Department of Psychiatry and Psychotherapy, University Medical Center (UMG), Georg-August-University, D37075 Göttingen, Germany.
IBL International GmbH, Tecan Group Company, D-22335 Hamburg, Germany.
Int J Mol Sci. 2020 Sep 8;21(18):6564. doi: 10.3390/ijms21186564.
The ratio of amyloid precursor protein (APP) (Aβ)/Aβ in blood plasma was reported to represent a novel Alzheimer's disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ and the development and "fit-for-purpose" technical validation of a sandwich immunoassay for the measurement of Aβ. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ with no appreciable cross reactivity with Aβ or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ with a quantitative assay range of 22 pg/mL-7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ/Aβ and Aβ/Aβ ratios were lower in patients with dementia of the Alzheimer's type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ/Aβ ratio as a novel biomarker candidate for Alzheimer's disease has been set.
血液血浆中淀粉样前体蛋白（APP）（Aβ）/Aβ 的比值被报道为一种新的阿尔茨海默病生物标志物。在这里，我们描述了两种针对 Aβ 氨基末端的抗体的特性，并开发了一种用于测量 Aβ 的夹心免疫测定法及其“适合目的”的技术验证。通过毛细管等电聚焦免疫测定、Western blot 分析和免疫组织化学评估了抗体的选择性。分析验证涉及测定范围、重复性、特异性、批间变异性、分析前样本处理程序的影响、测定干扰和分析物加标回收率。在通过两步免疫测定程序进行 Aβ 免疫沉淀后分析了血浆。两种单克隆抗体均检测到 Aβ，与 Aβ 或氨基末端截断的 Aβ 变体没有明显的交叉反应。然而，淀粉样前体蛋白也被识别。免疫测定法对 Aβ 具有高选择性，定量测定范围为 22 pg/mL-7.5 ng/mL。归一化后达到了完整两步免疫测定法可接受的中间精密度。在一个小的临床样本中，与其他痴呆症相比，阿尔茨海默病痴呆症患者的 Aβ/Aβ 和 Aβ/Aβ 比值较低。总之，已经为进一步优化和未来研究建立了方法学基础，以探讨 Aβ/Aβ 比值作为阿尔茨海默病的新型生物标志物候选物。
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