Amyloid beta conformation-specific monoclonal antibodies

描述（由申请人提供）：可溶性低聚形式的淀粉样蛋白（A）已从脑、血浆和CSF中分离出来，这些聚集体似乎与阿尔茨海默病（AD）的严重程度相关。为了进一步了解A 3组装状态影响活性，我们已经开发了寡聚体和纤维状A 31-42的结构确定的制剂。在体外，这些制剂诱导不同的神经毒性，炎症和突触可塑性反应。然而，纤维状和寡聚体A？对AD发病机制的相对贡献仍未得到解决。我们假设A在脑中以至少两种状态存在：可扩散的寡聚状态和不溶性的纤维状（聚合）状态，并且这些构象种类的比例和清除在AD患者中改变，从而促进神经变性。为了剖析这种机制，可以区分肽的纤维状和寡聚体聚集状态的构象特异性抗体是至关重要的。我们使用寡聚体A 31-42作为抗原，抗原/抗体筛选产生了一种寡聚体特异性单克隆抗体（MOAB-1）。通过抗原/抗体印迹和ELISA，MOAB-1不识别原纤维。通过SDS-PAGE的Western分析，MOAB-1不识别未聚集或寡聚样品中的A？单体，并且在原纤维样品中检测到很少的免疫反应性。实现以下具体目标将进一步检验我们的假设： 1.我们建议分离和生化特性的寡聚体或纤维状的A1 -42特异性的额外的单克隆抗体。 2.我们将确定所选抗体对寡聚体或原纤维诱导的神经毒性、神经炎症和神经可塑性的影响。我们预期寡聚体特异性单克隆抗体将抑制寡聚体而不是原纤维诱导的效应，对于原纤维特异性抗体，反之亦然。 3.我们将在立体定位注射寡聚体或纤维状A？1-42的大鼠脑中测试选定抗体的免疫组织化学（IHC）特异性和适用性。我们期望寡聚抗体仅免疫标记注射A？寡聚体的大脑，而不是注射纤维的大脑，反之亦然。 4.所选抗体将用于定量IHC和激光扫描共聚焦显微镜检查，以通过Feinberg医学院的NU-CNADC神经病理学核心确定其在神经心理学充分表征的患者大脑海马切片中的定位。 5.将使用现有和新型抗体设计ELISA捕获测定法，以便于测量脑、CSF和血清提取物中的可溶性A？寡聚体。

DESCRIPTION (provided by applicant): Soluble oligomeric forms of amyloid-¿ (A¿) have been isolated from brain, plasma and CSF and these assemblies appear to correlate with the severity of Alzheimer's disease (AD). To further understand how A¿3 assembly state influences activity, we have developed structurally defined preparations of oligomeric and fibrillar A¿31-42. In vitro, these preparations induce distinct neurotoxic, inflammatory, and synaptic plasticity responses. However, the relative contribution of fibrillar and oligomeric A¿ to the pathogenesis of AD remains unresolved. We hypothesize that A¿ exists in at least two states in the brain a diffusible oligomeric state, and an insoluble fibrillar (polymeric) state, and the ratio and clearance of these conformational species is altered in AD patients, promoting neurodegeneration. To dissect this mechanism, conformational-specific antibodies that can distinguish between fibrillar and oligomeric aggregation states of the peptide are critical. We used oligomeric A¿31-42 as an antigen and an antigen/antibody screen has yielded one oligomer-specific monoclonal antibody (MOAB-1). MOAB-1 does not recognize fibrils by antigen/antibody blotting and ELISA. By Western analysis of SDS-PAGE, MOAB-1 does not recognize A¿ monomer in unaggregated or oligomeric samples, and little immunoreactivity is detected in the fibril samples. Accomplishing the following specific Aims will further test our hypotheses: 1. We propose to isolate and biochemically characterize additional monoclonal antibodies specific for oligomeric or fibrillar A¿1-42. 2. We will determine the effect of selected antibodies on oligomer- or fibril-induced neurotoxicity, neuroinflammation, and neuroplasticity. We expect that oligomer-specific monoclonal antibodies will inhibit oligomer and not fibril-induced effects and vice versa for the fibril-specific antibodies. 3. We will test the specificity and suitability for immunohistochemistry (IHC) of selected antibodies in rat brains that have been stereotaxically injected with oligomeric or fibrillar A¿1-42. We expect oligomeric antibodies to immunolabel only the brains injected with A¿ oligomers and not fibril-injected brains, and vice versa. 4. Selected antibodies will be utilized in quantitative IHC and Laser Scanning Confocal Microscopy to determine their localization in hippocampal sections from the brains of neuropsychologically well-characterized patients through the NU-CNADC Neuropathology Core at the Feinberg School of Medicine. 5. An ELISA capture assay will be designed, using existing and novel antibodies, to facilitate measurement of soluble A¿ oligomers in extracts of brain, CSF, and sera.