FLUORESCENCE OVERLAY ANTIGEN MAPPING OF THE EPIDERMAL BASEMENT-MEMBRANE ZONE .3. TOPOGRAPHIC STAINING AND EFFECTIVE RESOLUTION

In this third study on the fluorescence overlay antigen mapping (FOAM) technique, we have addressed the question of which differences of ant igen distributions dose to the resolving power of the light microscope can be distinguished, An answer to this question should provide clues to future applications of the technique aiming at the topographic dif ferentiation of IgG deposits displayed at the epidermal basement membr ane zone (EBMZ) in certain bullous skin disorders. For the present pur pose we have developed a topographic staining model in human skin, usi ng structural EBMZ antigens as topographic reference markers. The dist ribution of these markers relative to one another is visualized in FOA M images obtained by selective double immunofluorescence tracing and v ideomicroscopic overlay imaging. The theoretical resolution limit of t he technique is discussed and suggests an effective lower limit of som e 60-65 nm, Although this limit is not reached under present condition s, our results show that it is possible to distinguish topographic dif ferences of antigen distributions with an upper resolution limit of 20 0 +/- 50 nm. Furthermore, our findings indicate that collagen Type VII and beta 4 integrin are the most suitable molecules to serve as topog raphic reference markers in future applications of the technique aimin g at the differentiation of bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA), Preliminary results on this topic are most pr omising indeed.