Aggregation and deposition of amyloid-β (Aβ) peptides in extracellular plaques and in the cerebral vasculature are prominent neuropathological features of Alzheimer’s disease (AD) and closely associated with the pathogenesis of AD. Amyloid plaques in the brains of most AD patients and transgenic mouse models exhibit heterogeneity in the composition of Aβ deposits, due to the occurrence of elongated, truncated, and post-translationally modified Aβ peptides. Importantly, changes in the deposition of these different Aβ variants are associated with the clinical disease progression and considered to mark sequential phases of plaque and cerebral amyloid angiopathy (CAA) maturation at distinct stages of AD. We recently showed that Aβ phosphorylated at serine residue 26 (pSer26Aβ) has peculiar characteristics in aggregation, deposition, and neurotoxicity. In the current study, we developed and thoroughly validated novel monoclonal and polyclonal antibodies that recognize Aβ depending on the phosphorylation-state of Ser26. Our results demonstrate that selected phosphorylation state-specific antibodies were able to recognize Ser26 phosphorylated and non-phosphorylated Aβ with high specificity in enzyme-linked immunosorbent assay (ELISA) and Western Blotting (WB) assays. Furthermore, immunofluorescence analyses with these antibodies demonstrated the occurrence of pSer26Aβ in transgenic mouse brains that show differential deposition as compared to non-phosphorylated Aβ (npAβ) or other modified Aβ species. Notably, pSer26Aβ species were faintly detected in extracellular Aβ plaques but most prominently found intraneuronally and in cerebral blood vessels. In conclusion, we developed new antibodies to specifically differentiate Aβ peptides depending on the phosphorylation state of Ser26, which are applicable in ELISA, WB, and immunofluorescence staining of mouse brain tissues. These site- and phosphorylation state-specific Aβ antibodies represent novel tools to examine phosphorylated Aβ species to further understand and dissect the complexity in the age-related and spatio-temporal deposition of different Aβ variants in transgenic mouse models and human AD brains.

淀粉样蛋白-β（Aβ）肽在细胞外斑块和脑血管中的聚集和沉积是阿尔茨海默病（AD）的突出神经病理学特征，并且与AD的发病机理密切相关。大多数AD患者和转基因小鼠模型的大脑中的淀粉样斑块由于伸长，截短和翻译后修饰的Aβ肽的出现而在Aβ沉积物的组成上表现出异质性。重要的是，这些不同的Aβ变体的沉积变化与临床疾病的发展有关，并被认为可以标志AD各个阶段斑块和脑淀粉样血管病（CAA）成熟的连续阶段。我们最近发现，丝氨酸残基26磷酸化的Aβ（pSer26Aβ）在聚集，沉积和神经毒性方面具有独特的特征。在当前的研究中，我们开发并充分验证了根据Ser26的磷酸化状态识别Aβ的新型单克隆和多克隆抗体。我们的结果表明，在酶联免疫吸附测定（ELISA）和蛋白质印迹（WB）分析中，选定的磷酸化状态特异性抗体能够以高特异性识别Ser26磷酸化和非磷酸化的Aβ。此外，用这些抗体进行的免疫荧光分析表明，与未磷酸化的Aβ（npAβ）或其他修饰的Aβ物种相比，pSer26Aβ在转基因小鼠脑中的沉积显示出差异。值得注意的是，pSer26Aβ物种在细胞外Aβ斑块中微弱地被检测到，但最明显的发现是在神经内和脑血管中。结论，我们开发了新的抗体，根据Ser26的磷酸化状态来特异性区分Aβ肽，可用于ELISA，WB和小鼠脑组织的免疫荧光染色。这些位点和磷酸化状态特异性的Aβ抗体代表了一种新颖的工具，可以检查磷酸化的Aβ物种，以进一步了解和分析转基因小鼠模型和人类AD脑中不同Aβ变体的年龄相关性和时空沉积的复杂性。