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Chapter 9 : Antibody-Antigen Interactions and Measurements of Immunologic Reactions

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Abstract:

Antibody-antigen interactions are in many respects comparable to enzyme-substrate interactions (immunologic reactions), in that both are highly specific, reversible, and based on noncovalent intermolecular interactions. The lock-and-key model describes antibody-antigen binding as the perfect fit of two rigid, complementary shapes. An important implication of the induced-fit model is a reduction in antibody-antigen specificity, since the ability of an antibody to subtly change the shape of its antigen-binding site might allow the site to bind multiple antigens. This could explain the frequently observed phenomenon of cross-reactivity, in which an antibody originally generated to one antigen is also capable of binding other, unrelated antigens. Affinity is one of the most important concepts that define the antibody-antigen interaction. The reversibility of antibody-antigen interactions implies that the binding conferred by the noncovalent forces generally is very weak and highly dependent on the distance between the partners. Electrostatic forces are the strongest of all noncovalent bonds but are relatively uncommon in naturally occurring antibody-antigen interactions. Tests based on antigen-antibody interactions are used widely in many fields because of their sensitivity, simplicity, and universal application of the concept that antibodies bind tightly and specifically to antigens. Large variety of laboratory techniques for determining antigen-antibody reactions and cellular responses to antigens have been developed, and sensitive analyses of single cell responses of immunologic relevance are now routinely made.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9

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Major Histocompatibility Complex
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Cytotoxic T Cell
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Enzyme-Linked Immunosorbent Assay
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Sodium Dodecyl Sulfate
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MHC Class I
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Figures

Image of Figure 9.1
Figure 9.1

The lock-and-key, induced-fit, and equilibrium models help explain the interaction of antibodies with antigen. According to the lock-and-key model, the CDRs of the antibody (black and dark blue triangles and rectangles) must be a perfect complement for the antigen's epitope for binding to occur. In contrast, the induced-fit model states that the CDRs of the antibody are able to alter their shape to some degree to adopt a conformation that is complementary to the epitope of the antigen. According to this model, the CDRs do not have to initially match the epitope perfectly for high-affinity binding to occur. In the equilibrium model one antibody exists in two isomeric forms that are both always available because they are in equilibrium, with each form able to bind to different and exclusive epitopes. When one isomeric form binds to its cognate antigen, it can no longer switch back to the other form; thus, to maintain the equilibrium, more of the form binding to the antigen is formed. This continues until all of the antigen is bound and the equilibrium can be maintained. Panel B is reprinted with permission from J. Foote, 1327–1328, 2003.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.2
Figure 9.2

The molecular bases of cross-reactivity. Cross-reactivity can occur if the same antigen is present on different cell types or in different tissues. In the example shown, an antibody that is meant to bind to T lymphocytes and that recognizes an epitope on the CD4 protein (orange) will cross-react with neurons, since the latter also express the CD4 protein. There are other potential antigens (green circle and red triangle) on T cells and neurons that will not be cross-reactive, since they are expressed on either T lymphocytes or neurons. Cross-reactivity can also result from the presence of identical epitopes on the surface of two nonidentical antigens. Serum albumins from chimpanzees and humans are closely related and thus share a large number of epitopes (red rectangles) that elicit antibodies (orange) that bind to this shared epitope on both albumins. Both human and chimpanzee albumins contain a number of unique epitopes that are not cross-reactive. Albumin in chicken blood is much more distantly related and, therefore, there are no shared antigenic epitopes and little or no reactivity of antibodies to human or chimpanzee albumin with chicken albumin. When human albumin is added to antibodies generated against human albumin all of the antialbumin antibodies will be bound to and precipitated by the antigen. In contrast, when chimpanzee albumin is added to the antibodies generated against human albumin only the antibodies in the antiserum that recognize the cross-reactive epitopes (orange antibodies) are bound to the chimpanzee albumin. If these immune complexes are separated from the unbound antibodies by centrifugation (precipitation), what remains soluble in the supernatant is antibodies specific to human serum albumin epitopes (pink antibodies). Cross-reactivity can result when two antigens possess similar (but not identical) epitopes. In the example shown, a monoclonal antibody is intended to be specific for “epitope 1.” However, in this case monoclonal antibody 1 cross-reacts with “epitope 2,” since epitope 2 is very similar to epitope 1. It is likely that the antibody will bind to epitope 2 more weakly than it binds to epitope 1. Two chemically unrelated entities, such as a protein antigen and a carbohydrate antigen, can have similar, cross-reactive epitopes.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.3
Figure 9.3

The application of cross-reactivity as a diagnostic tool. Individuals infected with EBV produce antibodies to EBV that are cross-reactive with an antigen on the surface of sheep red blood cells (RBCs). Therefore, if serum from an EBV-infected individual is mixed with sheep RBCs (), the antibodies to EBV will agglutinate (or clump) the RBCs, giving a positive reaction.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.4.
Figure 9.4.

The use of a biosensor device for the real-time measurement of antibody-antigen interactions. Antigen is covalently conjugated to the surface of a chip, and antibody is added to the solution above the surface of the chip.Meanwhile, a laser beam is bounced off the bottom surface of the chip. Binding or dissociation of the antibody to the surface of the chip results in mass changes near the surface, which alters its refractive index. This changes the angle of the reflected beam, which can be detected by a panel of photomultiplier tubes (right). A sample readout from a biosensor device in a hypothetical experiment similar to that depicted in panel A. Antibody is added at the time point marked by the arrow labeled “a,” and the binding event is measured over time. The change in the refractive index of the laser light is measured as a shift in the angular distance of the refracted light and is reported in arc seconds (60 arc seconds in an arc minute; 60 arc minutes equal 1 degree on a 360-degree circle; 3,600 arc seconds/degree). After a period of seconds to minutes, the binding event is complete, and the change in the refractive index levels off. If the antibody solution is then removed and the chip is washed using a solution that lacks antibody (time point marked with arrow b), the antibody that is already bound to the chip will dissociate. High-affinity antibodies bind rapidly (fast change in refractive index), lower-affinity antibodies bind more slowly. Similarly, antibodies with a low dissociation constant that stay bound to the antigen are removed slowly and there is a slower change in the refractive index, whereas antibodies with a high dissociation constant that are quickly washed off the antigen have a more rapid decrease in the refractive index.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.5
Figure 9.5

Equilibrium dialysis to measure antibody affinity. A small antigen can diffuse freely between the inside and outside of the dialysis tubing made of a semipermeable membrane, and the concentration of the antigen on both sides of the membrane eventually reaches equilibrium. If antibody molecules are placed inside the tubing, the large antibodies cannot diffuse through the dialysis membrane. The affinity of the antibody for the antigen corresponds to the amount of antigen that is kept inside the tubing compared with the concentration of free antigen outside. If the antigen is detectable by some tag such as a radioactive label, the amount of free antigen outside the membrane can be measured. It is assumed that the concentration of free antigen inside the membrane equals the concentration of free antigen outside the membrane. Any additional radioactivity inside the membrane represents the amount of bound antigen or the amount of antigen complexed to antibody (Ag – Ab). These data can be transformed by the Scatchard equation to estimate the affinity of an antibody for its antigen. If the value [Ag – Ab]/[Ag] (the concentration of antigen-antibody complexes divided by the concentration of free antigen) is plotted as a function of [Ag – Ab] (the concentration of antigen-antibody complexes), then the slope of the graph corresponds to (the dissociation constant).

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.6
Figure 9.6

Chemical forces in antigen-antibody interactions. From top to bottom in the figure, hydrogen bonds form when a single hydrogen atom is shared between highly electronegative atoms such as oxygen and nitrogen. The figure depicts a hydrogen bond between a threonine on the antigen and an asparagine on the antibody. Electrostatic interactions represent simple attraction between oppositely charged chemical groups on the two binding molecules. A positively charged amine group on a histidine molecule on the antigen is attracted to a negatively charged carboxyl group on an aspartic acid on the antibody. Hydrophobic interactions represent an energetically favorable juxtaposition of hydrophobic molecules (or hydrophobic regions of molecules), where the hydrophobic chemical groups get close enough to each other that water molecules are excluded from the space between them. The hydrophobic (aliphatic) regions of leucine and threonine amino acids on the antigen come extremely close to alanine and leucine amino acids on the antibody. While water molecules surround the aliphatic groups, all water is excluded from the space between the aliphatic groups. van der Waals forces (enlarged area) are weak electrostatic attractions that occur when two molecules (such as an antibody and antigen) get very near each other. The proximity of the two molecules causes local distortions in the electron clouds (gray) of both molecules, producing regions of higher electron (e) density (darker gray) and regions of lower electron density (lighter gray). The areas of higher electron density have a partial negative charge (δ), while the areas of lower electron density have a partial positive charge (δ). Electrostatic attractions occur between partial positive charges and partial negative charges.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.7
Figure 9.7

Antibody precipitation reactions. With increasing amounts of antigen added to a constant amount of antibody, the amount of precipitate will increase until the point of antigen excess, which prevents efficient cross-linking of antibody-antigen complexes. When antibody predominates (antibody excess, low antigen concentration), very few of the antibodies bind antigen, and those that do usually bind it monovalently. As antigen concentrations increase, a high level of cross-linking between the antigen and antibody can occur, giving rise to large, insoluble precipitates. The optimal ratio of antibody to antigen occurs in the zone of equivalence. As the amount of antigen increases further, all of the antibody is saturated and again large complexes cannot form, reducing the size of the precipitates (antigen excess). Fluid-phase immunoprecipitation. An antibody solution is overlaid onto a solution that contains antigen. Over time, antibodies and antigens that diffuse across the interface form lattices, which appear as an opaque ring. Single radial immunodiffusion, also called the Mancini test, starts with antibodies added to the gel matrix. The antigen is applied into a hole punched into the gel. As the antigen diffuses into the gel, a precipitation ring forms when the optimal ratio between the reactants is achieved. Beyond the outer edge, the reaction is in antibody excess and no precipitate forms. The diameter of the precipitation ring is proportional to the amount of antigen added. The Ouchterlony double immunodiffusion test with antigen and antibody applied into different holes diffusing toward each other to form a precipitin line at the zone of equivalence. The Ouchterlony test can be used to determine the identities of antigens in an unknown solution. Antigen solutions are placed into wells cut into agar plates. An additional well that is equidistant from all antigen-containing wells is filled with an antibody solution (for example, antiserum). Visible lines of precipitation (ppt) form where diffused antibody and antigen bind to each other and form lattices. If two adjacent wells contain the identical antigen, then a single precipitation line will form (far left panel). If two adjacent wells contain different antigens, each antigen will be precipitated by a different antibody in the antiserum. The result will be two lines of precipitation that cross each other (second panel from left). If one well contains several antigens, then multiple lines of precipitation are possible, some of which may demonstrate identity with antigens in adjacent wells (second panel from right). Last, when more than one antigen is present in a well it is possible that one of the antigens will possess some epitopes that are cross-reactive with epitopes on the other antigen but possess other epitopes that are not cross-reactive. In this case, antibodies that can react with epitopes common to both antigens will form a precipitation line of identity, while other antibodies that bind non-cross-reactive epitopes will diffuse past the line of identity and form a precipitation line with the specific epitope, resulting in a “spur” on the precipitation line (far right panel).

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.8
Figure 9.8

Immunoelectrophoresis. A mixture of antigens (A and B represent negatively charged antigens, while C represents a positively charged antigen) is added to a hole punched into an agarose or similar gel. An electric field is applied; it separates the different antigens by their charge and size. Although antigens A and B are both negatively charged, antigen A is larger and hence moves more slowly through the gel toward the positive pole. After the electric field is shut off, two troughs (dark blue and orange) are cut into the gel and are filled with antisera to the various antigens. In this example, a mixture of antibodies to antigens A and C is added to the lower trough, and antiserum directed to antigen B is added to the upper trough. After the electrical field is shut off, the migrated antigens diffuse out concentrically from the location to which they electrophoresed. The antibodies in each antiserum also diffuse away from the troughs. An antibody-antigen precipitation reaction occurs in the gel at each equivalence zone, allowing the simultaneous identification of different antigens.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.9
Figure 9.9

Western blot assay. A mixture of different antigens is combined with the negatively charged detergent sodium dodecyl sulfate and is applied to the top of a thin polyacrylamide gel. An electric field is applied. Because of the negatively charged sodium dodecyl sulfate, all antigens migrate toward the positive electrode at a rate that is inversely related to their molecular sizes. Large antigens (for example, antigen 2) migrate more slowly, while small antigens (for example, antigen 1) migrate faster. The separated antigens are transferred to a flexible membrane made of nitrocellulose or polyvinylidene fluoride (PVDF) by overlaying the membrane on the gel and applying an electric field. The membrane is then immersed in a solution containing antibody specific for one or more of the antigens bound to the membrane. The bound antibody can then be detected with an enzyme-linked secondary antibody and an enzyme substrate (not shown). The result is a visible line at the place in the gel to which the antigen had migrated. A sample Western blot.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.10
Figure 9.10

Rocket immunoelectrophoresis. This is a combination of single immunodiffusion ( Fig. 9.7C ) and immunoelectrophoresis ( Fig. 9.8 ). Antiserum is mixed into a gel such as agarose, as is done in single immunodiffusion. Next, an antigen solution is applied to wells and then an electrical field is applied to move the antigen through the gel (as in immunoelectrophoresis). A precipitation line will form at the equivalence point between antigen and antibody concentration. The height of the rocket-shaped precipitation line corresponds to the amount of antigen applied.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.11
Figure 9.11

Principles of agglutination and complement fixation tests. Direct agglutination. A latex bead is coated with an antigen and a test serum is added. Visible agglutination of the latex bead is indicative of a positive reaction well. Negative results are shown in wells b and c. The hemagglutination inhibition test can be used to detect and measure antibodies directed against viruses that agglutinate erythrocytes (red blood cells [RBCs]). If antibodies to a virus that can agglutinate erythrocytes are present in a serum sample, they will inhibit hemagglutination induced by the virus. Hemagglutination is detected by the formation of a lattice of RBCs coating the wells.Wells with a pellet of RBCs at the bottom indicate the presence of antibodies, which prevents viral hemagglutination, allowing the RBCs to roll to the bottom of the wells. Serial dilutions of sera give titers that are able to prevent agglutination (i.e., >1:512 in column 1, 1:16 in column 2, etc.). Photo courtesy of A. Angulo, Texas Veterinary Medical Center. Complement fixation. After an RBC is coated with an antigen (yellow semicircles) and specific antibody is added, a complement source can also be added. During an incubation step, antibody-coated red cells are lysed by complement activation on or near the cell surface. The hemoglobin released from the lysed cells can be measured spectrophotometrically. The remnants of the lysed RBCs are referred to as “ghosts”; they are transparent because of their lack of hemoglobin.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.12
Figure 9.12

ELISA. Indirect ELISA.Wells of microtiter plates are coated with antigen (green circles). A test sample is then applied; it may or may not contain antibody that binds specifically to the immobilized antigen. After unbound primary antibody is washed away, a secondary antibody that is specific for the primary antibody is added. If the primary antibody is specific for the immobilized antigen, and if it binds to the immobilized antigen (left wells), then the primary antibody will still be present after the washing step and can be bound by the secondary antibody. If the primary antibody is not specific for the coated antigen (middle wells) or if no primary antibody is added (right wells), then no secondary antibody will be bound. The secondary antibody is chemically conjugated to an enzyme that leads to a color reaction after the application of an appropriate chromogenic substrate. In the example shown, the secondary antibody is conjugated to the enzyme alkaline phosphatase (blue circles), which catalyzes the conversion of -nitrophenylphosphate (pNPP) to -nitrophenol (pNP). The optical density of the color reaction can be detected quantitatively with a spectrophotometric reader. Sandwich ELISAs are used to detect or measure antigen. The wells of a microtiter plate are coated with a capture antibody. The test solution containing the antigen is applied. If antigen reactive with the immobilized capture antibody is present (left wells), it will be bound. If the antigen is not specifically bound by the capture antibody (middle wells), or if no antigen is added (right wells), then no antigen will be bound. A secondary antibody, which recognizes an epitope on the antigen that is different than the epitope recognized by the capture antibody, is then used. This secondary antibody is conjugated with an enzyme that catalyzes a color reaction when the chromogenic substrate is applied. ELISPOT test to detect ASC.Wells of a microtiter plate are coated with antigen, and a source of ASC (such as a single cell suspension derived from an immune spleen) is applied. If a given cell is an ASC, the antibody produced by the cell will bind to the antigen surrounding the ASC. After incubation and washing, enzyme-linked secondary antibodies to the secreted antibodies are applied, and after washing a chromogenic substrate that gives rise to an insoluble product is added. The precipitate will form a spot that can be counted as being derived from one ASC. Courtesy of BD Biosciences; original photo © 2003 BD Biosciences. RIA. Before the RIA is performed, a radioactive label must be applied to an antigen (for example, a protein antigen might be synthesized in the presence of a radiolabeled amino acid). The radiolabeled antigen is then reacted with a test antiserum. If antigen-specific antibodies are present, they bind to the labeled antigen. The antibodies are then precipitated by addition of an antibody-binding protein such as protein A, and the protein A-antibody complex is pelleted by centrifugation. In a positive test the radioactive label is detected in the pellet, while in a negative test the radioactive label remains in solution.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.13
Figure 9.13

Flow cytometry and FACS can be used to measure subpopulation frequencies of cells in suspension and can separate the subpopulations. Cells can be identified in a number of ways, all based on expression or labeling with a fluorescent molecule. Recombinant cells can be made to express fluorescing proteins, and levels of fluorescence are determined under different conditions of cell stimuli. Also, cells can be reacted with fluorescently tagged antibodies that specifically bind to antigens of interest. These methods can identify the cells in the overall population that express that antigen. The cells are then passed through a nozzle that creates a flow of single cells. Separation (or “sorting”) of different cell subpopulations is achieved by deflecting the flow of cells into one of several pathways. Flow cytometric measurements are then made when the cells pass through a laser beam. Depending on how much of the laser light is adsorbed and scattered, certain characteristics of the single cells can be measured and analyzed by a computer system connected to the apparatus. For larger cells, the light passing through is scattered over a wider angle, producing a high level of “forward scatter.” For cells with complex structures in their cytoplasm, such as granules, there is a greater degree of side or 90° scatter. The data can be depicted graphically by using a relative scale of intensities. Thus, neutrophils, which are the largest and most granular of the leukocytes, have high levels of forward and 90° scatter. Monocytes are somewhat smaller and less granular, giving an intermediate level of forward and 90° scatter, and lymphocytes are the smaller leukocytes with the least granular cytoplasm. In addition, the amount of fluorescence given off by each cell is measured. A dual-parameter dot plot using two different antibodies (anti-CD4 and anti-CD8) labeled with two different fluorophores (the green dye fluorescein isothiocyanate [FITC] and the red dye Texas red) shows the types of T cells one would find in a thymus. Cells expressing neither CD4 nor CD8 (double negative [DN] cells) would be plotted in the lower left quadrant, CD4 T cells would be plotted in the lower right quadrant, CD8 T cells would be plotted in the upper left quadrant, and double-positive (DP) (CD4+ CD8) cells would be plotted in the upper right quadrant.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.14
Figure 9.14

The use of antibodies for the microscopic detection of antigens. Fluorescent immunohistochemistry allows for detection of antigens by light microscopy. In this case, the antigen of interest (an ionchannel protein) is detected in epithelium with the use of an antibody that is covalently coupled to the green fluorescent dye fluorescein isothiocyanate. Immunogold electron microscopy is a process whereby antigens are bound by antibodies covalently coupled to gold particles. The primary antibody in this instance is directed against a capsular or surface polysaccharide of a bacterium () and the binding of this IgG antibody is detected by addition of protein A (an IgG-binding protein) conjugated to 20-nanometer gold particles.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.15
Figure 9.15

Biologically relevant immunoassays. Toxin neutralization. In this assay, toxin (green triangles) produced by a microbe is given to an animal that will die from exposure to the toxin. If a specific immune serum is added, the toxin will be neutralized and the animal protected from the toxic effects. Virus neutralization. A suspension of virus particles treated with a control or test serum is given to an animal. If the test serum can neutralize the virus, the animals will be protected, while animals treated only with control serum will die. The reduction in this level of infectivity can be quantified in the virus neutralization assay by varying the amount of neutralizing antibody added to the viral inoculum. Opsonophagocytic killing assay. In the presence of phagocytic cells (granulocytes), antibody, and complement (left), bacteria are opsonized by the antibody and complement split products (middle, after 30 minutes). Receptors on the surface of the phagocyte bind to the immobilized complement and immunoglobulin, which facilitates ingestion and killing of the microbe. By 60 to 90 minutes (right), the majority of the bacteria are opsonized, phagocytosed, and killed in the presence of an immune serum with a large amount of antibody. Almost all antibodies mediating protective immunity to bacterial infections have this property.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.16
Figure 9.16

Lymphocyte proliferation or activation assay. Antigenpresenting cells (yellow) take up, process, and present antigen (red dots) on MHC class II to antigen-specific CD4 T cells (blue). The specific T cells proliferate in response to the presentation of the antigen and release IL-2 (T-cell growth factor) (green dots), which can be measured by ELISA. Alternately, [3H]thymidine can be added 12 to 24 hours before the end of the assay, and actively proliferating T cells will incorporate it into their newly synthesized DNA. Increases in nuclear [H]thymidine can be detected by scintillation counting.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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Image of Figure 9.17
Figure 9.17

Cytotoxicity assay. An epithelial cell (beige) presents an antigen (red dot) on MHC class I; CD8 T cells (blue) recognize the antigen as foreign and lyse the epithelial cell in response to the presentation of the antigen on MHC class I molecules. The lysis of the epithelial cells is accompanied by the release of cytoplasmic lactate dehydrogenase (LDH; green triangles) that can be measured to quantify the cytotoxic effect. Alternatively (not shown), the target cell can be labeled with 51Cr, and this radioactive marker is released when the cell is killed. The cytotoxicity assay can be made quantitative by varying the ratio of effector cytotoxic cells to target cells. Populations of cells with more potent cytotoxic activity will kill more targets at lower E:T ratios.

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
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References

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1. Bosshard, H. R. 2001. Molecular recognition by induced fit: how fit is the concept? News Physiol. Sci. 16:171173.
2. Djavadi-Ohaniance, L.,, M. E. Goldberg,, and B. Friguet,. 1996. Measuring antibody affinity in solution, p. 7797. In J. McCafferty,, H. R. Hoogenboom,, and D. J. Chiswell (ed.), Antibody Engineering, a Practical Approach. IRL Press, Oxford, United Kingdom.
3. Gabdoulline R. R.,, and R. C. Wade. 2002. Biomolecular diffusional association. Curr. Opin. Struct. Biol. 12:204213.
4. Padlan, E. A. 1994. Antibody-Antigen Complexes. R. G. Landes, Austin, Tex..
5. Rose, N. R.,, R. G. Hamilton,, and B. Detrick (ed.). 2002.Manual of Clinical Laboratory Immunology, 6th ed. American Society for Microbiology,Washington, D.C..
6. Zayats, M.,, O. A. Raitman,, V. I. Chegel,, A. B. Kharitonov,, and I. Willner. 2002. Probing antigen-antibody binding processes by impedance measurements on ion-sensitive field-effect transistor devices and complementary surface plasmon resonance analyses: development of cholera toxin sensors. Anal. Chem. 74:47634773.

Tables

Generic image for table
Table 9.1

Tests based on antigen-antibody interactions

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9
Generic image for table
Table 9.2

Identification of blood types by direct hemagglutination assays

Citation: Huebner J. 2004. Antibody-Antigen Interactions and Measurements of Immunologic Reactions, p 207-232. In Pier G, Lyczak J, Wetzler L (ed), Immunology, Infection, and Immunity. ASM Press, Washington, DC. doi: 10.1128/9781555816148.ch9

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