Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany.
Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany.
Anal Biochem. 2020 May 1;596:113646. doi: 10.1016/j.ab.2020.113646. Epub 2020 Feb 26.
Quantification of therapeutic antibodies is commonly based on physico-chemical assays such as enzyme-linked immunoabsorption assays (ELISA) and lately on mass spectrometry. However, the functional integrity of evaluated immunoglobulins is yet not assessed. Consequently, a commercially available reporter cell line was used to quantify the functional concentration of the anti-tumor necrosis factor alpha (TNF-α) antibody adalimumab present in serum of a healthy beagle dog treated with 3 mg intravenous adalimumab (Humira®). HEK-Blue™-hTLR3 cells express a secreted alkaline phosphatase under the control of a nuclear factor kappa B (NF-κB) response element. Its enzymatic activity can be recorded using colorimetry, which reports activity of extracellular NF-κB stimuli such as TNF-α. Using an adalimumab concentration-response calibration curve, the functional concentration of serum adalimumab was estimated to be 4.9 ± 1.4 μg/ml, which was in excellent agreement with ELISA results (4.8 μg/ml). The obtained data suggest that this simple, easy-to-handle reporter cell assay can be used for the functional quantification of adalimumab present in samples from in vitro or pre-clinical in vivo experiments. Moreover, this assay could be used in vitro to compare the pharmacodynamics of adalimumab biosimilars or different anti-TNF-α compounds, respectively.
治疗性抗体的定量分析通常基于物理化学分析方法，如酶联免疫吸附测定法（ELISA）和最近的质谱法。然而，评估的免疫球蛋白的功能完整性尚未得到评估。因此，使用市售的报告细胞系来定量评估在接受 3 毫克静脉注射阿达木单抗（Humira®）治疗的健康比格犬血清中存在的抗肿瘤坏死因子-α（TNF-α）抗体阿达木单抗的功能浓度。HEK-Blue™-hTLR3 细胞在核因子 kappa B（NF-κB）反应元件的控制下表达分泌型碱性磷酸酶。其酶活性可以通过比色法记录，该方法报告 TNF-α等细胞外 NF-κB 刺激物的活性。使用阿达木单抗浓度-反应校准曲线，估计血清阿达木单抗的功能浓度为 4.9±1.4μg/ml，与 ELISA 结果（4.8μg/ml）非常吻合。获得的数据表明，这种简单、易于操作的报告细胞测定法可用于定量分析来自体外或临床前体内实验的样本中阿达木单抗的功能。此外，该测定法可在体外用于比较阿达木单抗生物类似物或不同抗 TNF-α 化合物的药效动力学。
