Immunofluorescence techniques.

anTiBOdies An antibody is a protein complex produced by B cells that initiates an immune response against a target antigen. The basic organization of an antibody includes two functional domains that, together, resemble the letter Y (Figure 1, left). The Fab (fragment having the antigen binding site) domain makes up the arms of the Y, and at the end of each arm is a variable region responsible for antigen binding, called the antigen-binding site. The Fc (fragment that crystallizes) domain comprises the tail of the Y, which effector cells, immune proteins, and other antibodies recognize primarily. This unique structure allows direct detection of antigens in the skin using a single fluorophore-labeled antibody or indirect detection through binding of a fluorophore-labeled secondary antibody raised against the Fc domain of an unlabeled primary antibody (Figure 1, right). Because the Fc domain is conserved within a species, the labeled secondary antibody can be used to detect any primary antibody raised from a single species. This system is versatile and cost-effective because few labeled antibodies are required to detect many possible primary antibodies. For more information about laboratory procedures using antibodies, see Harlow and Lane (1999). In certain bullous diseases, connective-tissue diseases, and vasculitides, patients produce antibodies against an antigen in their own skin or blood vessels. Detection and characterization of the autoantibody–antigen complexes is accomplished by laboratory analysis of a skin biopsy and (possibly) a blood sample. Clinically, it is important to collect the biopsy from the appropriate location for the type of autoimmune disease under consideration. Because immune deposits are degraded in inflamed or blistered skin, bullous diseases require biopsy of normal-appearing skin immediately adjacent to a lesion, whereas connective tissue diseases and vasculitides can be evaluated by biopsy of the skin lesion itself. The biopsy can be stored temporarily in Michel’s transport medium (3.12 M ammonium sulfate, 5 mM N-ethylmaleimide, 5 mM magnesium sulfate heptahydrate, and 25 mM potassium citrate, pH 7.0) (Michel et al., 1972). The nearsaturating concentration of ammonium sulfate preserves autoantibody–antigen complexes of the specimen for days by precipitating the proteins, which prevents them from diffusing away from their original location in the tissue. However, the transport medium is not a fixative, so the integrity of cellular membranes will be lost over time (Vaughn Jones et al., 1995). WHAT IMMUNOFLUORESCENCE DOES