Detection Methods Using High-Affinity Interactions Between Molecules

A molecule present in a tissue section may be identified by using compounds that specifically interact with the molecule. The compounds that will interact with the molecule must be tagged with a label that can be detected under the light or electron microscope (Figure 1–20). The most commonly used labels are fluorescent compounds (which can be seen with a fluorescence or laser microscope), radioactive atoms (which can be detected with autoradiography), molecules of peroxidase (which can be detected after demonstration of the enzyme with hydrogen peroxide and 3,3'-diaminoazobenzidine) or other enzymes (which can be detected with their respective substrates), and metal (usually gold) particles that can be observed with light and electron microscopy. These methods are mainly used for detecting sugars, proteins, and nucleic acids.

Figure 1-20

Compounds that have affinity toward another molecule can be tagged with a label and used to identify that molecule. (1) Molecule A has a high and specific affinity toward a portion of molecule B. (2) When A and B are mixed, A binds to the portion of B it recognizes. (3) Molecule A may be tagged with a label that can be visualized with a light or electron microscope. The label can be a fluorescent compound, an enzyme such as peroxidase, a gold particle, or a radioactive atom. (4) If molecule B is present in a cell or extracellular matrix that is incubated with labeled molecule A, molecule B can be detected.

Phalloidin, protein A, lectins, and antibodies are examples of compounds that interact specifically with other molecules.

Phalloidin, which is extracted from a mushroom (Amanita phalloides), interacts strongly with actin and is usually labeled with fluorescent dyes to demonstrate actin filaments.

Protein A is a protein obtained from Staphylococcus aureus that binds to the Fc region of immunoglobulin (antibody) molecules. When protein A is tagged with a label, immunoglobulins can be detected.

Lectins are proteins or glycoproteins that are derived mainly from plant seeds and that bind with high affinity and specificity to carbohydrates. Different lectins bind to specific sequences of sugar molecules. They may bind to glycoproteins, proteoglycans, and glycolipids and are widely used to characterize membrane molecules containing defined sequences of sugars.

Immunocytochemistry

A highly specific interaction between molecules is that between an antigen and its antibody. For this reason, methods using labeled antibodies have proved most useful in identifying and localizing specific proteins and glycoproteins.

The body has cells that are able to distinguish its own molecules (self) from foreign ones. When exposed to foreign molecules—called antigens—the body may respond by producing proteins—antibodies—that react specifically and bind to the antigen, thus helping to eliminate the foreign substance. Antibodies are proteins of a large family, the immunoglobulin family.

In immunocytochemistry, a tissue section (or cells in culture) that may contain a certain protein is incubated in a solution containing an antibody to this protein. The antibody binds specifically to the protein, whose location can then be seen with either the light or electron microscope, depending on the type of compound used to label the antibody.

One of the most important requirements for immunocytochemistry is the availability of an antibody against the protein that is to be detected. This means that the protein must have been previously purified and isolated so that antibodies can be produced. Some methods for protein isolation can be seen in Figures 1–21 and 1–22.

Figure 1-21

Ultracentrifugation (A) and chromatography (B): methods of protein isolation. A: A mixture of proteins obtained from homogenized cells or tissues is submitted to centrifugation at high speed for several hours. The proteins separate into several bands, depending on the size and density of the protein molecules. The ultracentrifugation medium is drained and collected in several fractions that contain different proteins, which can be analyzed further. B: A solution containing a mixture of proteins obtained from homogenized cells or tissues is added to a column filled with beads that have different chemical properties. For instance, the beads may have different electrostatic charges (attracting proteins according to their charge) or different sizes of pores (acting as sieves for different-sized molecules). As the proteins migrate through the column, their movement is slowed according to their interaction with the particles. When the effluent is recovered, the different groups of proteins may be collected separately.

Figure 1-22

Gel electrophoresis: a method of protein isolation. A: Isolation of proteins. (1) Mixtures of proteins are obtained from homogenized cells or tissues. They are usually treated with a strong detergent (sodium dodecyl sulfate) and with mercaptoethanol to unfold and separate the protein subunits. (2) The samples are put on top of a slab of polyacrylamide gel, which is submitted to an electrical field. The proteins migrate along the gel according to their size and shape. (3) A mixture of proteins of known molecular mass is added to the gel as a reference to identify the molecular mass of the other proteins. B: Detection and identification of the proteins. (1) Staining. All proteins will stain the same color. The color intensity is proportional to the protein concentration. (2) Autoradiography. Radioactive proteins can be detected by autoradiography. An x-ray film is apposed to the gel for a certain time and then developed. Radioactive proteins will appear as dark bands in the film. (3) Immunoblotting. The proteins can be transferred from the gel to a nitrocellulose membrane. The membrane is incubated with a labeled antibody made against proteins that may be present in the sample.

Polyclonal and Monoclonal Antibodies

Let us suppose that our objective is to produce antibodies against protein x of a certain animal species (eg, a rat or a human). If protein x is already isolated, it is injected into an animal of another species (eg, a rabbit or a goat). If the protein is sufficiently different for this animal to recognize it as foreign—that is, as an antigen—the animal will produce antibodies against the protein (eg, rabbit antibody against rat x or goat antibody against human x). These antibodies are collected from the animal's plasma and used for immunocytochemistry.

Several groups (clones) of lymphocytes of the animal that was injected with protein x may recognize different parts of protein x and each group produces an antibody against each part. These antibodies constitute a mixture of polyclonal antibodies.

It is possible, however, to furnish protein x for lymphocytes maintained in cell culture (actually, lymphocytes fused with tumor cells). The different clones of lymphocytes will produce different antibodies against the several parts of protein x. Each clone can be isolated and cultured separately so that the different antibodies against protein x can be collected separately. Each of these antibodies is a monoclonal antibody. There are several advantages to using a monoclonal antibody rather than a polyclonal antibody: for instance, a monoclonal antibody can be selected to be highly specific and to bind strongly to the protein to be detected. Therefore, there will be less nonspecific binding to other proteins similar to the one being looked for.

In the direct method of immunocytochemistry, the antibody (either monoclonal or polyclonal) must be tagged with an appropriate label. A tissue section is incubated with the antibody for some time so that the antibody interacts with and binds to protein x. The section is then washed to remove the unbound antibody (Figure 1–23). Depending on the label that was used (fluorescent compound, enzyme, gold particles), the section can be observed with a light or electron microscope. If peroxidase or another enzyme was used as a label, the enzyme must be detected before the tissue section is observed in the microscope (see Histochemistry & Cytochemistry). The areas of the tissue section that contain protein x will become fluorescent or will be covered by gold particles or by a dark precipitate if an enzyme was used as a marker.

Figure 1-23

Direct method of immunocytochemistry. (1) Immunoglobulin molecule (Ig). (2) Production of a polyclonal antibody. Protein x from a rat is injected into a rabbit. Several rabbit Igs are produced against protein x. (3) Labeling the antibody. The rabbit Igs are tagged with a label. (4) Immunocytochemical reaction. The rabbit Igs recognize and bind to different parts of protein x.

The indirect method of immunocytochemistry is more sensitive but requires more steps. Let us suppose that our objective is to detect protein x, present in rats. Before proceeding to the immunochemical reaction, two procedures are needed: (1) antibodies (polyclonal or monoclonal) to rat protein x must first be produced in an animal of another species (eg, a rabbit); (2) in a parallel procedure, immunoglobulin from a normal (noninjected) rabbit must be injected into an animal of a third species (eg, a goat). Rabbit immunoglobulins are considered foreign by a goat and are thus capable of inducing the production of an antibody (an antiantibody or antiimmunoglobulin) in that animal.

Indirect immunocytochemical detection is performed by initially incubating a section of a rat tissue believed to contain protein x with rabbit anti-x antibody. After washing, the tissue sections are incubated with labeled goat antibody against rabbit antibodies. The antiantibodies will bind to the rabbit antibody that had previously recognized protein x (Figure 1–24). Protein x can then be detected by using a microscopic technique appropriate for the label used in the secondary antibody. There are other indirect methods that involve the use of other intermediate molecules, such as the biotin-avidin technique.

Figure 1-24

Indirect method of immunocytochemistry. (1) Production of a primary polyclonal antibody. Protein x from a rat is injected into a rabbit. Several rabbit immunoglobulins (Ig) are produced against protein x. (2) Production of secondary antibody. Ig from a nonimmune (normal) rabbit is injected into a goat. Goat Igs against rabbit Ig are produced. The goat Igs are then isolated and tagged with a label. (3) First step of the immunocytochemical reaction. The rabbit Igs recognize and bind to different parts of protein x. (4) Second step of the immunocytochemical reaction. Labeled goat Igs recognize and bind to different parts of rabbit immunoglobulin molecules, therefore labeling protein x.

Medical Application

Immunocytochemistry has contributed significantly to research in cell biology and to the improvement of medical diagnostic procedures. Figures 1–25, 1–26, 1–27, and 1–28 show examples of immunocytochemical detection of molecules. Table 1–1 shows some of the routine applications of immunocytochemical procedures in clinical practice.

Table 1–1. Commonly Used Proteins (Antigens) Important for Immunocytochemical Diagnosis and Treatment of Disease.

Antigens Diagnosis Intermediate filament proteins  Cytokeratins Tumors of epithelial origin Glial fibrillary acid protein Tumors of some glial cells Vimentin Tumors of connective tissue Desmin Tumors of muscle Other proteins  Protein and polypeptide hormones Protein or polypeptide hormone–producing tumors Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the digestive tract and breast Steroid hormone receptors Breast duct cell tumors Antigens produced by viruses Specific virus infections

Figure 1-25

Photomicrograph of a mouse decidual cell grown in vitro. The protein desmin, which forms intermediate filaments, was detected with an indirect immunofluorescence technique. A mesh of fluorescent intermediate filaments occupies most of the cytoplasm. The nucleus (N) is stained blue. High magnification. (Courtesy of FG Costa.)

 

Figure 1-26

Photomicrograph of a section of small intestine in which an antibody against the enzyme lysozyme was applied to demonstrate lysosomes in macrophages and Paneth cells. The brown color, indicating the presence of lysozyme, results from the reaction done to show peroxidase, which was linked to the secondary antibody. Nuclei were counterstained with hematoxylin. Medium magnification.

Figure 1-27

Carcinoembryonic antigen is a protein present in several malignant tumors mainly of the breast and intestines. This photomicrograph is an immunocytochemical demonstration of carcinoembryonic antigen in a section of large intestine adenocarcinoma. The antibody was labeled with peroxidase and the brown precipitate indicates tumor cells. The counterstain was hematoxylin. Medium magnification.

Figure 1–28

Electron micrograph showing a section of a pancreatic acinar cell that was incubated with antiamylase antibody and stained by protein A coupled with gold particles. Protein A has high affinity toward antibody molecules. The gold particles appear as very small black dots over the secretory granules. (Courtesy of M Bendayan.)

Hybridization Techniques

The central challenge in modern cell biology is to understand the workings of the cell in molecular detail. This goal requires techniques that permit analysis of the molecules involved in the process of information flow from DNA to protein. Many techniques are based on hybridization. Hybridization is the binding between two single strands of nucleic acids (DNA with DNA, RNA with RNA, or RNA with DNA) that recognize each other if the strands are complementary. The greater the similarities of the sequences, the more readily complementary strands form "hybrid" double-stranded molecules. Hybridization thus allows the specific identification of sequences of DNA or RNA.

In Situ Hybridization

When applied directly to cells and tissue sections, smears, or chromosomes of squashed mitotic cells, the technique is called in situ hybridization. This technique is ideal for determining if a cell has a specific sequence of DNA (such as a gene or part of a gene), for identifying the cells in which a specific gene is being transcribed, or for determining the localization of a gene in a specific chromosome. The DNA inside the cell must be initially denatured by heat or by denaturing agents so that both strands of the DNA separate. They are then ready to be hybridized with a segment of single-stranded DNA or RNA that is complementary to the sequence to be detected. This sequence is called a probe. The probe may be obtained by cloning, by polymerase chain reaction (PCR) amplification of the target sequence, or by synthesis if the desired sequence is short. The probe must be tagged with a label, usually a radioactive isotope (which can be localized by autoradiography) or a modified nucleotide (digoxygenin), which can be identified by immunocytochemistry.

In in situ hybridization, the tissue section, cultured cells, smears, or chromosomes of squashed mitotic cells must first be heated to separate the double strands of their DNA. A solution containing the probe is then placed over the specimen for a period of time necessary for hybridization. After washing off the excess probe, the localization of the bound probe is revealed through its label (Figure 1–29).

Figure 1-29

Tissue section of a benign epithelial tumor (condyloma) submitted to in situ hybridization. The brown areas are places where DNA of human papillomavirus type 2 is present. The counterstain was hematoxylin. Medium magnification. (Courtesy of JE Levi.)

Hybridization can also be performed with purified DNA or RNA in solid supports. Mixtures of DNA or RNA are separated by electrophoresis in an agarose gel or a polyacrylamide gel. After electrophoresis, the fragments of nucleic acids are transferred to a nylon or nitrocellulose sheet by solvent drag: a buffer flows through the gel and membrane by capillarity, carrying the nucleic acid molecules that bind strongly to the nylon or nitrocellulose sheet, where the nucleic acids can be further analyzed. This technique of DNA identification is called Southern blotting. When electrophoresis of RNA is performed, the technique is called Northern blotting.

Hybridization techniques are highly specific and are routinely used in research, clinical diagnosis, and forensic medicine.

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