeficiency states and im- by rocket electrophoresis; nor is it possible to measure munoproliferative disorders the amounts of several antigens in a mixture at the same A related quantitative technique, rocket electrophore-time sis,does permit measurement of antigen levels. In rocket electrophoresis, a negatively charged antigen is elec trophoresed in a gel containing antibody. The precipitate Agglutination Reactions rocket, the height of which is proportional to the concen- The interaction between antibody and a particulate antigen tration of antigen in the well. One limitation of rocket sults in visible clumping called agglutination Antibodies that electrophoresis is the need for the antigen to be negatively produce such reaction are called agglutinins. Agglutination charged for electrophoretic movement within the agar reactions are similar in principle to precipitation reactions; matrix. Some proteins, immunoglobulins for example, they depend on the crosslinking of polyvalent antigens. Just as
of quantitative abnormalities only when the departure from normal is striking, as in immunodeficiency states and immunoproliferative disorders. A related quantitative technique, rocket electrophoresis, does permit measurement of antigen levels. In rocket electrophoresis, a negatively charged antigen is electrophoresed in a gel containing antibody. The precipitate formed between antigen and antibody has the shape of a rocket, the height of which is proportional to the concentration of antigen in the well. One limitation of rocket electrophoresis is the need for the antigen to be negatively charged for electrophoretic movement within the agar matrix. Some proteins, immunoglobulins for example, are not sufficiently charged to be quantitatively analyzed by rocket electrophoresis; nor is it possible to measure the amounts of several antigens in a mixture at the same time. Agglutination Reactions The interaction between antibody and a particulate antigen results in visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins. Agglutination reactions are similar in principle to precipitation reactions; they depend on the crosslinking of polyvalent antigens. Just as 144 PART II Generation of B-Cell and T-Cell Responses Antigens (a) Antibody FIGURE 6-6 Immunoelectrophoresis of an antigen mixture. (a) An antigen preparation (orange) is first electrophoresed, which separates the component antigens on the basis of charge. Antiserum (blue) is then added to troughs on one or both sides of the separated antigens and allowed to diffuse; in time, lines of precipitation (colored arcs) form where specific antibody and antigen interact. (b) Immunoelectrophoretic patterns of human serum from a patient with myeloma. The patient produces a large amount of a monoclonal IgG ( -light-chain-bearing) antibody. A sample of serum from the patient was placed in the well of the slide and electrophoresed. Then antiserum specific for the indicated antibody class or light chain type was placed in the top trough of each slide. At the concentrations of patient’s serum used, only anti-IgG and anti- antibodies produced lines of precipitation. [Part(b), Robert A. Kyle and Terry A. Katzman, Manual of Clinical Immunology, 1997, N. Rose, ed., ASM Press, Washington, D.C., p. 164.] Ig A Ig G Ig M κ λ (b) 8536d_ch06_137-160 8/1/02 9:01 AM Page 144 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch06_137-160 8/1/02 9: 01 AM Page 145 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e Antigen-Antibody Interactions: Principles and Applications CHAPTER 6 145 an excess of antibody inhibits precipitation reactions, such antigens, RBCs are mixed on a slide with antisera to the a excess can also inhibit agglutination reactions; this inhibition or B blood-group antigens. If the antigen is present on the is called the prozone effect. Because prozone effects can be en- cells, they agglutinate, forming a visible clump on the slide. countered in many types of immunoassays, understanding the Determination of which antigens are present on donor and basis of this phenomenon is of general importance. recipient RBCs is the basis for matching blood types for Several mechanisms can cause the prozone effect. First, at transfusions high antibody concentrations, the number of antibody bind ing sites may greatly exceed the number of epitopes. As a re sult, most antibodies bind antigen only univalently instead Bacterial Agglutination Is Used of multivalently. Antibodies that bind univalently cannot To Diagnose Infection crosslink one antigen to another. Prozone effects are readily a bacterial infection often elicits the production of antibodies specific for surface antigens on the bacterial cells. (or antigen)concentrations. As one dilutes to an optimum The presence of such antibodies can be detected by bacterial ntibody concentration, one sees higher levels of agglutina tion or whatever parameter is measured in the assay being agglutination reactions. Serum from a patient thought to be infected with a given bacterium is serially diluted in an array used When one is using polyclonal antibodies, the prozone of tubes to which the bacteria is added. The last tube showing effect can also occur for another reason. The antiserum may visible agglutination will reflect the serum antibody titer of the patient. The agglutinin titer is defined as the reciprocal of antigen but do not induce agglutination; these antibodies, the greatest serum dilution that elicits a positive agglutina called incomplete antibodies, are often of the IgG class. At high concentrations of IgG, incomplete antibodies may oc- tion reaction. For example, if serial twofold dilutions of HArpy most of the antigenic sites, thus blocking access by IgM, Serum are prepared and if the dilution of 1/640 shows agglu- which is a good agglutinin. This effect is not seen with agglu- tination but the dilution of 1/1280 does not, then the agglu- tinating monoclonal antibodies. The lack of agglutinating tination titer of the patient's serum is 640. In some cases activity of an incomplete antibody may be due to restricted Serum can be diluted up to 1750,000 and still show agglutina flexibility in the hinge region, making it difficult for the anti tion of bacteria The agglutinin titer of an antiserum can be used to diag- body to assume the required angle for optimal cross-linking nose a bacterial infection. Patients with typhoid fever, for ex- of epitopes on two or more particulate antigens. Alterna- tively, the density of epitope distribution or the location of ample, show a significant rise in the agglutination titer to some epitopes in deep pockets of a particulate antigen may almonella typhi. Agglutination reactions also provide a way to type bacteria. For instance, different species of the bac- make it difficult for the antibodies specific for these epitopes terium Salmonella can be distinguished by to agglutinate certain particulate antigens. When feasible, the actions with a panel of typing antisera. aggiutnanon re- solution to both of these problems is to try different antibod- ies that may react with other epitopes of the antigen that do not present these limitations. Passive Agglutination Is Useful Hemagglutination Is Used in Blood Typing with Soluble Antigens Agglutination reaction The sensitivity and simplicity of agglutination reactions can s( Figure 6-7)are routinely performed be extended to soluble antigens by the technique of passive to type red blood cells (RBCs). In typing for the ABo hemagglutination. In this technique, antigen-coated red blood cells are prepared by mixing a soluble antigen with red blood cells that have been treated with tannic acid or chromium chloride, both of which promote adsorption of the antigen to the surface of the cells. Serum containing anti- body is serially diluted into microtiter plate wells, and the antigen-coated red blood cells are then added to each well; glutination is assessed by the size of the characteristic spread pattern of agglutinated red blood cells on the bottom FIGURE6-7 Demonstration of hemagglutination using antibodies of the well, like the pattern seen in agglutination reactions against sheep red blood cells(SRBCs). The control tube(10) con.(see Figure 6-7) tains only SRBCs, which settle into a solid"button. "The experime Over the past several years, there has been a shift aw tal tubes 1-9 contain a constant number of SRBCs plus serial from red blood cells to synthetic particles, such as latex two-fold dilutions of anti-SRBC serum. The spread pattern in the ex. beads, as matrices for agglutination reactions. Once the anti perimental series indicates positive hemagglutination through tube gen has been coupled to the latex beads, the preparation can 3. Louisiana State University Medical Center/MIP. Courtesy of Hariet either be used immediately or stored for later use. The use C W. Thompson of synthetic beads offers the advantages of consistency
an excess of antibody inhibits precipitation reactions, such excess can also inhibit agglutination reactions; this inhibition is called the prozone effect. Because prozone effects can be encountered in many types of immunoassays, understanding the basis of this phenomenon is of general importance. Several mechanisms can cause the prozone effect. First, at high antibody concentrations, the number of antibody binding sites may greatly exceed the number of epitopes. As a result, most antibodies bind antigen only univalently instead of multivalently. Antibodies that bind univalently cannot crosslink one antigen to another. Prozone effects are readily diagnosed by performing the assay at a variety of antibody (or antigen) concentrations. As one dilutes to an optimum antibody concentration, one sees higher levels of agglutination or whatever parameter is measured in the assay being used. When one is using polyclonal antibodies, the prozone effect can also occur for another reason. The antiserum may contain high concentrations of antibodies that bind to the antigen but do not induce agglutination; these antibodies, called incomplete antibodies, are often of the IgG class. At high concentrations of IgG, incomplete antibodies may occupy most of the antigenic sites, thus blocking access by IgM, which is a good agglutinin. This effect is not seen with agglutinating monoclonal antibodies. The lack of agglutinating activity of an incomplete antibody may be due to restricted flexibility in the hinge region, making it difficult for the antibody to assume the required angle for optimal cross-linking of epitopes on two or more particulate antigens. Alternatively, the density of epitope distribution or the location of some epitopes in deep pockets of a particulate antigen may make it difficult for the antibodies specific for these epitopes to agglutinate certain particulate antigens. When feasible, the solution to both of these problems is to try different antibodies that may react with other epitopes of the antigen that do not present these limitations. Hemagglutination Is Used in Blood Typing Agglutination reactions (Figure 6-7) are routinely performed to type red blood cells (RBCs). In typing for the ABO antigens, RBCs are mixed on a slide with antisera to the A or B blood-group antigens. If the antigen is present on the cells, they agglutinate, forming a visible clump on the slide. Determination of which antigens are present on donor and recipient RBCs is the basis for matching blood types for transfusions. Bacterial Agglutination Is Used To Diagnose Infection A bacterial infection often elicits the production of serum antibodies specific for surface antigens on the bacterial cells. The presence of such antibodies can be detected by bacterial agglutination reactions. Serum from a patient thought to be infected with a given bacterium is serially diluted in an array of tubes to which the bacteria is added. The last tube showing visible agglutination will reflect the serum antibody titer of the patient. The agglutinin titer is defined as the reciprocal of the greatest serum dilution that elicits a positive agglutination reaction. For example, if serial twofold dilutions of serum are prepared and if the dilution of 1/640 shows agglutination but the dilution of 1/1280 does not, then the agglutination titer of the patient’s serum is 640. In some cases serum can be diluted up to 1/50,000 and still show agglutination of bacteria. The agglutinin titer of an antiserum can be used to diagnose a bacterial infection. Patients with typhoid fever, for example, show a significant rise in the agglutination titer to Salmonella typhi. Agglutination reactions also provide a way to type bacteria. For instance, different species of the bacterium Salmonella can be distinguished by agglutination reactions with a panel of typing antisera. Passive Agglutination Is Useful with Soluble Antigens The sensitivity and simplicity of agglutination reactions can be extended to soluble antigens by the technique of passive hemagglutination. In this technique, antigen-coated red blood cells are prepared by mixing a soluble antigen with red blood cells that have been treated with tannic acid or chromium chloride, both of which promote adsorption of the antigen to the surface of the cells. Serum containing antibody is serially diluted into microtiter plate wells, and the antigen-coated red blood cells are then added to each well; agglutination is assessed by the size of the characteristic spread pattern of agglutinated red blood cells on the bottom of the well, like the pattern seen in agglutination reactions (see Figure 6-7). Over the past several years, there has been a shift away from red blood cells to synthetic particles, such as latex beads, as matrices for agglutination reactions. Once the antigen has been coupled to the latex beads, the preparation can either be used immediately or stored for later use. The use of synthetic beads offers the advantages of consistency, Antigen-Antibody Interactions: Principles and Applications CHAPTER 6 145 FIGURE 6-7 Demonstration of hemagglutination using antibodies against sheep red blood cells (SRBCs). The control tube (10) contains only SRBCs, which settle into a solid “button.” The experimental tubes 1–9 contain a constant number of SRBCs plus serial two-fold dilutions of anti-SRBC serum. The spread pattern in the experimental series indicates positive hemagglutination through tube 3. [Louisiana State University Medical Center/MIP. Courtesy of Harriet C. W. Thompson.] 8536d_ch06_137-160 8/1/02 9:01 AM Page 145 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch06_137-160 8/1/02 9: 01 AM Page 146 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e RT II Generation of B-Cell and T-Cell Responses uniformity, and stability. Furthermore, agglutination reac- types of home pregnancy test kits included latex particles ions employing synthetic beads can be read rapidly, often coated with human chorionic gonadotropin(HCG)and within 3 to 5 minutes of mixing the beads with the test sam- antibody to HCG(Figure 6-8). The addition of urine from ple. Whether based on red blood cells or the more convenient a pregnant woman, which contained HCG, inhibited agglu and versatile synthetic beads, agglutination reactions are tination of the latex particles when the anti-HCG antibody mple to perform, do not require expensive equipment, and was added; thus the absence of agglutination indicated can detect small amounts of antibody(concentrations as low pregnancy. as nanograms per milliliter Agglutination inhibition assays can also be used to deter mine whether an individual is using certain types of illegal In Agglutination Inhibition, Absence of drugs, such as cocaine or heroin. A urine or blood sample is Agglutination Is Diagnostic of Antigen first incubated with antibody specific for the suspected drug. Then red blood cells(or other particles)coated with the drug A modification of the agglutination reaction, called agglu- are added. If the red blood cells are not agglutinated by the tination inhibition, provides a highly sensitive assay for antibody, it indicates the sample contained an antigen recog small quantities of an antigen. For example, one of the early nized by the antibody, suggesting that the individual was KIT REAGENTS HCG Hapten carrier-conjugate Anti-HCG antibody TEST PROCEDURE Urine Anti-HCG HCG carTier Observe for visible POSSIBLE REACTIONS reaction: not pregnant Visible clumping HCG in No visible FIGURE6-8 The original home pregnancy test kit employed hap. kit would react, producing visible clumping. If a woman was preg ten inhibition to determine the presence or absence of human chori- nant, the HCG in her urine would bind to the anti-HCG antibodies, nic gonadotropin(HCG). The original test kits used the presence or thus inhibiting the subsequent binding of the antibody to the HCG- absence of visible clumping to determine whether HCG was present. carrier conjugate. Because of this inhibition, no visible clumping oc- If a woman was not pregnant, her urine would not contain HCG; in curred if a woman was pregnant. The kits currently on the market use this case, the anti-HCG antibodies and HCG-carrier conjugate in the ELISA-based assays (see Figure 6-10)
uniformity, and stability. Furthermore, agglutination reactions employing synthetic beads can be read rapidly, often within 3 to 5 minutes of mixing the beads with the test sample. Whether based on red blood cells or the more convenient and versatile synthetic beads, agglutination reactions are simple to perform, do not require expensive equipment, and can detect small amounts of antibody (concentrations as low as nanograms per milliliter). In Agglutination Inhibition, Absence of Agglutination Is Diagnostic of Antigen A modification of the agglutination reaction, called agglutination inhibition, provides a highly sensitive assay for small quantities of an antigen. For example, one of the early types of home pregnancy test kits included latex particles coated with human chorionic gonadotropin (HCG) and antibody to HCG (Figure 6-8). The addition of urine from a pregnant woman, which contained HCG, inhibited agglutination of the latex particles when the anti-HCG antibody was added; thus the absence of agglutination indicated pregnancy. Agglutination inhibition assays can also be used to determine whether an individual is using certain types of illegal drugs, such as cocaine or heroin. A urine or blood sample is first incubated with antibody specific for the suspected drug. Then red blood cells (or other particles) coated with the drug are added. If the red blood cells are not agglutinated by the antibody, it indicates the sample contained an antigen recognized by the antibody, suggesting that the individual was 146 PART II Generation of B-Cell and T-Cell Responses FIGURE 6-8 The original home pregnancy test kit employed hapten inhibition to determine the presence or absence of human chorionic gonadotropin (HCG). The original test kits used the presence or absence of visible clumping to determine whether HCG was present. If a woman was not pregnant, her urine would not contain HCG; in this case, the anti-HCG antibodies and HCG-carrier conjugate in the kit would react, producing visible clumping. If a woman was pregnant, the HCG in her urine would bind to the anti-HCG antibodies, thus inhibiting the subsequent binding of the antibody to the HCGcarrier conjugate. Because of this inhibition, no visible clumping occurred if a woman was pregnant. The kits currently on the market use ELISA-based assays (see Figure 6-10). KIT REAGENTS Hapten carrier–conjugate Anti–HCG antibody TEST PROCEDURE Urine Incubate HCG carrier conjugate Observe for visible clumping + Visible clumping reaction: pregnant No visible clumping HCG in urine + POSSIBLE REACTIONS reaction: not pregnant HCG and + Anti–HCG + – + + + 8536d_ch06_137-160 8/1/02 9:01 AM Page 146 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: