Laboratory Techniques in Immunology (World of Microbiology and Immunology)
Various laboratory techniques exist that rely on the use of antibodies to visualize components of microorganisms or other cell types and to distinguish one cell or organism type from another.
Electrophoresis is a technique whereby the protein or carbohydrate components of microorganisms can be separated based upon their migration through a gel support under the driving influence of electricity. Depending upon the composition of the gel, separation can be based on the net charge of the components or on their size. Once the components are separated, they can be distinguished immunologically. This application is termed immunoelectrophoresis.
Immunoelectrophoresis relies upon the exposure of the separated components in the gel to a solution that contains an antibody that has been produced to one of the separated proteins. Typically, the antibody is generated by the injection of the purified protein into an animal such as a rabbit. For example, the protein that comprises the flagellar appendage of a certain bacteria can be purified and injected into the rabbit, so as to produce rabbit anti-flagellar protein.
Immunoelectrophoresis can be used in a clinical immunology laboratory in order to diagnose illness, especially those that alter the immunoglobulin composition of body fluids. Research immunology laboratories also employ immunoelectrophoresis to analyze the components of organisms, including microorganisms.
One example of an immunoelectrophoretic technique used with microorganisms is known as the Western Blot. Proteins that have been separated on a certain type of gel support can be electrically transferred to a special membrane. Application of the antibody will produce binding between the antibody and the corresponding antigen. Then, an antibody generated to the primary antibody (for example, goat anti-rabbit antibody) is added. The secondary antibody will bind to the primary antibody. Finally, the secondary antibody can be constructed so that a probe binds to the antibody's free end. A chemical reaction produces a color change in the probe. Thus, bound primary antibody is visualized by the development of a dark band on the support membrane containing the electrophoretically separated proteins. Various controls can be invoked to ensure that this reaction is real and not the result of an experimental anomaly.
A similar reaction can be used to detect antigen in sections of biological material. This application is known as immunohistochemistry. The sections can be examined using either an electron microscope or a light microscope. The preparation techniques differ for the two applications, but both are similar in that they ensure that the antigen is free to bind the added antibody. Preservation of the antigen binding capacity is a delicate operation, and one that requires a skilled technician. The binding is visualized as a color reaction under light microscopic illumination or as an increased electron dense area under the electron beam of the electron microscope.
The binding between antigen and antibody can be enhanced in light microscopic immunohistochemistry by the exposure of the specimen to heat. Typically a microwave is used. The heat energy changes the configuration of the antigen slightly, to ease the fit of the antigen with the antibody. However, the shape change must not be too great or the antibody will not recognize the altered antigen molecule.
Another well-establish laboratory immunological technique is known as enzyme-linked immunosorbent assay. The technique is typically shortened to ELISA. In the ELISA technique, antigen is added to a solid support. Antibody is flooded over the support. Where an antibody recognizes a corresponding antigen, binding of the two will occur. Next an antibody raised against the primary antibody is applied, and binding of the secondary antibody to the primary molecule occurs. Finally, a substrate is bound to a free portion of the secondary antibody, and the binding can be subsequently visualized as a color reaction. Typically, the ELISA test is
The nature of the antibody can be important in laboratory immunological techniques. Antibodies such as those raised in a rabbit or a goat are described as being polyclonal in nature. That is, they do recognize a certain antigenic region. But if that region is present on different molecules, the antibody will react with all the molecules. The process of monoclonal antibody production can make antigenic identification much more specific, and has revolutionized immunological analysis.
Monoclonal antibodies are targeted against a single antigenic site. Furthermore, large amounts of the antibody can be made. This is achieved by fusing the antibody-producing cell obtained from an immunized mouse with a tumor cell. The resulting hybrid is known as a hybridoma. A particular hybridoma will mass-produce the antibody and will express the antibody on the surface of the cell. Because hybridoma cells are immortal, they grow and divide indefinitely. Hence the production of antibody can be ceaseless.
Monoclonal antibodies are very useful in a clinical immunology laboratory, as an aid to diagnose diseases and to detect the presence of foreign or abnormal components in the blood. In the research immunology laboratory, monoclonal technology enables the specific detection of an antigenic target and makes possible the development of highly specific vaccines.
One example of the utility of monoclonal antibodies in an immunology laboratory is their use in the technique of flow cytometry. This technique separates sample as individual sample molecules pass by a detector. Sample can be treated with monoclonal antibody followed by a second treatment with an antibody to the monoclonal to which is attached a molecule that will fluoresce when exposed to a certain wavelength of light. When the labeled sample passes by the detector and is illuminated (typically by laser light of the pre-determined wavelength), the labeled sample molecules will fluoresce. These can be detected and will be shunted off to a special collection receptacle. Many sorts of analyses are possible using flow cytometry, from the distinguishing of one type of bacteria from another to the level of the genetic material comprising such samples.
See also Antibody-antigen, biochemical and molecular reactions