Evaluation of molecular mechanisms of dialysis related amyloidosis and the diagnosis method of early detection

为探讨透析相关性淀粉样变性或终末期肾病的发病机制，制备了抗β-2-微球蛋白C-末端的新单抗(β-2 MG)，并鉴定了新的抗β-2-MG的单抗。从超滤液中提取的β-2 MG不与β-2 MG 92-99反应，而从超滤液中酸化的b2-M对β-2 MG 92-99有少量反应。免疫印迹显示β-2 MG92-99与腕部淀粉样蛋白组织匀浆呈强阳性反应。我们认为，单抗β2 MG 92-99不与β2 MG的天然形式反应，而是β2 MG、β2 MG 92-99单抗的展开和结构变化反应了β2 MG。在单核细胞系中，温度应激、渗透压应激、自由基应激对细胞表面β-2 MG的表达无明显影响。维持性血液透析患者细胞表面未折叠的β-2 MG表达明显高于正常对照组。此外，维持性血液透析患者GRP78mRNA的表达也高于正常对照组。免疫组织化学研究还显示，在维持性血液透析患者的亲和性组织中，mAb92-99也有明显的染色。更有趣的是，mAbGRP78mAb2 MG 92-99可以在大多数非亲和性组织中发现，尽管不是全部，但也可以在非亲和性组织中发现。抗β-2 MG 92-99 C-末端的单抗可在体外检测B2M淀粉样蛋白形成的构象中间体，证明β-2 MG在C-末端不仅在亲和区，甚至在非亲和区也能被发现。

New monoclonal antibody to c-terminal of β 2-microglobulin (β 2MG) was prepared and investigated the importance of pathogeneses of dialysis related amyloidosis (DRA) or end stage renal disease.We characterized new monoclonal antibody to c-terminal of β 2MG 92-99. β2MG extracted from ultrafiltrate showed no reaction form mAb β 2MG 92-99, whereas acidified b2mfrom ultrafiltrate showed a little reaction for form mAb β2MG 92-99. A homogenate of carpal amyloid tissues showed a strong reaction for mAb β2MG 92-99 on immunoblotting. We considered that mAb β2MG 92-99 didn't react native form of β2MG, but unfolded and change of structure β2MG, mAb β 2MG 92-99 react unfolded β2MG. In the monocyte cell line, temperature stress, osmotic pressure stress, radical stress unregulated the cell surface unfolded β2MG expression. The same stresses also unregulated the GRP78 mRNA expression.In the maintenance hemodialysis patients, cell surface unfolded β2MG expression was increased compare to normal subjects. Moreover, GRP78 mRNA expression was also increased in maintenance hemodialysis patients, compared to normal subjects.Immunohistochemical study showed also a distinct staining for mAb 92-99 in congophilic specimens from DRA patients more interestingly, staining form mAb β2MG 92-99 could be found in most, though not all, non-congophilic tissues. The monoclonal antibody specific to the C-terminal 92-99 of β2MG 92-99 can detect the conformational intermediate in amyloidogenesis of b2m ex vivo, and demonstrates that an unfolded β2MG at C-terminal could be found not only in congophilic area but even in non-congophilic area as well.