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Antibodies against desmoglein 3, generally IgG, can be found tissue bound, at the level of the spinous cells' membrane and circulating in the blood of patients with active PV. Fluorescent techniques are used for the detection and localization of the tissue bound antibodies. The use of fluorescent techniques in immunopathology is based on the possibility of labeling antibodies or immunoglobulins with a fluorescent dye, generally fluorescein isothiocyanate or FITC, without disturbing the specificity of the antibody. This combination is known as FITC conjugate. Technically, two methods are used, in vivo or direct, and in vitro or indirect.
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These drawings schematically illustrate the direct procedure. The upper drawing represents a frozen section of a vesicle from a patient with PV; Tzanck cells are also depicted. It is known that the autoantibodies, generally IgG and complement, are bound to desmoglein 3 in the desmosomes of the spinous cells in all PV lesions and adjacent skin or mucosal epithelium. This binding of desmoglein 3-antibodies is an antigen-antibody reaction (middle drawing).
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The visualization of this reaction is rendered possible by treating a human frozen section with FITC conjugate. This conjugate is commercially obtained and represents a combination of the fluorescent dye FITC and an antihuman antibody, generally obtained from rabbits. Binding of the antihuman antibody contained in the conjugate to the human IgG and complement bound to the cell membrane, will occur on contact. Due to the labelling with FITC, this end reaction will show a marked green fluorescence when observed with the proper equipment under the fluorescent light microscope (lower drawing).
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