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，从相对简单的可在大多数实验室使用的方法（预包埋过氧化物酶法）到主要与专业研究实验室相关的复杂程序（包埋后、冷冻固定和冷冻替代技术）。在本项目中，我们采用最新的低温免疫电镜技术研究皮肤水疱病，包括大疱性类天疱疮抗原和大疱性获得性表皮松解抗原。以前的免疫过氧化物酶方法只能相当准确地显示免疫反应物与真皮-表皮连接处亚组分的超微结构定位。因为过氧化物酶-二氨基联苯胺反应产物体积大，容易扩散，不可避免地掩盖了底层结构。我们使用冷冻固定和冷冻替代技术，而不使用化学固定剂与胶体金结合，我们已经证明大疱性表皮松解抗原（患者自身抗体的靶分子）定位于锚定原纤维的真皮和致密层端，但不定位于中心带状部分本身。锚定原纤维是一种长度为785nm的结构。胶体金而非过氧化物酶可以阐明靶表位在这种小结构上的精细超微结构定位。大疱性类天疱疮抗原由230-kD抗原和180-kD抗原两种主要抗原蛋白组成。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。针对每个抗原的自身抗体使用硝化纤维素膜亲和纯化，用SDS-PAGE从人表皮提取物中分离的抗原作为免疫吸收剂进行印迹。低温技术进行包埋后免疫金电镜观察。230-kD抗原仅定位于半脂小体的胞内结构域。相反，针对180-kD抗原的自身抗体定位在半脂酶的质膜上。这些结果提示，针对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起不同的作用。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