IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 29. Immunological Methods

■ Many immunological techniques are based on antigen-antibody interaction; the high Specificity of Antibodies allows the identification, isolation, or quantification of the target antigen.

■ Cell populations can be detected and identified by their surface markers using immunofluorescence and immunohistochemical Methods.

■ The isolation of cell populations bearing specific surface markers can be achieved through various techniques, including fluorescence-activated cell sorting (FACS), panning, and density gradient ultracentrifugation.

■ The key indicators of the functional activity of lymphocytes include antibody or cytokine production, proliferative response to an antigen, or cytotoxic activity.

Immunology employs a wide range of experimental techniques borrowed from other biological disciplines. For instance, the isolation of Antigens and antibodies is performed using biochemical protein Fractionation Methods, while the genes encoding immunologically relevant molecules are sequenced using standard Molecular Genetics techniques. At the same time, immunology has developed its own specialized research Methods based on antigen-antibody interactions. These immunological techniques have, in turn, found Applications across various fields of biology. Specifically, any molecules possessing antigenic properties can be detected in Tissues via immunohistochemical staining. To quantify such molecules when present at extremely low concentrations, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are employed. Today, hundreds of diverse immunological methods exist; the most widely used ones are described in this chapter.

ANTIGEN-ANTIBODY INTERACTION

Precipitation Reaction

One of the earliest described manifestations of the antigen-antibody interaction is The formation of a precipitate when both reactants are mixed in equivalent or near-equivalent proportions. This phenomenon is observed in the classical precipitation reaction using soluble antigens and antibodies (Fig. 29.1). Performing this reaction in an Agar gel makes it possible to differentiate individual antigen-antibody reactions caused by various antibody populations present in the serum. This method, known as double immunodiffusion, is also used to assess the degree of relatedness between different antigens (Fig. 29.2).

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Fig. 29.1. The classical example of an in vitro antigen-antibody interaction is the precipitation reaction. To demonstrate this, increasing concentrations of an antigen are added to a solution of antibodies. The amount of precipitating immune complexes initially increases and then decreases. Thus, the precipitation curve exhibits three distinct zones.

Zone of antibody excess: the amount of antigen is insufficient to react with all the antibodies; free antibodies can be detected in the supernatant.

Zone of equivalence: the amount of antigen is sufficient to bind and precipitate all available antibodies; free antigens and antibodies are absent in the supernatant.

Zone of antigen excess: the amount of antigen exceeds what is required to bind all the antibodies, leading to a decrease in the amount of antibody in the precipitate. This is due to the solubilization of antigen-antibody complexes caused by antigen excess. The prominence of this phenomenon varies depending on the type of antibody and the animal species from which the antibodies were derived.

Fig. 29.2. For double immunodiffusion, agar gel is poured onto Glass slides, allowed to solidify, and wells are punched out and filled with the test solutions of antigen (Ag) and antibodies (Ab). The solutions diffuse into the gel, and a precipitation line (arc) forms at the intersection where Ag and Ab interact (with cross-linking and precipitation of immune complexes). This can be visualized by washing the gel (to remove soluble Proteins) and staining it with a protein-binding dye, such as Coomassie Brilliant Blue. This method can be used to determine the relatedness between antigens (blue circles) and their reaction with reference test antibodies (yellow circle). Three MAIN TYPES OF results are possible (numbers in the blue circles indicate antigen epitopes). In reaction (1), the precipitation arcs formed by the antibodies and the two test antigens fuse, indicating that the antibodies interact with identical epitopes on both antigens (epitope 1). This result does not necessarily imply the complete identity of the antigens themselves, but merely their identity in the sense that the given antibodies cannot distinguish between them. In reaction (2), the antibodies detect 3 different antigens that form independent precipitation arcs. In reaction (3), the antigens share a common epitope 1, but one of them carries an additional epitope 2. This case differs from reaction (1) in that here the antibodies can distinguish between the antigens because they react with both epitopes. The interaction with anti-epitope 1 forms a line of identity, whereas the reaction of anti-epitope 2 with epitope 2 produces a "spur", indicating only partial identity between the two antigens.

However, some antigen mixtures are too complex to be resolved simply by diffusion and precipitation. To analyze such mixtures, Immunoelectrophoresis was developed—before visualizing the antigens via the precipitation reaction, they are separated according to their electrical charge using this technique (Fig. 29.3).

Fig. 29.3. Immunoelectrophoresis allows the Separation of complex antigen mixtures, such as serum antigens (1). The separation is carried out in an agar gel placed within an electric field, with the pH of the gel adjusted so that positively charged proteins migrate toward the cathode and negatively charged ones toward the anode. (2) A trough is then cut between the wells and filled with an antibody solution, which diffuses into the gel. (3) Antigens and antibodies form precipitation arcs.

Methods based on gel diffusion allow only qualitative detection of antigens and antibodies, whereas the quantitative Assessment of the reacting components is carried out by the subsequently developed method of single radial immunodiffusion (Fig. 29.4).

Fig. 29.4. Single radial immunodiffusion makes it possible to quantify antigens (Ag). Antibodies (Ab) are mixed into an agar gel, which is then poured onto glass slides and allowed to solidify. Wells are punched into the solidified gel and filled with a standard volume of antigen solutions at various concentrations. The slides are left for at least 24 hours. During this time, the antigen diffuses into the gel and forms soluble complexes with the antibodies (in antigen excess). The complexes continue to diffuse, binding more and more antibodies until the equivalence point is reached and the complexes precipitate, forming a ring. The area is determined from the radius of the zone bounded by the precipitation ring. This area is proportional to the antigen concentration, which is calculated using a calibration curve (graph at the bottom). The same method can be applied in reverse to determine antibody concentration—that is, by incorporating the antigen into the gel and placing the antibodies into the wells.

Immunodiffusion in an electric field can be performed with the simultaneous counter-current movement of antigens and antibodies; this approach is termed counter-current Electrophoresis. A similar modification of single radial immunodiffusion is known as rocket electrophoresis (Fig. 29.5).

The described methods are used to detect antigens and antibodies at concentrations ranging from 20 µg/ml to 2 mg/ml.

Fig. 29.5. Countercurrent electrophoresis is performed in an agar gel adjusted to a pH such that the antibodies (Ab) carry a net negative charge and the test antigen (Ag) a positive charge. In an electric field, the antigen and antibodies migrate toward each other and form a precipitate. The principle is identical to that of double immunodiffusion, but the sensitivity is 10–20 times higher. Rocket immunoelectrophoresis allows Quantitative determination of an antigen in an antibody-containing gel, with the pH chosen such that the antibodies do not migrate while the antigen carries a net negative charge. The precipitation line traces a rocket-shaped pattern whose length is proportional to the antigen concentration, which can be determined from a calibration curve. Bottom right: a photograph of the stained gel. Both methods rely on differences in the net charges of the antigen and antibodies at a selected pH; such differences exist for most antigens because antibodies have a higher isoelectric point (i.e., they are neutral at a higher pH than most antigens). If the charges of the antigen and antibodies do not differ significantly, the antigen can be chemically modified to shift its isoelectric point. Rocket electrophoresis can also be performed in reverse to determine antibody concentration; in this case, it is crucial to properly select the gel and pH to immobilize the antigen without disrupting its Structure or hindering the Antigen-Antibody Reaction. (R is the precipitation ring diameter.)

Hemagglutination and Complement Fixation Assays

If antibodies are present in concentrations too low to be detected and quantified by countercurrent or rocket electrophoresis, the hemagglutination assay is used. This method is based on the ability of antibodies to cross-link red Blood Cells by interacting with their surface antigens (Fig. 29.6).

Fig. 29.6. Active hemagglutination assay (top left) is used to detect antibodies against erythrocyte antigens. The serum is serially diluted (usually twofold dilutions) with saline and added to the wells of a microplate (bottom; rows 1–10 from left to right). Rows 11 and 12 serve as positive and negative controls. In this example, 8 different antisera (A–H) are tested. A suspension of erythrocytes (containing a specific protein to prevent nonspecific red blood cell agglutination) is added to each well to a final cell concentration of 1%. If the antibody concentration in the well is sufficient to agglutinate (cross-link) all the erythrocytes, they settle to the bottom of the well, forming a diffuse mat. If there are insufficient antibodies, the cells slide down the walls of the well to the bottom, forming a small, dense "button." Some antibodies agglutinate red blood cells poorly, requiring an indirect hemagglutination assay for their detection: secondary antibodies are added to the wells to bind to the non-agglutinating antibodies already attached to the erythrocytes. Hemagglutination can also be used to detect non-erythrocyte antigens covalently or non-covalently coupled to red blood cells. To attach an antigen to the erythrocyte surface (sensitize them), chromium chloride, tannic acid, glutaraldehyde, and A number of other chemical agents are employed.

The antigen-antibody reaction leads to the formation of immune complexes that fix complement upon activation via the classical pathway, which forms The basis of one of the quantitative Methods for determining antigens and antibodies (Fig. 29.7). Hemagglutination and complement fixation assays make it possible to detect antibodies present at concentrations of <1 µg/ml.

Fig. 29.7. Determination of antibodies based on the complement fixation test. 1. Serial twofold dilutions of the test serum are prepared, distributed into tubes (or wells), and a fixed amount of antigen is added to each. If the serum contains antibodies, immune complexes are formed. 2. Complement is added to the mixture. If complexes are present, they bind and "consume" the complement. 3. At The final stage of the assay, indicator cells (erythrocytes) along with a subagglutinating amount of anti-erythrocyte antibodies are introduced into the mixture. If any complement remains in the mixture, the cells will be lysed; however, if the complement was bound by immune complexes in step (2), there will not be enough left to lyse the erythrocytes. An amount of complement is used that is just sufficient to lyse the indicator cells in the absence of complement consumption by immune complexes. The test is frequently performed on plastic microplates. This reaction can also be used to measure antigens by using a fixed amount of antibodies and various dilutions of the antigen. In this case, proper controls are particularly important, as some antibody preparations consume complement even before the antigen is added—for example, if the serum already contains immune complexes. Certain antigens also exhibit anticomplementary activity. Therefore, two control sets are required, containing only antibodies and only antigen respectively; neither reagent alone should fix complement.

Direct and Indirect Immunofluorescence

Immunofluorescence methods are widely used to detect autoantibodies and antibodies against tissue and cellular antigens (Fig. 29.8). Although technically more demanding than the methods described above, they offer a distinct advantage when multiple antibody specificities must be identified. Using tissue sections (containing a vast array of antigens) on a single glass slide, antibodies against several different antigens can be visualized while determining their intertissue (cellular) or intracellular distribution.

Fig. 29.8. Immunofluorescence is used to determine antigen localization in situ. To achieve this, sections are cut in a cryostat from a snap-frozen tissue block. This Procedure preserves labile antigens that might otherwise be destroyed by fixatives.

Direct immunofluorescence method. A solution of antibodies labeled with a fluorescent dye is applied to The surface of the tissue section; the specimen is incubated and then washed to remove unbound antibodies. The bound antibodies are subsequently visualized using a fluorescence Microscope. A beam of UV light directed onto the section through the objective lens reveals a dark field with bright green fluorescent areas where the bound antibodies are localized. The distribution of fluorescence on the section has a characteristic pattern for each tissue antigen.

Indirect immunofluorescence method. A solution of unlabeled antibodies is applied to the tissue section and subsequently detected using fluorochrome-labeled anti-immunoglobulin antibodies.

Indirect complement-fixation immunofluorescence method. This variation was developed to detect complement-fixing antibodies (see Fig. 29.7). Following Treatment of the tissue section with antibodies, fresh serum is applied as a source of complement. Complement binds to the sites where antibodies have localized. Due to the signal Amplification provided by the activation of the classical complement pathway (see Chapter 5), a single antibody molecule can cause the deposition of numerous C3b molecules on the section, which are then detected by treating the section with fluorescently labeled anti-C3b antibodies.

In addition, immunofluorescence tests can identify individual cells within a cell suspension, i.e., detect antigens On the surface of living cells. For this purpose, a suspension of viable cells fluorescently stained with specific Reagents is passed through a flow cytometer—an instrument that measures the fluorescence intensity of each cell across different spectral regions and sorts the cells based on their fluorescence parameters. This method makes it possible to isolate distinct cell populations, separating cells bearing specific surface antigens and correspondingly stained with different fluorescently labeled antibodies (Fig. 29.9). Chapter 13 details how this technique is applied to define subpopulations of developing thymocytes (see Fig. 13.19).

Fig. 29.9. Cells in the test sample are stained with specific fluorescent reagents to identify surface molecules and injected into the flow chamber of the instrument. Cells exiting the chamber enter a stream of sheath buffer solution. As the cells pass through a laser beam, detectors measure the size (forward scatter detector) and granularity (side scatter detector) of each cell, as well as the fluorescence colors (red and green) corresponding to two different surface markers. Due to vibration, The Cell-bearing liquid stream breaks into droplets; these droplets are given an electrical charge, allowing computer-controlled deflection plates to sort and collect distinct cell populations According to the measured parameters. The three-dimensional plot illustrates the size (s), cell count (n), and fluorescence (f) of lymphocytes from a whole cell population and a CD8⁺ subset isolated using a cell sorter with anti-CD8 antibodies.

Immunoassay of Antigens and Antibodies Using Labeled Reagents

Methods in this category are characterized by exceptionally high sensitivity and economical reagent consumption (Fig. 29.10). The most common of all immunological techniques is undoubtedly the enzyme-linked immunosorbent assay (ELISA) using radioisotope- or enzyme-labeled ligands (Fig. 29.11); it enables the analysis of large numbers of samples in a relatively short time. (Nowadays, fluorescent or chemiluminescent markers are increasingly used instead of radioactive labels.) Antigen quantities can be measured using a "sandwich" (double-antigen bridge) assay or a competitive immunoassay with any suitable detection marker (Fig. 29.12).

Fig. 29.10. Immunoassay for antibodies. 1. The antigen in a saline solution is incubated on a plastic solid support or in tubes, resulting in the adsorption of a small amount of antigen onto the plastic surface. 2. Unbound antigen is removed by washing. (The support can then be treated with an excess of an irrelevant protein to prevent subsequent nonspecific protein binding.) 3. Test antibodies are added and bind to the antigen. 4. Unbound proteins are washed away. 5. Antibodies are detected using a labeled Ligand. The ligand may be, for example, staphylococcal protein A, which binds to the Fc region of IgG; more commonly, other antibodies specific to the test antibodies are used. By employing a ligand that binds to a specific antibody class or subclass, antibody isotypes can be differentiated. 6. Unbound antibodies are removed by washing. 7. The bound label is quantified. A typical titration curve is shown at the bottom. As the amount of antibody increases, the signal intensity rises linearly from Background to a plateau level. The antibody titer can be accurately determined only within this linear range. The plateau level is typically 20–100 times higher than the background. The sensitivity of the method is usually approximately 1–50 ng of specific antibodies per ml. The Specificity of the assay can be verified by adding increasing concentrations of free test antigen to the test antibodies in step (3). The antigen binds to the antibodies and blocks their attachment to the solid-phase antigen. The addition of escalating amounts of free antigen results in a decrease in signal intensity.

Fig. 29.11. The ELISA microplate (solid support) is prepared in exactly the same manner as for the antibody immunoassay (see Fig. 29.10) up to step 4. In this system, the ligand is a molecule capable of detecting antibodies and covalently linked to an enzyme, such as peroxidase. The ligand binds to the test antibodies, and following the preliminary removal of unbound ligand by washing (6), the bound ligand is visualized by adding a chromogen (7)—a colorless substrate that is converted into a colored reaction product by the enzyme attached to the ligand. Bottom: a photograph of a "developed" microplate (8). The amount of test antibodies is quantified by measuring the optical density of the colored reaction product.

Fig. 29.12. 1. Competitive immunoassay. The test antigen, along with a labeled antigen, is applied to a solid support coated with specific antibodies. The higher the concentration of the test antigen in the solution, the lower the amount of standard labeled antigen that binds. This type of assay is frequently used to determine antigens present at relatively high concentrations, or Hormones that possess only a single antigen-binding site. 2. Double-antigen sandwich assay. The test solution is applied to a support with Immobilized Antibodies; if the antigen is present, it is captured by the antibodies. After washing away unbound material, the captured antigen is detected using labeled antibodies directed against a different epitope of that same antigen. Because the antigen is identified using two distinct types of antibodies—with the second added in excess—this method is highly specific and sensitive.

Immunoblotting and Immunoprecipitation

While the methods described above are generally used to quantify specific, known antigens and antibodies, it is often necessary to identify and characterize unknown antigens present in a multicomponent mixture. Immunoblotting is particularly well-suited for this purpose.

In immunoblotting, a complex mixture of antigens is first subjected to gel electrophoresis, after which the fractionated Peptides are transferred (blotted) onto a nitrocellulose membrane to identify individual antigens using specific antisera. By performing preliminary separation in sodium dodecyl sulfate polyacrylamide gels or isoelectric focusing gels, valuable data can be obtained regarding the molecular weight and isoelectric point of the target antigens, as well as their structural relatedness (Fig. 29.13).

Fig. 29.13. For immunoblotting, the target antigens are first separated by gel electrophoresis, such as sodium dodecyl sulfate Polyacrylamide gel electrophoresis or isoelectric focusing. The resulting fractions are electroblotted onto a nitrocellulose sheet placed within a specialized transfer chamber. The blots are then incubated with antibodies specific to the target antigen, washed, and treated with a radiolabeled conjugate to detect the bound antibodies. The underlying principle is analogous to that of RIA or ELISA. Following a subsequent wash, the nitrocellulose sheet is exposed to an X-ray film for autoradiography; the developed film reveals the band positions of the antigen that bound the labeled antibodies. This method can be modified by employing a chemiluminescent label or an enzyme-linked antibody conjugate (as in ELISA), which visualizes the bound material upon the direct addition of a chromogen to the nitrocellulose sheet.

In some cases, the Procedures of gel electrophoresis and blotting cause the antigen to denature, destroying certain epitopes and abolishing their ability to bind specific antibodies. Under such circumstances, immunoprecipitation should be used instead of blotting to determine which antigen is bound by the antibodies. This technique can be applied to the detection of both soluble and membrane-associated antigens (Fig. 29.14).

Fig. 29.14. In an immunoprecipitation assay, the target antigens are radiolabeled with ¹³¹I and mixed with antibodies that bind exclusively to the specific antigen of interest. The resulting complexes are precipitated by adding coprecipitating agents, such as anti-immunoglobulin antibodies or staphylococcal Protein A. The insoluble complexes are then pelleted by centrifugation, and the pellet is washed extensively to completely remove unbound labeled antigens. The precipitate is resolubilized (e.g., in a sodium dodecyl sulfate solution) and subsequently resolved by gel electrophoresis. Following washing, the gels are subjected to autoradiographic analysis to detect the specific labeled antigen. Frequently, the study objects are surface antigens from radiolabeled cells that have been pre-solubilized using detergents. Alternatively, biotin can be used as a label; detection is then carried out chromatographically using streptavidin (a biotin-binding protein) conjugated to an enzyme, such as peroxidase (cf. ELISA).

(SDS – sodium dodecyl sulfate.)