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: