Radboud University Nijmegen Medical Centre, Department of Neurology, Department of Laboratory Medicine, Donders Institute for Brain, Cognition and Behaviour, Alzheimer Centre Nijmegen, Nijmegen, The Netherlands.
J Immunol Methods. 2012 Jul 31;381(1-2):1-8. doi: 10.1016/j.jim.2012.04.001. Epub 2012 Apr 19.
The measurement of proteins in cerebrospinal fluid (CSF) by enzyme-linked immunosorbent assays (ELISAs) is becoming increasingly important in the diagnosis of many neurodegenerative diseases such as Alzheimer's Disease. However, detection of proteins in these immunoassays can be hampered by confounding factors either present in the sample matrix or inherent to the protein of interest. These confounding factors may, for example, include protein aggregation or binding to other proteins resulting in epitope masking. Furthermore, the pH of CSF may vary considerably amongst different samples which may limit standardisation of CSF analysis. Pre-treatment of CSF to liberate epitopes or optimise conditions for antibody binding may enhance protein detection. In the current study we investigated whether CSF acidification followed by neutralisation (in short: AFBN) or neutralisation alone prior to measurement might improve the detection of a panel of brain-specific proteins. We demonstrate that the AFBN pre-treatment protocol for CSF significantly enhances the measurement of glutamine synthetase (GS) and myelin basic protein (MBP) in CSF but does not affect detection of glial fibrillary protein (GFAP), amyloid β 42 (Aβ₄₂), total tau (t-tau) or phosphorylated tau (p-tau). Neutralisation alone did not improve detection of any of the proteins tested. Based on our results, we suggest including the AFBN protocol in the evaluation of new biomarker development protocols to avoid confounders such as CSF pH or epitope-masking of the target protein.
酶联免疫吸附测定法（ELISA）测量脑脊液（CSF）中的蛋白质，在诊断阿尔茨海默病等许多神经退行性疾病方面变得越来越重要。然而，在这些免疫测定中，蛋白质的检测可能会受到样本基质中存在的或感兴趣蛋白质固有的混杂因素的阻碍。例如，这些混杂因素可能包括蛋白质聚集或与其他蛋白质结合，从而导致表位掩盖。此外，CSF 的 pH 值在不同样本之间可能有很大差异，这可能会限制 CSF 分析的标准化。CSF 预处理以释放表位或优化抗体结合条件可能会增强蛋白质的检测。在当前的研究中，我们研究了 CSF 酸化后再中和（简称：AFBN）或单独中和预处理是否可以提高一组脑特异性蛋白质的检测效果。我们证明 CSF 的 AFBN 预处理方案可显著提高 CSF 中谷氨酰胺合成酶（GS）和髓鞘碱性蛋白（MBP）的检测效果，但不影响对神经胶质纤维酸性蛋白（GFAP）、β淀粉样蛋白 42（Aβ₄₂）、总tau（t-tau）或磷酸化 tau（p-tau）的检测。单独中和处理并不能提高任何测试蛋白质的检测效果。基于我们的研究结果，我们建议在新生物标志物开发方案的评估中纳入 AFBN 方案，以避免 CSF pH 或目标蛋白质表位掩盖等混杂因素。
