ADC抗体内吞检测试剂

抗体内化，也可以理解为抗体内吞，是指在细胞表面的抗体与相应的抗原结合后，通过自身分子运输体系，将抗体和抗原复合物带入细胞内部，并在内部起到特定的生物学作用。抗体内化是大多数抗体偶联药物（Antibody-drug conjugates，ADC）分子进入细胞的方式。常规内吞作用可分为三个阶段：芽形成；膜弯曲和囊泡成熟；以及膜分裂并释放到细胞质中。更多关于抗体内吞原理>>

ADC主要由单克隆抗体、连接子和细胞毒素（又称为有效载荷或Payload）三部分构成。其中抗体作为ADC药物的“导弹”，具有靶向功能，可以特异性识别肿瘤细胞表面抗原，与细胞表面抗原结合后发生内吞，将细胞毒素带到肿瘤细胞并发挥毒性效应。由于一些抗体与抗原结合后不一定会被靶细胞内吞，所以抗体内化能力是ADC药物早期筛选的一项重要指标。

缔码生物已开发出两种抗体内吞检测试剂，兼具高灵敏度、稳定性、成本效益及广泛的IgG亚型兼容性。

pH敏感型IgG标记试剂：利用pH敏感荧光标记的Fc结合蛋白与不同物种的IgG抗体结合，形成荧光复合物。抗体被细胞内吞后，酸性环境会增强荧光信号，其强度可直接反映内吞效率。
载荷偶联型IgG标记试剂：通过将常用的ADC药物载荷偶联至IgG分子，模拟ADC机制，实现抗体内吞检测与细胞毒性评估的结合。

DiTag™ pH-sensitive IgG labeling reagents offer a convenient way to measure antibody internalization. These reagents use a pH-sensitive fluorescent dye conjugated Fc binding protein, which forms a complex with IgG antibodies. Upon internalization, the acidic environment enhances the fluorescence signal, indicating internalization activity. By measuring fluorescence intensity, researchers can assess internalization efficiency and gain insights into antibody uptake mechanisms, helping optimize antibody-based therapies and drug delivery systems.

The fluorescent signal from GPRC5D ADC BMK-AME100002 conjugate is only detected in GPRC5D positive cells (K562-GPRC5D stable expression cell line) and not in parental K562 cells, indicating specific internalization.

Comparison of internalization efficiency between AME100002 and a competitor reagent on GPRC5D-positive cells (K562-GPRC5D stable expression cell line). AME100002 requires less reagent and shorter incubation time while demonstrating higher sensitivity. It is the most cost-effective pH-sensitive fluorescence IgG labeling reagent available. This pH-sensitive IgG labeling reagent is compatible with human IgG1, IgG2, IgG3, IgG4, rabbit IgG, and mouse IgG subtypes (IgG1, IgG2a, IgG2b, IgG3).

Payload-conjugated IgG labeling reagents use a payload-conjugated Fc binding protein to label various IgG antibodies. Once the IgG-payload complex is added to the cells, the cell inhibition rate for each sample is determined using the CCK8 method. This approach mimics the real ADC mechanism, where the antibody delivers the payload into the cells, resulting in cell death. By measuring the cell inhibition, researchers can evaluate the effectiveness of the candidate mAbs and gain insights into the potential of ADCs for therapeutic applications.

Cell inhibition rate of HeLa cells detected using the CCK8 method. B7H3 ADC BMK was mixed with DiTag™ MMAE IgG labeling reagent (Cat. No. AME100003) to form a complex, which was applied to HeLa cells for the inhibition test. The results were compared to the control, where Isotype Control IgG was mixed with AME100003 and applied to the cells. The IC50 of B7H3 ADC BMK is 66 ng/ml.

Accelerated stability test of AME100003. Following lyophilization, samples were stored at -20°C (black), 37°C for 1 day (red), 37°C for 2 days (blue), and 37°C for 3 days (green), separately. Upon reconstitution, the cell inhibition rate of each sample was determined using the CCK8 method. The data indicate excellent stability for all samples.