Application of up-to date immunoelectron microscopy for the study of pathogenesis of skin blistering disease.

许多不同类型的免疫电镜技术已被引入皮肤病学研究中，从相对简单的，可用于大多数实验室（包埋前过氧化物酶法）的复杂程序，主要是专门的研究实验室（包埋后，冷冻固定和冷冻置换技术）。在本计画中，我们应用最新的低温免疫电子显微镜技术来研究大疱性类天疱疮抗原及获得性大疱性表皮抗原。以前的免疫过氧化物酶法可以证明的免疫反应物的超微结构定位只有相当准确的真皮表皮交界处的亚组分。由于过氧化物酶-二氨基联苯胺反应产物的尺寸较大且易于扩散，不可避免地掩盖了底层结构。在不使用化学固定剂的情况下， 关于我们 结合胶体金，我们已经证明，大疱性表皮获得性抗原，病人自身抗体的靶分子，定位于真皮和致密层的锚定纤维的末端，但不是中央带状部分本身。包围原纤维是一种长度为785 nm的结构。大疱性类天疱疮抗原是由230 kD和180 kD两种主要抗原蛋白组成的。我们已经使用这种新的免疫电镜技术来定位针对每种抗原的自身抗体的表位。使用硝酸纤维素膜亲和纯化针对每种抗原的自身抗体，所述硝酸纤维素膜用来自人表皮提取物的SDS-PAGE分级抗原作为免疫吸附剂进行印迹。采用低温技术进行着床后免疫金电镜检查。230-kD抗原仅定位于半桥粒的胞内区。而抗180 kD抗原的自身抗体则沿着半桥粒的质膜分布。这些结果表明，抗180和230-kD大疱性类天疱疮抗原的自身抗体可能在大疱性类天疱疮患者的水疱形成中发挥不同的作用。少

Many different types of immuno EM techniques have been introduced in dermatological research varing from relatively simple ones which could be used in most laboratories ( pre-embedding peroxidase method) to complex procedure relevant mainly to the specialized research laboratory (post-embedding, cryofixation and cryosubstitution technique). In this project, we have employed up-to date low temperature immunoelectron microscopic techniques for the study of skin blistering disease including bullous pemphigoid antigen and epidermolysis bullosa acquisita antigen. Previous immuno-peroxidase method could demonstrate the ultrastructural localization of the immunoreactants only fairly accurately in relation to the subcomponents of the dermo-epidermal junction. Because the size of peroxidase-diaminobenzidine reaction products is large and prone to diffuse, and inevitably obscure the underlying structure. Applying cryofixation and cryosubstitution techniques without using chemical fixatives in co … More njunction with colloidal gold, we have demonstrated that epidermolysis bullosa acquisita antigen, a target molecule of the autoantibodies of the patient, localized to both dermal and lamina densa ends of anchoring fibrils, but not to the central banded portion itself. Anchoring fibrils is a structure of 785nm in length. Colloidal gold but not peroxidase can elucidate the fine ultrastructural localization of the target epitope on such small structure.Bullous pemphigoid antigen is composed of two major antigenic proteins of the 230-kD antigen and the 180-kD antigen. We have used this new immunoelectron microscopic technique to localize the epitopes for autoantibodies against each antigen. Autoantibodies against each antigen were affinity-purified using nitrocellulose membrane, which was blotted with SDS-PAGE fractionated antigens from human epidermal extract as the immunoabsorbent. Postembedding immunogold electron microscopy was performed using low temperature techniques. The 230-kD antigen only localized to the intracellular domain of hemidesmosome. In contrast, the autoantibodies against the 180-kD antigen localized along the plasma membrane of hemidesmosome. These result suggest that the autoantibodies against the 180 and 230-kD bullous pemphigoid antigen may play different roles in the blister formation in patients with bullous pemphigoid. Less