Promega Corporation

ADCC Bioassay Effector Cells, Propagation Model

ADCC Bioassay Effector Cells, Propagation Model, allows for propagation and banking of the engineered Jurkat effector cells developed for the ADCC Reporter Bioassay line of products.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism of action (MOA) of antibodies through which virus-infected or other diseased cells are targeted for destruction by components of the cell-mediated immune system. The ADCC Reporter Bioassay is a bioluminescent assay for quantifying Fc effector function of therapeutic antibodies as measured by activation of NFAT signaling pathway (Figure 1). The assay includes effector cel...

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ADCC Bioassay Effector Cells, Propagation Model

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ADCC Bioassay Effector Cells, Propagation Model

 Components

  • ADCC Bioassay Effector Cells

    G701A2 x .65ml
1 each
- G7102 Please Enquire


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Storage Conditions

Upon arrival, immediately transfer vials of ADCC Bioassay Effector Cells for long-term storage below –140°C (freezer or liquid nitrogen vapor phase). For safety reasons do not store cell vials submerged in liquid nitrogen.

Product includes two vials at 2 × 107 cells/ml and 0.65ml/vial. One vial should be thawed, propagated and cells frozen to create a cell bank. The remaining vial should be reserved as backup.

For product intended use please see Patents & Disclaimers tab.

Representation of the ADCC Reporter Bioassay.Figure 1. Representation of the ADCC Reporter Bioassay.

Readout is luminescence signal from NFAT response element driving expression of firefly luciferase.

Readout is luminescence signal from NFAT response element driving expression of firefly luciferase.

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Schematic protocol for the ADCC Reporter Bioassay.Figure 2. Schematic protocol for the ADCC Reporter Bioassay.

For the Cell Propagation Model, we recommend 16–24 hours for induction.

Specificity of the ADCC Reporter Bioassay.Figure 3. Specificity of the ADCC Reporter Bioassay.

Serial dilutions of rituximab (anti-CD20 chimeric monoclonal antibody drug), trastuzumab (anti-Her2 humanized monoclonal antibody drug) or assay medium control (no antibody) were incubated for 6 hours of induction at 37°C with engineered Jurkat effector cells (ADCC Bioassay Effector Cells) with or without ADCC Bioassay Target Cells (WIL2-S), as indicated. Luciferase activity was quantified using Bio-Glo™ Reagent. Data were fitted using 4PL curve fitting of GraphPad Prism® software.

Serial dilutions of rituximab (anti-CD20 chimeric monoclonal antibody drug), trastuzumab (anti-Her2 humanized monoclonal antibody drug) or assay medium control (no antibody) were incubated for 6 hours of induction at 37°C with engineered Jurkat effector cells (ADCC Bioassay Effector Cells) with or without ADCC Bioassay Target Cells (WIL2-S), as indicated. Luciferase activity was quantified using Bio-Glo™ Reagent. Data were fitted using 4PL curve fitting of GraphPad Prism® software.

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11167LAFigure 4. Bioassay characterization.

The ADCC Reporter Bioassay was characterized in studies that evaluated accuracy, repeatability, intermediate precision and linearity across the 50–150% relative potency range. Dilution ranges for Control Antibody, Anti-CD20, were selected to ensure good coverage of upper and lower asymptotes and sufficient points in the intermediate dose-range for accurate slope and EC50 determinations. A series of relative potency samples, of 50%, 75%, 125% and 150% theoretical relative potency, were evaluated as triplicate dilution series of antibody dose on each of 3 different days. The effector-to-target cell ratio (E:T ratio) was 6:1. The ADCC Reporter Bioassay was characterized using ADCC Bioassay Target Cells (WIL2-S) and ADCC Bioassay Target Cells (Raji). 4PL curve fitting analysis was performed with GraphPad Prism® software, and relative potencies were calculated after parallelism determination using SAS Institute JMP software. Relative potencies were calculated using the 100% reference sample run as a triplicate dilution series in the same assay plate as the test sample.

The ADCC Reporter Bioassay was characterized in studies that evaluated accuracy, repeatability, intermediate precision and linearity across the 50–150% relative potency range. Dilution ranges for Control Antibody, Anti-CD20, were selected to ensure good coverage of upper and lower asymptotes and sufficient points in the intermediate dose-range for accurate slope and EC50 determinations. A series of relative potency samples, of 50%, 75%, 125% and 150% theoretical relative potency, were evaluated as triplicate dilution series of antibody dose on each of 3 different days. The effector-to-target cell ratio (E:T ratio) was 6:1. The ADCC Reporter Bioassay was characterized using ADCC Bioassay Target Cells (WIL2-S) and ADCC Bioassay Target Cells (Raji). 4PL curve fitting analysis was performed with GraphPad Prism® software, and relative potencies were calculated after parallelism determination using SAS Institute JMP software. Relative potencies were calculated using the 100% reference sample run as a triplicate dilution series in the same assay plate as the test sample.

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Detection of antibody glycosylation.Figure 5. Detection of antibody glycosylation.

Rituximab-blended samples containing mixes of fully deglycosylated and fully N-glycosylated antibody were assayed as serial dilutions against serial dilutions of a 100% reference sample of fully N-glycosylated rituximab using the ADCC Reporter Bioassay. Target cells were ADCC Bioassay Target Cells (WIL2-S), and the E:T ratio was 6:1. Biological activity was expressed relative to the 100% control run in the same assay plate and plotted against the % of N-glycosylation present. Linear regression analysis was performed to determine correlation.

ADCC Reporter Bioassay provides antibody activity ranking equivalent to classic LDH release ADCC bioassay.Figure 6. ADCC Reporter Bioassay provides antibody activity ranking equivalent to classic LDH release ADCC bioassay.

The graph shows correlation of relative ADCC activity with fraction of trastuzumab N-glycosylation. For the experiment, trastuzumab was N-deglycosylated using PNGase F, blended with fully N-glycosylated parent preparations to create test samples representing different % N-glycosylation (indicated on the X-axis) and assayed using either the ADCC Reporter Bioassay or a lytic LDH release ADCC bioassay in which PBMCs were used as effector cells. Target cells were SK-BR-3. For the ADCC Reporter Bioassay, ADCC pathway activation was measured by quantification of luciferase activity in the effector cell; for classic ADCC bioassay, LDH release from target cells was measured. For both assays, biological activity reflects downstream effects of effector cell FcγRIIIa crosslinking by antibody bound to target cells. Biological activity was determined and expressed relative to fully N-glycosylated trastuzumab, then plotted against percent N-glycosylated trastuzumab.

The graph shows correlation of relative ADCC activity with fraction of trastuzumab N-glycosylation. For the experiment, trastuzumab was N-deglycosylated using PNGase F, blended with fully N-glycosylated parent preparations to create test samples representing different % N-glycosylation (indicated on the X-axis) and assayed using either the ADCC Reporter Bioassay or a lytic LDH release ADCC bioassay in which PBMCs were used as effector cells. Target cells were SK-BR-3. For the ADCC Reporter Bioassay, ADCC pathway activation was measured by quantification of luciferase activity in the effector cell; for classic ADCC bioassay, LDH release from target cells was measured. For both assays, biological activity reflects downstream effects of effector cell FcγRIIIa crosslinking by antibody bound to target cells. Biological activity was determined and expressed relative to fully N-glycosylated trastuzumab, then plotted against percent N-glycosylated trastuzumab.

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Use Restrictions

G7102 Not For Medical Diagnostic Use.

Patents - Disclaimers

G7102 NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.
This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) drug discovery and development of biologic drugs, (3) quality assurance testing of biologic drugs, and (4) product release assays for biologic drugs. No other commercial use is allowed. "Commercial use" means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to modify, derivatize, genetically engineer or otherwise create variations of the Effector Cells or the luciferase gene stably transfected within the Effector Cells, except that recipient may propagate and store Effector Cells for use in Antibody-Dependent Cell-Mediated Cytotoxicity assays. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all determinations of luminescence activity of this product, or contact Promega to obtain a license for use of this product with reagents other than Promega's. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

G7102 U.S. Pat. No. 5,670,356.

G7102 U.S. Pat. No. 8,008,006 and European Pat. No. 1341808.

G7102 The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673. A license (from Promega for research reagent products and from The Regents of the University of California for all other fields) is needed for any commercial sale of nucleic acid contained within or derived from this product.

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