Impact of cellular states on the activity detection of anti-RANKL antibody using a reporter gene assay:method optimization
ZHU Weifan
YUAN Haiyan
HOU Sheng
GUO Qingcheng
XU Jin
ZHANG Dapeng
LI Jun
ZHUANG Huangzhen
REN Yule
GAO Tianyu
PAN Zhiyuan
CHEN Yi
GUO Menghui
QIAN Weizhu
GUO Huaizu
Abstract：Reporter gene assays are widely used for evaluating the bioactivity of anti-RANKL antibodies(e.g,denosumab)due to their high sensitivity and operational simplicity.However,the non-classical Z-shaped inflection in dose-response curves frequently observed in experiments compromises data reliability.This study systematically investigated variables such as serum sources,trypsinization time,and pH val-ues,and identified the state of the Raw264.7 cells modified with NF-κB response element-luciferase as the key factor causing the Z-shaped inflection.Results demonstrated that adherent culture or trypsin digestion can alter the differentiation state of the cells,altering the dynamic response of signaling pathway and lead-ing to abrupt transitions in curve inflection points.By contrast,using suspension-state cells eliminated this phenomenon,restoring the typical sigmoidal curve.Further validation experiments confirmed that the op-timized method significantly improved reproducibility and stability.This study clarifies the critical role of the Raw264.7 cells modified with NF-κB response element-luciferase state in anti-RANKL antibody detec-tion systems and proposes a cell-state-based optimization strategy,providing both theoretical and practical guidance for precise bioactivity evaluation and experimental design.
Keywords：reporter gene assaycellular statebioactivityanti-RANKL antibodymethod optimization
Publication Date：2026-06-25
Online Publishing Date：2026-08-28(First online date of this platform, not the publication date of the document)
Pages：7( 457-463 )