A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody
A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody
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DOI：
10.1016/s0022-1759(97)00002-1
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发表时间：
1997-03-28
影响因子：
2.2
通讯作者：
Mukku, VR
中科院分区：
文献类型：
--
作者：
GazzanoSantoro, H;Ralph, P;Mukku, VR
开发了一种简单的非放射性补体依赖性细胞毒性测定法来确定抗 CD20 mAb IDEC-C2B8 的相对效力。该测定根据氧化还原染料阿拉玛蓝的吸收和代谢来测量活细胞的相对数量。证明了产生的相对荧光单位和活细胞数量之间的线性关系。该测定方法简单，通量高（在 96 孔微量滴定板中进行），并在 0.02-3.3 μg/ml 的浓度范围内显示出可重复的剂量反应曲线。测定内变异性为 5-12%，测定间变异性为 6-10%，加标回收率为 101-109%，该测定具有高精度和准确度。特异性通过不结合CD20的免疫球蛋白或不结合补体的抗CD20抗体同种型(γ4)缺乏活性来证明。该测定能够检测由热、光和蛋白水解处理引起的分子降解变化，表明其可用作稳定性指示方法。最后，与用于评估细胞活力的其他更常规方法相比，阿拉玛蓝方法具有优势。该测定具有用作抗 CD20 mAb 质量控制测试的效力测定所需的特性。
A simple and non-radioactive complement-dependent cytotoxicity assay was developed to determine the relative potency of an anti-CD20 mAb, IDEC-C2B8. The assay measures the relative number of viable cells based on the uptake and metabolism of the redox dye, Alamar blue. A linear relationship between the relative fluorescence unit generated and the number of viable cells was demonstrated. The assay is simple, has high throughput (performed in 96-well microtiter plates), and shows reproducible dose-response curves in the concentration range of 0.02-3.3 mu g/ml. With intra-assay variability of 5-12%, interassay variability of 6-10% and spike recoveries of 101-109%, the assay has high precision and accuracy. Specificity was demonstrated by the lack of activity of immunoglobulins that do not bind CD20, or anti-CD20 antibody isotype (gamma 4) which does not bind complement. The assay is able to detect degradative changes in the molecule caused by heat, light and proteolytic treatments, suggesting its use as a stability-indicating method. Finally, the Alamar blue method compared favorably with other more conventional methods used to assess cell viability, The assay has the desired properties for use as a potency assay for quality control testing of anti-CD20 mAb.