Lumit® hKi-67 immunoassay for cell proliferation with optimized performance for screening applications

Ki-67 is a well-established marker of cell proliferation and a valuable readout for evaluating anticancer agents across diverse biological models. Here, we describe an improved homogeneous luminescent immunoassay for detection of human Ki-67 using Lumit® technology. The assay incorporates optimized chemistry and a streamlined workflow and can be multiplexed with a same-well cytotoxicity readout. These features make the platform well suited for screening of proliferative and antiproliferative agents in primary immune cells, immortalized cell lines, and 3D cancer spheroids. To enable use in automated and screening environments, we developed a robust detection reagent for room-temperature “on-deck” stability. This NanoBiT® substrate and buffer formulation yielded a detection reagent that retained ~90% brightness after a 4-h room temperature hold, with no loss in signal-to-background. The reagent also delivered stable glow kinetics, with a signal half-life of ~6 h. Assay robustness was demonstrated by treating Jurkat cells for 48 h with palbociclib (CDK4/6 inhibitor) or BAY-1895344 (ATR kinase inhibitor). Palbociclib reduced Ki-67 levels with an IC₅₀ of 164 nM, while BAY-1895344 showed slightly more potent antiproliferative activity (IC₅₀ = 94 nM). Normalized dose-response curves were unchanged whether the detection reagent was freshly prepared or held 4 h at room temperature, and results remained consistent when luminescence was measured between 5 min and 6 h after reagent addition. Same-well multiplexing with a cell-impermeant fluorogenic DNA dye revealed that palbociclib induced no cell death, whereas the ATR inhibitor was cytotoxic, enabling clear resolution of proliferative versus cytotoxic drug effects. To increase ease of use and workflow flexibility, the assay protocol was optimized. Following the addition of an optimized lysis buffer, a brief shake and 10-min incubation produced maximal Ki-67 recovery in both adherent and suspension cell lines. Under these conditions, anti-CD3/CD28 stimulation of primary human CD8⁺ T cells increased Ki-67 levels by 57-fold after 72 h, whereas total ATP levels increased by ~2-fold, highlighting the superior sensitivity of Ki-67 to changes in proliferative activity. The assay also performed robustly in 3D models: HCT116 spheroids generated over 3 days displayed >90% reductions in Ki-67 following 48-h treatment with BAY-1895344 or nutlin-3a (MDM2/p53 inhibitor), with the ATR kinase inhibitor demonstrating ~20-fold greater potency. Together, these results demonstrate that the Lumit® hKi-67 immunoassay is a stable, scalable, and automation-friendly platform suitable for high-throughput screening and for profiling proliferative and antiproliferative mechanisms across 2D, 3D, and primary immune cell systems.