Page 126 Figure 8.3 Diagrammatic representation of the avidity concept. The binding of antigen molecules by several antibodies of different specificities (A) stabilizes the immune complex, since it is highly unlikely that all Ag·Ab reactions dissociate simultaneously at any given point of time (B). Redrawn from Roitt, I. Essential Immunology, 4th ed. Blackwell Scientific Publications, Oxford, 1980. 1. Antisera containing polyclonal antibodies can often be found to cross-react with immunogens partially related to that used for immunization, due to the existence of common epitopes or of epitopes with similar configurations. 2. Less frequently, a cross-reaction may be totally unexpected, involving totally unrelated antigens that happen to present epitopes whose whole spatial configuration may be similar enough to allow the cross-reaction. 3. The avidity of a cross-reaction depends on the degree of structural similarity between the shared epitopes; when the avidity reaches a very low point, the cross-reaction will no longer be detectable (Fig. 8.4). 4. The differential avidity of given antiserum for the original immunogen and for other immunogens sharing epitopes of similar structure is responsible for the specificity of the antiserum (i.e., its ability to recognize only one single immunogen or a few, very closely related immunogens). Figure 8.4 Diagrammatic representation of the concept of cross-reaction between complex antigens. An antiserum containing several antibody populations to the determinants of a given antigen will react with other antigens sharing common or closely related determinants. The avidity of the reaction will decrease with decreasing structural closeness, until it will no longer be detectable. The reactivity of the same antiserum with several related antigens is designated as a cross-reaction. Redrawn from Roitt, I. Essential Immunology, 4th ed., Blackwell Scientific Publications, Oxford, 1980. Page 127 II. Specific Types of Antigen-Antibody Reactions A. Precipitation. When antigen and antibody are mixed in a test tube, one of two things may happen. Both components will remain soluble, or variable amounts of Ag·Ab precipitate will be formed. 1. The precipitin curve. If progressively increasing amounts of antigen are mixed with a fixed amount of antibody, a precipitin curve can be constructed (Fig. 8.5). There are three areas to consider in a precipitin curve. a. Antibody excess —Free antibody remains in solution after centrifugation of Ag·Ab complexes. b. Equivalence —No free antigen or antibody remains in solution. The amount of precipitated Ag·Ab complexes reaches its peak at this point. c. Antigen excess —Free antigen is detected in the supernatant after centrifugation of Ag·Ab complexes. 2. The lattice theory explains why different amounts of precipitation are observed at different antigen-antibody ratios. Figure 8.5 The precipitin curve. When increasing amounts of antigen are added to a fixed concentration of antibody, increasing amounts of precipitate appear as a consequence of the antigen-antibody interaction and, after a maximum precipitation is reached, the amounts of precipitate begin to decrease. Analysis of the supernatants reveals that at low antigen concentrations there is free antibody left in solution (antibody excess), at the point of maximal precipitation, neither antigen nor antibody are detected in the supernatant (equivalence zone); with greater antigen concentrations, antigen becomes detectable in the supernatant (antigen excess). Page 128 Figure 8.6 The lattice theory explaining precipitation reactions in fluid media: At great antigen excess (A), each antibody molecule has all its binding sites occupied. There is free antigen in solution, and the antigen-antibody complexes are very small (Ag 2 ·Ab 1 , Ag 1 ·Ab 1 ). The number of epitopes bound per antibody molecule at great antigen excess corresponds to the antibody valency. With increasing amounts of antibody (B), larger Ag·Ab complexes are formed (Ag 3 ·Ab 2 , etc.), but there is still incomplete precipitation and free antigen in solution. At equivalence, large Ag·Ab complexes are formed, in which virtually all Ab and Ag molecules in the system are cross-linked (C). Precipitation is maximal, and no free antigen or antibody are left in the supernatant. With increasing amounts of antibody (D), all antigen binding sites are saturated, but there is free antibody left without binding sites available for it to react. The Ag·Ab complexes are larger than at antigen excess [Ag 1 ·Ab 4,5,6(n) ], but usually soluble. The number of antibody molecules bound per antigen molecule at great antibody excess allows an estimate of the antigen valency. a. The equivalence point is characterized by maximum cross-linking between Ag and Ab (Fig. 8.6). b. At great antibody excess, each antigen will tend to have its binding sites saturated, with antibody molecules bound to all its exposed determinants. The number of antibody molecules bound to one single antigen molecule gives a rough indication of the valency of the antigen. c. At great antigen excess, the binding sites of the antibody molecule will be saturated by different antigen molecules and not much cross-linking will take place. 3. Precipitation in agar. Semisolid supports, such as agar gel, in which a carbohydrate matrix functions as a container for buffer that fills the interstitial spaces left by the matrix, have been widely used for the study of antigen-antibody reactions. Antigen and antibody are placed in wells carved in the semisolid agar, and allowed to passively diffuse. The diffusion of antigen and antibody is unrestricted, and in the area that separates antigen from antibody, the two reactants will mix in a gradient of concentrations. When the optimal proportions for Ag·Ab binding are reached, a precipitate will be formed, appearing as a sharp, linear opacity (Fig. 8.7). B. Agglutination. When bacteria, cells, or large particles in suspension are mixed with antibodies directed to their surface determinants, one will observe the formation of large clumps; this is known as an agglutination reaction. 1. Agglutination reactions result from the cross-linking of cells and insoluble particles by specific antibodies. Due to the relatively short distance between Page 129 Figure 8.7 Diagrammatic representation of a reaction of double immunodiffusion. Antigen and antibody are placed in opposite wells carved in a semisolid medium (e.g., agarose gel). Both antigen and antibody diffuse in all directions and toward each other, reacting and eventually reaching equivalence, at which point a linear precipitate appears between the antigen and antibody wells. the two Fab fragments, 7S antibodies (such as IgG) are usually unable to bridge the gap between two cells, each of them surrounded by an electronic “cloud” of identical charge that will tend to keep them apart. IgM antibodies, on the other hand, are considerably more efficient in inducing cellular agglutination (Fig. 8.8). 2. The visualization of agglutination reactions differs according to the technique used for their study. In slide tests, the nonagglutinated cell or particulate antigen appears as a homogeneous suspension, while the agglutinated antigen will appear irregularly clumped. If antibodies and cells are mixed in a test tube, the cross-linking of cells and antibodies will result in the diffuse deposition of cell clumps in the bottom and walls of the test tube, while the nonagglutinated red cells will sediment in a very regular fashion, forming a compact red button on the bottom of the tube. 3. Agglutination reactions follow the same basic rules of the precipitation reaction. a. When cells and antibody are mixed at very high antibody concentrations (low dilutions of antisera), antibody excess may result, no significant cross-linking of the cells is seen, and, therefore, the agglutination reaction may appear to be negative. Those dilutions at which antibody excess prevents agglutination constitutes the prozone. b. With increasing antibody dilutions, more favorable rations for cross-linking are reached, and very fine clumps cover the walls of the test tube or microtitration wells. c. When equivalence is approached, larger clumps of cells can be distinguished. d. At still higher dilutions, when the concentration of antibody is very low, the zone of antigen excess is reached, and agglutination is no longer seen (Fig. 8.9). III. Biological Consequences of the Antigen-Antibody Reaction A. Opsonization. After binding to particulate antigens or after forming large molecular aggregates, antibodies unfold and may interact with Fc receptors on Page 130 Figure 8.8 IgM antibodies are more efficient in inducing red cell agglutination. Red cells remain at the same distance from each other due to their identical electrical charge (zeta potential). IgG antibodies are not large enough to bridge the space between two red cells, but IgM antibodies, due to their polymeric nature and size, can induce red blood cell agglutination with considerable ease. phagocytic cells (see Chap. 6). Such interaction is followed by ingestion by the phagocytic cell (phagocytosis). Substances that promote phagocytosis are known as opsonins. B. Fc-Receptor-Mediated Cell Activation. The interaction of antigen-antibody complexes with phagocytic cells through their Fc receptors results in the delivery of activating signals to the ingesting cell. Figure 8.9 Diagrammatic representation of an hemagglutination reaction performed in a microtiter plate. The objective of the study is to determine the existence and titer of hemagglutinating antibodies in three different samples. In the first step, each sample is sequentially diluted from 1/10 to 1/20480 in a separate row of wells (A,B,C). In a second step, a fixed amount of red cells is added to each serum dilution with saline, a negative control. A, no agglutination can be seen. With patient A, the first three dilutions do not show agglutination (prozone), but the next dilutions, up to 1/5120 are positive; this sample is positive, and the tier is 5120. With patient B, the agglutination is positive until the 1/320 dilution; the titer of the sample is 320. Page 131 1. When the Fc-receptor-bearing cell is a phagocyte, the activation is usually associated with enhancement of its microbicidal activity. 2. Some of the toxic mediators generated in the phagocytic cell spill during phagocytosis. Spillage is maximal if the antigen-antibody complex is immobilized along a basement membrane or a cellular surface. The spillage of enzymes and oxygen active radicals can trigger an inflammatory reaction and cause tissue damage (see Chaps. 17 and 25). 3. The interaction of an antigen with IgE immobilized on a Fc receptor of a basophil or mast cell activates the release of potent mediators and triggers an allergic reaction (see Chap. 23). C. Complement Activation. One of the most important consequences of antigen-antibody interactions is the activation (or “fixation) of the complement system (see Chap. 9). 1. The activation sequence induced by antigen-antibody reactions is known as the “classical” pathway. This pathway is initiated by the binding of C1q to the CH 2 domain of the Fc region of IgG and equivalent regions of IgM. 2. The complement binding sequences in IgG and IgM are usually not exposed in free antibody molecules. The antigen-antibody interaction causes configurational changes in the antibody molecule and the complement binding regions become exposed. 3. The activation of C1q requires simultaneous interaction with two complement binding immunoglobulin domains. This means that when IgG antibodies are involved, relatively large concentrations are required, so that antibody molecules coat the antigen in very close apposition allowing C1q to be fixed by IgG duplets. On the other hand, IgM molecules, by containing five closely spaced monomeric subunits, can fix complement at much lower concentrations. One IgM molecule bound by two subunits to a given antigen will constitute a complement binding duplet. 4. After binding of C1q, a cascade reaction takes place, resulting in the successive activation of eight additional complement components. a. Some of the components generated during complement activation are recognized by receptors on phagocytic cells and promote phagocytosis. C3b is the complement fragment with greater opsonizing capacity. An antigen coated with opsonizing antibodies and C3b is taken up with maximal efficiency by phagoctic cells (see Chaps. 9 and 13). b. The terminal complement components bind to cell membranes where they polymerize, forming transmembrane channels and eventually inducing cell lysis. These reactions have great biological significance and have been adapted to a variety of serological tests for diagnosis of infectious diseases, as will be discussed in Chapter 14. c. The activation of the complement system may have adverse effects, if it results in the destruction of host cells or if it promotes inflammation, which is beneficial with regard to the elimination of infectious organisms, but always has the potential for causing tissue damage. D. Neutralization. The binding of antibodies to bacteria, toxins, and viruses has protective effects because it prevents the interaction of the microbial agents or their products with the receptors that mediate their ineffectiveness or toxic Page 132 effects. As a consequence, the infectious agent or the toxin become harmless, or, in other words, are neutralized. Self-Evaluation Questions Choose the ONE best answer. 8.1 The following precipitation curve is prepared by adding variable amounts of tetanus toxoid to a series of tubes containing 0.7 mg of antibody each. Precipitation observed in Supernatant from tube # supernatant after addition of 1 2 3 4 5 6 7 8 9 Antibody - - - - + + + + + Antigen + + ± - - - - - - (+ means visible precipitation; - means no visible precipitation) What tube(s) correspond to the antigen excess zone: A. 1 to 3 B. 1 to 4 C. 4 D. 4 to 9 E. 5 to 9 8.2 Using the data shown in Question 8.1, what was the concentration of antigen added to the tube corresponding to the equivalence point? A. 0.005 mg B. 0.01 mg C. 0.05 mg D. 0.1 mg E. 0.5 mg Page 133 8.3 The affinity of an antigen-antibody reaction depends primarily on the: A. Activation of the complement system B. Antibody isotype C. Closeness of fit between antibody-binding site and antigen epitope D. Nature of the antigen E. Valency of the antibody 8.4 Dr. I.M. Smart immunized a rabbit with sheep red cells and managed to separate anti-sheep red cell antibodies of several different isotypes. He then proceeded to mix 1 ml of saline containing 5 × 10 9 red cells and guinea pig complement with equimolecular concentrations of antibodies to sheep red cells of each different isotype. After incubating the mixture of red cells and anti-red cell antibodies for 5 minutes, he measured the amount of free hemoglobin in each tube. Which antibody was he likely to have added to the tube in which the concentration of free hemoglobin was highest? A. IgA B. IgD C. IgG1 D. IgG4 E. IgM 8.5 Dr. Smart then proceeded to perform another experiment in which he incubated 1 ml of saline containing 5 × 10 9 red cells with equimolecular concentrations of antibodies to sheep red cells of each different isotype, in the complete absence of complement. After incubating the mixture of red cells and anti-red cell antibodies for 1 hour, he washed the red cells, added them to human monocytes, incubated for another hour, and then examined the monocytes microscopically to determine whether they had ingested the sheep red cells. Which antibody was most likely to have been added to the red cells that were more efficiently ingested? A. IgA B. IgD C. IgE D. IgG1 E. IgM 8.6 The protective effect of preformed antiviral antibodies is related to their ability to: A. Agglutinate circulating viral particles B. Form soluble immune complexes with viral antigens C. Induce phagocytosis of the virus D. Lyse the virus E. Prevent the virus from infecting its target cell(s) 8.7 Which of the following hypotheses would sufficiently explain the nonprecipitation of an antigen-antibody system? A. The antigen has only two determinants B. The antigen has multiple, closely repeated determinants C. The antibody has been cleaved with papain D. The antibody has been cleaved with pepsin E. Both C and D are correct For Questions 8.8–8.10 find the ONE lettered sentence most closely related to it. An animal is immunized with DNP- ovalbumin and 2 weeks later the animal is bled and the [...]... surface that retards its rapid inactivation by Factor I and its cofactors (Factor H or CR1) 3 Binding of Factor B to C3b leading to C3bB 4 Activation of the bound B by D leading to C3bBb 5 C3bBb activation of more C3, leading to formation of C3bnBb (and liberation of more C3a) 6 Binding of properdin to C3b and stabilization of the association of Bb on C3b 7 C3bnBb activation of C5 (with liberation of C5a);... ligand-receptor interactions plays a major role in the effector stages of CD8-mediated cytotoxicity? A CD2 : CD58 (LFA -3 ) B CD8 : MHC-I C CD95 (Fas) : Fas ligand D IL-2 : IL-2R E Interferon-γ (IFN-γ) : IFN-γR 11 .3 The development of antigen-specific anergy is believed to result from: A Apoptosis of the antigen-stimulated T lymphocytes B Lack of delivery of co-stimulating signals by antigen-binding cells... or lack of activation of cytotoxic pathways depends on the balance between stimulatory and inhibitory receptors If the inhibitory receptor is not triggered (due to either presentation of a non-self peptide, lack of interaction of the inhibitory receptor with a non-self MHC-I-peptide complex, or to down-regulation of expression of MHC-I molecules on the cell membrane), stimulatory activity prevails and... yet, iC3b remains covalently bound to the foreign activator b Other very important reactive sites become exposed when Factor I cleaves C3b The newly exposed regions on antigen-bound iC3b react with other cell receptors, CR2 and CR3 CR3, which avidly binds to iC3b, is not a ubiquitous complement receptor (like CR1); rather, it is expressed on phagocytes and is very important in enhancing phagocytosis... active mediators and upregulation of CR1 and CR3, leading to enhanced phagocytosis, etc 2 Phagocytic cells, in addition to CR1, CR3, and CR4, have receptors for complement regulatory factors, such as C1 inhibitor Their physiological role has yet to be defined Page 209 CD2 reverts to resting levels The cytotoxic T cell can then move on to another antigen-bearing target with which it will develop the... enhancing phagocytosis c As iC3b continues to be degraded by Factor I, it breaks into two major fragments: C3dg and C3c The C3dg fragment remains bound to the antigen and retains the site that interacts with CR2 With time, C3dg is further degraded into C3d by the continued action of plasma or tissue proteases; this fragment remains bound to the antigen and, like C3dg, continues to express the site for interaction... to be host cell protective mechanisms, which probably explain how a phagocyte approaching a complement-activating immune complex is itself resistant to bystander damage initiated by activated complement fragments (C3b and C4b) being formed on and near its surface 1 CR1 receptor This glycoprotein binds to activated C3b and acts as a co-factor for Factor I, which cleaves C3b into inactivated C3b (iC3b)... C3dg, and C3d) remain associated to the antigen and react with other receptors in phagocytic cells (Fig 9.7) a A serum protease known as Factor I slowly cleaves the alpha chain of bound C3b, transforming this component into inactivated C3b (iC3b) This fragment has lost the sites that allow C3b to bind to other complement components; consequently, iC3b has irreversibly lost the ability to participate... that they are able to bind to C3b/C4b receptors (currently designated as CR1 receptors) located on almost all host cells, most notably phagocytes The increased affinity of phagocytic cells for C3b (or iC3b)/C4b-coated particles is known as immune adherence, and its main consequence is a significant enhancement of phagocytosis, which is Page 1 43 Figure 9.5 Binding of C3 fragments to cell membranes one... chelation of Ca2+ (to disrupt C1q, C1r2, and C1s2) and the addition of sufficient Mg2+ to allow activation of the alternative pathway 6 Factor B within the C3bB complex is activated by a plasma enzyme, Factor D, to yield activated C3bBb C3bBb is a C3 convertase that activates more C3, leading to the formation C3bnBb, which in turn is capable of activating C5 and the membrane attack complex 7 C3bnBb is stabilized . of 1 2 3 4 5 6 7 8 9 Antibody - - - - + + + + + Antigen + + ± - - - - - - (+ means visible precipitation; - means no visible precipitation) What tube(s) correspond to the antigen excess zone: A. 1 to 3 B. 1 to 4 C. 4 D. 4 to 9 E. 5 to 9 8.2 Using. I cleaves C3b. The newly exposed regions on antigen-bound iC3b react with other cell receptors, CR2 and CR3. CR3, which avidly binds to iC3b, is not a ubiquitous complement receptor (like CR1);. important in enhancing phagocytosis. c. As iC3b continues to be degraded by Factor I, it breaks into two major fragments: C3dg and C3c. The C3dg fragment remains bound to the antigen and retains