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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 4.3Immunology Provides Important Techniques with Which to Investigate Proteins

Immunological methods have become important tools used to purify a protein, locate it in the cell, or quantify how much of the protein is present. These methods are predicated on the exquisite specificity of antibodies for their target proteins. Labeled antibodies provide a means to tag a specific protein so that it can be isolated, quantified, or visualized.

4.3.1. Antibodies to Specific Proteins Can Be Generated

Immunological techniques begin with the generation of antibodies to a particular protein. An antibody (also called an immunoglobulin, Ig) is a protein synthesized by an animal in response to the presence of a foreign substance, called an antigen, and normally functions to protect the animal from infection (Chapter 33). Antibodies have specific and high affinity for the antigens that elicited their synthesis. Proteins, polysaccharides, and nucleic acids can be effective antigens. An antibody recognizes a specific group or cluster of amino acids on a large molecule called an antigenic determinant, or epitope (Figures 4.30 and 4.31). Small foreign molecules, such as synthetic peptides, also can elicit antibodies, provided that the small molecule contains a recognized epitope and is attached to a macromolecular carrier. The small foreign molecule itself is called a hapten. Animals have a very large repertoire of antibody-producing cells, each producing an antibody of a single specificity. An antigen acts by stimulating the proliferation of the small number of cells that were already forming an antibody capable of recognizing the antigen (Chapter 33).

Figure 4.30. Antibody Structure.

Figure 4.30

Antibody Structure. Image mouse.jpg (A) IgG antibodies consist of four chains, two heavy chains (blue) and two light chains (red), linked by disulfide bonds. The heavy and light chains come together to form Fab domains, which have the antigen-binding sites at the ends. (more...)

Figure 4.31. Antigen-Antibody Interactions.

Figure 4.31

Antigen-Antibody Interactions. Image mouse.jpg A protein antigen, in this case lysozyme, binds to the end of an Fab domain from an antibody. The end of the antibody and the antigen have complementary shapes, allowing a large amount of surface to be buried on binding. (more...)

Immunological techniques depend on our being able to generate antibodies to a specific antigen. To obtain antibodies that recognize a particular protein, a biochemist injects the protein into a rabbit twice, 3 weeks apart. The injected protein stimulates the reproduction of cells producing antibodies that recognize the foreign substance. Blood is drawn from the immunized rabbit several weeks later and centrifuged to separate blood cells from the supernatant, or serum. The serum, called an antiserum, contains antibodies to all antigens to which the rabbit has been exposed. Only some of them will be antibodies to the injected protein. Moreover, antibodies of a given specificity are not a single molecular species. For instance, 2,4- dinitrophenol (DNP) has been used as a hapten to generate antibodies to DNP. Analyses of anti-DNP antibodies revealed a wide range of binding affinities—the dissociation constants ranged from about 0.1 nM to 1 μM. Correspondingly, a large number of bands were evident when anti-DNP antibody was subjected to isoelectric focusing. These results indicate that cells are producing many different antibodies, each recognizing a different surface feature of the same antigen. The antibodies are heterogeneous, or polyclonal (Figure 4.32). This heterogeneity is a barrier, which can complicate the use of these antibodies.

Figure 4.32. Polyclonal and Monoclonal Antibodies.

Figure 4.32

Polyclonal and Monoclonal Antibodies. Most antigens have several epitopes. Polyclonal antibodies are heterogeneous mixtures of antibodies, each specific for one of the various epitopes on an antigen. Monoclonal antibodies are all identical, produced by (more...)

4.3.2. Monoclonal Antibodies with Virtually Any Desired Specificity Can Be Readily Prepared

The discovery of a means of producing monoclonal antibodies of virtually any desired specificity was a major breakthrough that intensified the power of immunological approaches. Just as working with impure proteins makes it difficult to interpret data and understand function, so too does working with an impure mixture of antibodies. The ideal would be to isolate a clone of cells that produce only a single antibody. The problem is that antibody-producing cells isolated from an organism die in a short time.

Immortal cell lines that produce monoclonal antibodies do exist. These cell lines are derived from a type of cancer, multiple myeloma, a malignant disorder of antibody-producing cells. In this cancer, a single transformed plasma cell divides uncontrollably, generating a very large number of cells of a single kind. They are a clone because they are descended from the same cell and have identical properties. The identical cells of the myeloma secrete large amounts of normal immunoglobulin of a single kind generation after generation. A myeloma can be transplanted from one mouse to another, where it continues to proliferate. These antibodies were useful for elucidating antibody structure, but nothing is known about their specificity and so they are useless for the immunological methods described in the next pages.

Cesar Milstein and Georges Köhler discovered that large amounts of homogeneous antibody of nearly any desired specificity could be obtained by fusing a short-lived antibody-producing cell with an immortal myeloma cell. An antigen is injected into a mouse, and its spleen is removed several weeks later (Figure 4.33). A mixture of plasma cells from this spleen is fused in vitro with myeloma cells. Each of the resulting hybrid cells, called hybridoma cells, indefinitely produces homogeneous antibody specified by the parent cell from the spleen. Hybridoma cells can then be screened, by using some sort of assay for the antigen-antibody interaction, to determine which ones produce antibody having the desired specificity. Collections of cells shown to produce the desired antibody are subdivided and reassayed. This process is repeated until a pure cell line, a clone producing a single antibody, is isolated. These positive cells can be grown in culture medium or injected into mice to induce myelomas. Alternatively, the cells can be frozen and stored for long periods.

Figure 4.33. Preparation of Monoclonal Antibodies.

Figure 4.33

Preparation of Monoclonal Antibodies. Hybridoma cells are formed by fusion of antibody-producing cells and myeloma cells. The hybrid cells are allowed to proliferate by growing them in selective medium. They are then screened to determine which ones produce (more...)

The hybridoma method of producing monoclonal antibodies has opened new vistas in biology and medicine. Large amounts of homogeneous antibodies with tailor-made specificities can be readily prepared. They are sources of insight into relations between antibody structure and specificity. Moreover, monoclonal antibodies can serve as precise analytical and preparative reagents. For example, a pure antibody can be obtained against an antigen that has not yet been isolated (Section 4.4). Proteins that guide development have been identified with the use of monoclonal antibodies as tags (Figure 4.34). Monoclonal antibodies attached to solid supports can be used as affinity columns to purify scarce proteins. This method has been used to purify interferon (an antiviral protein) 5000-fold from a crude mixture. Clinical laboratories are using monoclonal antibodies in many assays. For example, the detection in blood of isozymes that are normally localized in the heart points to a myocardial infarction (heart attack). Blood transfusions have been made safer by antibody screening of donor blood for viruses that cause AIDS (acquired immune deficiency syndrome), hepatitis, and other infectious diseases. Monoclonal antibodies are also being evaluated for use as therapeutic agents, as in the treatment of cancer. Furthermore, the vast repertoire of antibody specificity can be tapped to generate catalytic antibodies having novel features not found in naturally occurring enzymes.

Figure 4.34. Fluorescence Micrograph of a Developing Drosophila Embryo.

Figure 4.34

Fluorescence Micrograph of a Developing Drosophila Embryo. The embryo was stained with a fluorescent-labeled monoclonal antibody for the DNA-binding protein encoded by engrailed, an essential gene in specifying the body plan. [Courtesy of Dr. Nipam Patel (more...)

4.3.3. Proteins Can Be Detected and Quantitated by Using an Enzyme-Linked Immunosorbent Assay

Antibodies can be used as exquisitely specific analytic reagents to quantify the amount of a protein or other antigen. The technique is the enzyme-linked immunosorbent assay (ELISA). In this method, an enzyme, which reacts with a colorless substrate to produce a colored product, is covalently linked to a specific antibody that recognizes a target antigen. If the antigen is present, the antibody-enzyme complex will bind to it, and the enzyme component of the antibody-enzyme complex will catalyze the reaction generating the colored product. Thus, the presence of the colored product indicates the presence of the antigen. Such an enzyme-linked immunosorbent assay, which is rapid and convenient, can detect less than a nanogram (10-9 g) of a protein. ELISA can be performed with either polyclonal or monoclonal antibodies, but the use of monoclonal antibodies yields more reliable results.

Image caduceus.jpg We will consider two among the several types of ELISA. The indirect ELISA is used to detect the presence of antibody and is the basis of the test for HIV infection. In that test, viral core proteins (the antigen) are absorbed to the bottom of a well. Antibodies from a patient are then added to the coated well and allowed to bind to the antigen. Finally, enzyme-linked antibodies to human antibodies (for instance, goat antibodies that recognize human antibodies) are allowed to react in the well and unbound antibodies are removed by washing. Substrate is then applied. An enzyme reaction suggests that the enzyme-linked antibodies were bound to human antibodies, which in turn implies that the patient had antibodies to the viral antigen (Figure 4.35).

Figure 4.35. Indirect ELISA and Sandwich ELISA.

Figure 4.35

Indirect ELISA and Sandwich ELISA. (A) In indirect ELISA, the production of color indicates the amount of an antibody to a specific antigen. (B) In sandwich ELISA, the production of color indicates the quantity of antigen. [After R. A. Goldsby, T. J. (more...)

The sandwich ELISA allows both the detection and the quantitation of antigen. Antibody to a particular antigen is first absorbed to the bottom of a well. Next, the antigen (or blood or urine containing the antigen) is added to the well and binds to the antibody. Finally, a second, different antibody to the antigen is added. This antibody is enzyme linked and is processed as described for indirect ELISA. In this case, the extent of reaction is directly proportional to the amount of antigen present. Consequently, it permits the measurement of small quantities of antigen (see Figure 4.35).

4.3.4. Western Blotting Permits the Detection of Proteins Separated by Gel Electrophoresis

Often it is necessary to detect small quantities of a particular protein in the presence of many other proteins, such as a viral protein in the blood. Very small quantities of a protein of interest in a cell or in body fluid can be detected by an immunoassay technique called Western blotting (Figure 4.36). A sample is subjected to electrophoresis on an SDS-polyacrylamide gel. Blotting (or more typically electroblotting) transfers the resolved proteins on the gel to the surface of a polymer sheet to make them more accessible for reaction. An antibody that is specific for the protein of interest is added to the sheet and reacts with the antigen. The antibody-antigen complex on the sheet then can be detected by rinsing the sheet with a second antibody specific for the first (e.g., goat antibody that recognizes mouse antibody). A radioactive label on the second antibody produces a dark band on x-ray film (an autoradiogram). Alternatively, an enzyme on the second antibody generates a colored product, as in the ELISA method. Western blotting makes it possible to find a protein in a complex mixture, the proverbial needle in a haystack. It is the basis for the test for infection by hepatitis C, where it is used to detect a core protein of the virus. This technique is also very useful in the cloning of genes.

Figure 4.36. Western Blotting.

Figure 4.36

Western Blotting. Proteins on an SDS-polyacrylamide gel are transferred to a polymer sheet and stained with radioactive antibody. A band corresponding to the protein to which the antibody binds appears in the autoradiogram.

4.3.5. Fluorescent Markers Make Possible the Visualization of Proteins in the Cell

Biochemistry is often performed in test tubes or polyacrylamide gels. However, most proteins function in the context of a cell. Fluorescent markers provide a powerful means of examining proteins in their biological context. For instance, cells can be stained with fluorescence-labeled antibodies or other fluorescent proteins and examined by fluorescence microscopy to reveal the location of a protein of interest. Arrays of parallel bundles are evident in cells stained with antibody specific for actin, a protein that polymerizes into filaments (Figure 4.37). Actin filaments are constituents of the cytoskeleton, the internal scaffolding of cells that controls their shape and movement. By tracking protein location, fluorescent markers also provide clues to protein function. For instance, the glucocorticoid receptor protein is a transcription factor that controls gene expression in response to the steroid hormone cortisone. The receptor was linked to green fluorescent protein (GPF), a naturally fluorescent protein isolated from the jellyfish Aequorea victoria (Section 3.6.5). Fluorescence microscopy revealed that, in the absence of the hormone, the receptor is located in the cytoplasm (Figure 4.38A). On addition of the steroid, the receptor is translocated to the nucleus, where it binds to DNA (Figure 4.38B).

Figure 4.37. Actin Filaments.

Figure 4.37

Actin Filaments. Fluorescence micrograph of actin filaments in a cell stained with an antibody specific to actin. [Courtesy of Dr. Elias Lazarides.]

Figure 4.38. Nuclear Localization of a Steroid Receptor.

Figure 4.38

Nuclear Localization of a Steroid Receptor. (A) The receptor, made visible by attachment of the green fluorescent protein, is located predominantly in the cytoplasm of the cultured cell. (B) Subsequent to the addition of corticosterone (a glucocorticoid (more...)

The highest resolution of fluorescence microscopy is about 0.2 μm (200 nm, or 2000 Å), the wavelength of visible light. Finer spatial resolution can be achieved by electron microscopy by using antibodies tagged with electron-dense markers. For example, ferritin conjugated to an antibody can be readily visualized by electron microscopy because it contains an electron-dense core rich in iron. Clusters of gold also can be conjugated to antibodies to make them highly visible under the electron microscope. Immunoelectron microscopy can define the position of antigens to a resolution of 10 nm (100 Å) or finer (Figure 4.39).

Figure 4.39. Immunoelectron Microscopy.

Figure 4.39

Immunoelectron Microscopy. The opaque particles (150-Å, or 15-nm, diameter) in this electron micrograph are clusters of gold atoms bound to antibody molecules. These membrane vesicles from the synapses of neurons contain a channel protein that (more...)

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22420


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