In order for the antigen–antibody immunoreaction to be seen in the microscope, the antibody must take a label – enzyme, fluorophore, colloidal gold or biotin. An enzyme label can be visualized in the light microscope by means of enzyme histochemical methods via chromogenic reactions. A fluorophore label can be directly visualized in a fluorescent microscope. Electron-dense labels such as colloidal gold are visible in the electron microscope without further treatment. Biotin label can be exploited in light, fluorescence and electron microscopy in combination with ABC technique. Like biotin, some other haptens, such as digoxigenin (DIG) or dinitrophenol(DNP), can also be coupled to antibodies. For their visualization, enzymeor fluorophore-conjugated secondary antibodies are affordable.
Labels can be chemically introduced into antibodies via a variety of functional groups on the antibody using appropriate group-specific reagents. Many fluorophores bear a reactive group through which they readily combine covalently with antibodies in alkaline solutions (pH 9–10). Labeling with an enzyme requires an additional large molecule such as glutaraldehyde to cross-link the enzyme to the antibody.
The most widely applied principle is haptenylation of amino groups via N-hydroxysuccinimide esters (NHS-ES). For convenient protein-labeling procedures,a number of haptens (various fluorophores, enzymes, biotin, DIG, etc.) are now commercially available as activated NHS-ES from various vendors.
There are two complex steps in the manufacture of antibody for immunolabeling. The first is producing the antibody that binds specifically to the antigen of interest and the second is fusing the tag to the antibody. Since it is impractical to fuse a tag to every conceivable antigen-specific antibody, most immunolabeling processes use an indirect method of detection. This indirect method employs a primary antibody that is antigen-specific and a secondary antibody fused to a tag that specifically binds the primary antibody. This indirect approach permits mass production of secondary antibody that can be bought off the shelf. Pursuant to this indirect method, the primary antibody is added to the test system. The primary antibody seeks out and binds to the target antigen. The tagged secondary antibody, designed to attach exclusively to the primary antibody, is subsequently added.
Typical tags include: a fluorescent compound, gold beads, a particular epitope tag, or an enzyme that produces a colored compound. The association of the tags to the target via the antibodies provides for the identification and visualization of the antigen of interest in its native location in the tissue, such as the cell membrane, cytoplasm, or nuclear membrane. Under certain conditions the method can be adapted to provide quantitative information.
Immunolabeling can be used in pharmacology, molecular biology, biochemistry and any other field where it is important to know of the precise location of an antibody-bindable molecule.