Petersen Elijah J, Uhl Richard, Toman Blaza, Elliott John T, Strickland Judy, Truax James, Gordon John
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), 100 Bureau Drive, Gaithersburg, MD 20899, USA.
Division of Laboratory Sciences, Chemistry, US Consumer Product Safety Commission (CPSC), 5 Research Place, Rockville, MD 20850, USA.
Toxics. 2022 May 17;10(5):257. doi: 10.3390/toxics10050257.
The Electrophilic Allergen Screening Assay (EASA) has emerged as a promising method to detect the first key event in the adverse outcome pathway (AOP) for skin sensitization. This assay functions by assessing the depletion of one of two probe molecules (4-nitrobenzenethiol (NBT) and pyridoxylamine (PDA)) in the presence of a test compound (TC). The initial development of EASA utilized a cuvette format resulting in multiple measurement challenges such as low throughput and the inability to include adequate control measurements. In this study, we describe the redesign of EASA into a 96-well plate format that incorporates in-process control measurements to quantify key sources of variability each time the assay is run. The data from the analysis of 67 TCs using the 96-well format had 77% concordance with animal data from the local lymph node assay (LLNA), a result consistent with that for the direct peptide reactivity assay (DPRA), an OECD test guideline (442C) protein binding assay. Overall, the measurement science approach described here provides steps during assay development that can be taken to increase confidence of assays by attempting to fully characterize the sources of variability and potential biases and incorporate in-process control measurements into the assay.
亲电变应原筛选测定法（EASA）已成为一种很有前景的方法，用于检测皮肤致敏不良结局途径（AOP）中的首个关键事件。该测定法通过在测试化合物（TC）存在的情况下评估两种探针分子（4-硝基苯硫醇（NBT）和吡哆胺（PDA））之一的消耗情况来发挥作用。EASA的最初开发采用比色皿形式，导致了多种测量挑战，如通量低以及无法进行充分的对照测量。在本研究中，我们描述了将EASA重新设计为96孔板形式，该形式在每次运行测定时纳入过程控制测量，以量化关键变异来源。使用96孔形式对67种测试化合物进行分析得到的数据与局部淋巴结测定法（LLNA）的动物数据有77%的一致性，这一结果与经合组织测试指南（442C）蛋白质结合测定法——直接肽反应性测定法（DPRA）的结果一致。总体而言，此处描述的测量科学方法在测定法开发过程中提供了一些步骤，可通过尝试全面表征变异来源和潜在偏差，并将过程控制测量纳入测定法来提高测定法的可信度。
