Bio-Firefly Luciferase Reporter Gene Assay Application - ADCC Cytotoxicity Assay Protocol
The ADCC (Antibody-dependent cellular cytotoxicity (ADCC)) reporter gene assay is a bioluminescent reporter gene assay used to quantify the biological activity of antibodies . The assay uses effector cells, Jurkat cells, which stably express the human FcγRIIIa receptor and the NFAT response element (NFAT-RE) that drives the expression of firefly luciferase. When the antibody binds to the antigen on the target cell and the FcγRIIIa receptor on the ADCC bioassay effector cell, it leads to the activation of FcγRIIIa and the subsequent expression of luciferase in the ADCC bioassay effector cell.
YEASEN launches Bio-Firefly Firefly Luciferase Reporter Gene Assay Kit (Cat. No.11409ES60), with high sensitivity and stable luminescent signal , suitable for reporter gene-based biological activity detection and high-throughput detection, such as: Fc effector detection, T cell activation detection, immune checkpoint detection, as well as cytokine and growth factor detection, etc., which can meet the needs of most high-throughput experiments. In addition, YEASEN also provides Jurkat effector cell lines for ADCC detection (Cat. No.42001ES03) , the Jurkat effector cell line we developed is sterile and free of mycoplasma contamination. The cell line is a monoclonal cell line and can be used for applications such as ADCC mechanism verification, glycosylation level detection, and antibody screening.
YEASEN launches Bio-Firefly Firefly Luciferase Reporter Gene Assay Kit (Cat. No.11409ES60), with high sensitivity and stable luminescent signal , suitable for reporter gene-based biological activity detection and high-throughput detection, such as: Fc effector detection, T cell activation detection, immune checkpoint detection, as well as cytokine and growth factor detection, etc., which can meet the needs of most high-throughput experiments. In addition, YEASEN also provides Jurkat effector cell lines for ADCC detection (Cat. No.42001ES03) , the Jurkat effector cell line we developed is sterile and free of mycoplasma contamination. The cell line is a monoclonal cell line and can be used for applications such as ADCC mechanism verification, glycosylation level detection, and antibody screening.
ADCC Experiment Principle & Operation
ADCC is the main mechanism of action of many antibody-based therapeutic molecules. Antibody-dependent cellular cytotoxicity (ADCC) is induced by binding of the Fc domain of antibodies to Fcγ receptors (FcγRs) on effector cells, while antibodies simultaneously bind to tumor antigens on target cells through the Fab domain, which triggers cross-linking and activation of Fc receptors, leading to the formation and release of cytotoxic granules of cytokines, perforins, and granzymes, ultimately resulting in target cell killing.
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Figure 1. ADCC experimental principle
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Figure 2. ADCC experimental process
Figure 3. Antibody dilution reference
Key experimental parameters
- Effector cell number : The effector cell number is 25,000-150,000 cells/well. The target cell number can be adjusted according to the experiment.- Effector cell to target cell ratio (E:T) : Generally, it is 1:3-15:1.- Antibody dilution gradient : Generally, 3-5 times serial dilution.- Other influencing factors include assay buffer and incubation time. These parameters may vary depending on the specific antibody and target cell and should be critically evaluated.
Experimental Results
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Figure 4. Representative data of the CD20 antibody rituximab ADCC reporter bioassay. WIL2-S cells were plated in a 96-well white assay plate, followed by serial titration of rituximab. ADCC bioassay effector cells (150,000 per well) were then added to the assay plate. The E:T ratio was 6:1. Chemiluminescence values were measured after 20 hours of induction at 37°C.
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Figure 5. Representative data for the ADCC reporter bioassay of the anti-HER2 antibody trastuzumab. SK-BR-3 cells were plated in a 96-well assay plate. On the day of the assay, the medium was replaced with assay buffer and trastuzumab was titrated continuously. ADCC bioassay effector cells (150,000 per well) were then added to the assay plate. The E:T ratio was 15:1. Chemiluminescence values were measured after 20 hours of induction at 37°C .
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Figure 6. ADCC effect detection of CD20 antibody using Bio-Firefly reporter gene detection kit (11409). Target cells are Raji, and effector cells are Jurkat-luc2p-NFAT-RE- FcγRIIIa-high affinity (V158). The ratio of effector cells to target cells is 6:1, and EC50 = 34.53ng/ml.
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Figure 7. Luciferase luminescence was detected using the Bio-Firefly reporter gene assay kit (11409). Jurkat-luc2p-NFAT-RE-hyg cells were treated with different concentrations of lonomycin, and the luminescence of firefly luciferase was detected, and the EC50 was 3.465μg/mL.