Institut des Biomolécules Max Mousseron-IBMM, Université de Montpellier, CNRS, ENSCM, 34093 Montpellier Cedex 5, France.
Laboratoire d'astrophysique de Bordeaux, Univ. Bordeaux, CNRS, B18N, allée Geoffroy Saint-Hilaire, 33615 Pessac, France.
J Pharm Biomed Anal. 2018 Apr 15;152:17-24. doi: 10.1016/j.jpba.2018.01.041. Epub 2018 Feb 3.
The scope of this paper is to present a gold standard method to evaluate functional activity of antibody (Ab)-based materials during the different phases of their development, after their exposure to forced degradations or even during routine quality control. Ab-based materials play a central role in the development of diagnostic devices, for example, for screening or therapeutic target characterization, in formulation development, and in novel micro(nano)technology approaches to develop immunosensors useful for the analysis of trace substances in pharmaceutical and food industries, clinical and environmental fields. A very important aspect in diagnostic device development is the construction of its biofunctional surfaces. These Ab surfaces require biocompatibility, homogeneity, stability, specificity and functionality. Thus, this work describes the validation and applications of a unique ligand binding assay to directly perform the quantitative measurement of functional Ab binding sites immobilized on the solid surfaces. The method called Antibody Anti-HorseRadish Peroxidase (A2HRP) method, uses a covalently coated anti-HRP antibody (anti-HRP Ab) and does not need for a secondary Ab during the detection step. The A2HRP method was validated and gave reliable results over a wide range of absorbance values. Analyzed validation criteria were fulfilled as requested by the food and drug administration (FDA) and European Medicines Agency (EMA) guidance for the validation of bioanalytical methods with 1) an accuracy mean value within +15% of the nominal value; 2) the within-assay precision less than 7.1%, and 3) the inter-day variability under 12.1%. With the A2HRP method, it is then possible to quantify from 0.04 × 10 to 2.98 × 10 functional Ab binding sites immobilized on the solid surfaces. A2HRP method was validated according to FDA and EMA guidance, allowing the creation of a gold standard method to evaluate Ab surfaces for their resistance under laboratory constraints. Stability testing was described through forced degradation studies after exposure of Ab-surfaces to storage, pH and aqueous-organic solvent mixture stresses.
本文的范围是提出一种金标准方法，以评估抗体 (Ab)-基于材料在其开发的不同阶段的功能活性，在它们暴露于强制降解后，甚至在常规质量控制期间。Ab-基于材料在诊断设备的开发中起着核心作用，例如，用于筛选或治疗靶标特征描述、制剂开发以及新型微（纳）技术方法，以开发用于分析制药和食品工业、临床和环境领域痕量物质的免疫传感器。在诊断设备开发中非常重要的一个方面是构建其生物功能表面。这些 Ab 表面需要生物相容性、均一性、稳定性、特异性和功能性。因此，本工作描述了一种独特的配体结合测定的验证和应用，以直接对固定在固体表面上的功能性 Ab 结合位点进行定量测量。该方法称为抗体抗辣根过氧化物酶 (A2HRP) 方法，使用共价涂层的抗 HRP 抗体（抗 HRP Ab），并且在检测步骤中不需要第二抗体。A2HRP 方法经过验证，在很宽的吸光度范围内给出可靠的结果。分析验证标准符合食品和药物管理局 (FDA) 和欧洲药品管理局 (EMA) 对生物分析方法验证的指南要求，满足以下标准：1）准确度平均值在标称值的 +15% 以内；2）日内精密度小于 7.1%；3）日间变异性小于 12.1%。使用 A2HRP 方法，可以定量固定在固体表面上的 0.04×10 至 2.98×10 个功能性 Ab 结合位点。A2HRP 方法经过 FDA 和 EMA 指南的验证，允许创建一种金标准方法来评估 Ab 表面在实验室限制下的抗性。通过暴露于储存、pH 值和水-有机溶剂混合物应激下的 Ab 表面的强制降解研究来描述稳定性测试。
