Selective recognition of human small transthyretin aggregates by a novel monoclonal antibody

Biochemical characterisation of transthyretin variant TTR Y78F showed that this variant adopts a tetrameric conformation as normal TTR but exhibits some of the characteristics of an intermediate structure in the fibrillogenesis pathway. It was hypothesised that native Y78F might represent an early event in TTR amyloidogenesis. We immunised TTR knock out mice with recombinant variant TTR Y78F. One stable hybridoma named CE11, of the IgM isotype, was tested for reactivity towards several soluble recombinant TTR variants both amyloidogenic and non-amyloidogenic. CE11 only recognises the highly amyloidogenic TTR variants L55P, S52P, A97S, Y78F or acidified TTR wt preparations. At the same time, this clone was negative for TTR V30M, soluble wild type protein or TTR T119M. The reactivity increased with oligomer formation and decreased as mature fibrils grow. After size exclusion chromatography (SEC) followed by sandwich ELISA and native immunoblotting, the mAb recognised two peaks (i) peak 1 present in acidified and in soluble variant proteins preparations with material above 146 KDa (ii) peak 2 only present in soluble L55P and S52P TTR preparations with material between 66 and 146 KDa. mAb CE11 may be a potential tool to survey therapeutical agents against TTR aggregation.针对运甲状腺素蛋白（transthyretin, TTR）突变体Y78F的生化表征结果显示，该突变体与野生型TTR一样采用四聚体构象，但同时表现出淀粉样纤维形成通路中中间结构的部分特征。研究人员据此提出假说：天然状态的Y78F突变体可能代表了TTR淀粉样变性过程中的早期事件。我们以重组突变型TTR Y78F免疫TTR基因敲除小鼠，获得了一株稳定的IgM型杂交瘤细胞株CE11。针对多种可溶性重组TTR突变体（包括致淀粉样变性与非致淀粉样变性型）的反应性检测结果显示，CE11仅能识别高致淀粉样变性的TTR突变体L55P、S52P、A97S、Y78F，以及酸化处理的野生型TTR样品；与此同时，该细胞株对TTR V30M、可溶性野生型TTR蛋白及TTR T119M均无反应性。其反应性随寡聚体形成而增强，随成熟淀粉样纤维的生成而减弱。经尺寸排阻色谱法（size exclusion chromatography, SEC）分离后，再通过夹心酶联免疫吸附测定与天然免疫印迹检测，该单克隆抗体（monoclonal antibody, mAb）可识别两个特征峰：（i）峰1：存在于酸化处理样品与可溶性突变体蛋白样品中，对应分子量大于146 kDa的组分；（ii）峰2：仅存在于可溶性L55P与S52P型TTR样品中，对应分子量介于66 kDa至146 kDa之间的组分。单克隆抗体CE11有望成为检测抗TTR聚集治疗药物的潜在工具。