Amyloid beta conformation-specific monoclonal antibodies

描述（由申请人提供）：已从脑、血浆和脑脊液中分离出淀粉样蛋白-¿（A¿）的可溶性寡聚物形式，这些组合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组装状态如何影响活性，我们开发了结构明确的低聚物和原纤维A¿31-42的制备。在体外，这些制剂可诱导不同的神经毒性、炎症和突触可塑性反应。然而，纤维蛋白和寡聚A¿在AD发病机制中的相对作用仍未得到解决。我们假设A¿在大脑中至少以两种状态存在：扩散低聚状态和不溶性纤维（聚合物）状态，并且这些构象物种的比例和清除在AD患者中发生改变，促进神经变性。为了剖析这种机制，能够区分原纤维和寡聚肽聚集状态的构象特异性抗体是至关重要的。我们使用寡聚物A¿31-42作为抗原，抗原/抗体筛选得到一种寡聚物特异性单克隆抗体（MOAB-1）。通过抗原/抗体印迹和ELISA检测，MOAB-1不识别原纤维。通过SDS-PAGE的Western分析，MOAB-1不识别未聚集或低聚样品中的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：

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.