Epidermolysis bullosa acquisita antigen and the carboxy terminus of type VII collagen have a common immunolocalization to anchoring fibrils and lamina densa of basement membrane

Abstract

In this study a variety of immunoelectron microscopic methods were used to define the precise ultrastructural binding site of epidermolysis bullosa acquisita antibodies (EBA-Ab). We used two EBA sera which immunoblotted with the same skin-extracted protein as that labelled by a monoclonal antibody (LH7.2) which is known to react with the carboxy terminus of type VII collagen. Gold-conjugated antibodies were used in two different immunoelectron microscopic procedures to compare the labelling characteristics of EBA-Ab and LH7.2 in normal human skin. Antibody incubations were performed using ultra-thin cryosections of unfixed skin and thin slices of fresh skin (en bloc technique) before conventional fixation and embedding in Epon. Both methods showed similar labelling features for both EBA-Ab and LH7.2. With ultra-thin cryosections there was labelling of the lamina densa and an undefined component of the sublamina densa region. With the en bloc technique, labelling of dermal ends of anchoring fibrils and of amorphous material recently defined as 'anchoring plaques' was evident. There was no labelling of the central banded portions of anchoring fibrils. We conclude that EBA-Ag is localized to the dermal ends of anchoring fibrils in addition to the lamina densa and possibly anchoring plaques, and thus has the same distribution as the carboxy terminus of type VII collagen.

Colocalization of Multiple Laminin Isoforms Predominantly beneath Hemidesmosomes in the Upper Lamina Densa of the Epidermal Basement Membrane

“…Four samples of human skin were cryofixed and processed for postembedding IEM according to the previously described methods (Shimizu et al 1989(Shimizu et al ,1990. Samples were washed in PBS and cryoprotected in 20% glycerol (in PBS) for up to 1 hr at 4C.…”

Fluorescence overlay antigen mapping of the epidermal basement membrane zone: II. Color fidelity.

“…With these reservations in mind, the results presented in Table 3 show enough promise to use collagen Type VI1 as the prime TRM for differentiating between BP and EBA. It can be expected that the staining pattem of this TRM will show substantial similarity and will overlap with that of skin-bound IgG deposits in EBA (18,23). By contrast, the staining pattern of IgG deposits in BP (20,22) is likely to be distinctive from that of collagen Type VII.…”

Molecular Biological Aspects of Acquired Bullous Diseases

“…By the use of gold-conjugated antibodies and electron microscopy, the epitopes were demonstrated at the dermal ends of anchoring fibrils and in the anchoring plaques (Fig. 2) (Shimizu et al, 1990).…”