目的 探讨不同的细胞消化方式对靶向CD3L1的人源化单克隆抗体（代号：5H）结合肿瘤细胞的影响。 方法 使用胰蛋白酶溶液或胶原酶与中性蛋白酶混合液，在室温和37 ℃两种消化温度下对不同贴壁肿瘤细胞系进行离解、传代和收集。随后，通过流式细胞术和共聚焦显微镜成像，比较不同消化条件下肿瘤细胞与5H抗体的结合差异。 结果 使用胰蛋白酶溶液在室温离解和传代的细胞仅表现出与5H抗体的微弱结合，而相同的细胞系用胶原酶与中性蛋白酶混合液在室温消化后，则显示出与5H抗体的明显结合，两组之间抗体结合信号及抗体染色阳性细胞比例的差异有统计学意义（P<0.01）。胶原酶与中性蛋白酶混合液处理的细胞与抗体的结合信号呈现出抗体浓度依赖性，可用于受体占位分析。相比室温，37 ℃的条件下消化后略微削弱了靶细胞与5H抗体的结合，但该差异无统计学意义。 结论 胰蛋白酶溶液消化方式显著削弱了抗CD3L1抗体与肿瘤细胞的结合，而胶原酶与中性蛋白酶混合液的消化方式能有效保护该结合。在选择适宜的消化酶处理细胞的前提下，消化温度的影响相对次要。在研究抗体药物与细胞表面抗原相互作用时，应采用合理且经过优化的细胞消化方案。

Objective To investigate the impact of different cell digestion methods on the binding of a humanized monoclonal antibody targeting CD3L1 （designated as 5H） to tumor cells. Methods Trypsin solution and a collagenase/neutral protease cocktail， combined with two digestion temperatures （room temperature and 37°C）， were used to dissociate， passage， and collect various adherent tumor cell lines. Flow cytometry and confocal microscopy were then employed to compare differences in 5H antibody binding under different cell digestion conditions. Results Cells dissociated and passaged with trypsin solution at room temperature exhibited only weak binding to the 5H antibody， whereas those treated with the collagenase/neutral protease cocktail at room temperature showed significantly stronger binding. There were notable differences in the binding signal and the proportion of antibody-stained positive cells between the two groups （P<0.01）. Additionally， cells treated with the collagenase/neutral protease cocktail exhibited antibody binding signals that were dependent on the concentration of the 5H antibody， enabling receptor occupancy analysis. Compared to digestion at room temperature， treatment at 37°C resulted in a slight reduction in the binding of target cells to the 5H antibody， however， this difference was not statistically significant. Conclusion Trypsin digestion significantly reduces the binding of the anti-CD3L1 antibody to tumor cells， whereas the collagenase/neutral protease cocktail effectively preserves it. With an appropriate choice of digestion enzymes， the impact of digestion temperature is relatively minor. These findings suggest that optimized cell digestion protocols should be employed when studying interactions between antibody drugs and cell surface antigens.