Membrane RANKL Target Cells

Measure Effector Functions and Antibody Binding Affinity

Receptor activator of nuclear factor κB (RANK/TRANCE receptor, TNFRSF11A) is a member of the tumor necrosis factor receptor (TNFR) family. Binding of its ligand RANKL to the receptor regulates osteoclast formation, activation and survival in bone modeling and remodeling as well as several pathologic conditions characterized by increased bone turnover. RANKL is initially produced as a type II transmembrane protein (mRANKL) that is cleaved by proteases, resulting in soluble RANKL. Binding of RANKL-targeted antibodies to mRANKL-expressing cells may induce effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) to destroy the mRANKL-expressing cells.

mRANKL Target Cells are a genetically engineered cell line stably expressing a cleavage-resistant form of mRANKL that enforces its surface expression. They are designed to be used as target cells in assays that measure the effector functions, such as ADCC and CDC, of anti-RANKL blockers. In addition, they can be used to measure antibody binding affinity to mRANKL.

Assay Components

Membrane RANKL Target Cells are provided in thaw-and-use format (Cat.# J3381, J3385). These cryopreserved cells are ready to be thawed for use in assays that measure the effector functions of anti-RANKL blockers.

Membrane RANKL Target Cells are also available in Cell Propagation Model (CPM) format (Cat.# J3362). These cryopreserved cells can be thawed, propagated and banked for long-term use.

Workflow

Standardized Reagents Improve Results

Thaw-and-use format provides significant time and labor savings over traditional methods while reducing variability.
Easily amenable to high-throughput workflows.

Assay Principle

17870ma — **mRANKL Target Cells used to measure ADCC with the ADCC Reporter Bioassay.** Following engagement with the Fc region of a relevant antibody bound to a Target Cell, ADCC Bioassay Effector Cells expressing the FcγR will transduce intracellular signals, resulting in NFAT-mediated luciferase activity that can be easily quantified.

17871ma — **mRANKL Target Cells used to measure CDC with the CytoTox-Glo™ Assay.** The assay measures dead-cell protease activity, and the amount of luminescence directly correlates with the percentage of cells undergoing cytotoxic stress.

Assay Performance Data

Accuracy and precision are critical to bioassays and the development of biologics. This assay was developed and prequalified according to ICH guidelines for accuracy and precision, giving it the performance characteristics required for many applications of a bioassay used for antibody drug discovery research and development.

Accuracy: Measure of how close results are to a reference or "target" value.

IntraLab Precision: Measure of repeatability from multiple assays of the same homogeneous sample under the same operating conditions over a short time (same analyst; same lab; same day).

Intermediate Precision: Measure of repeatability of multiple assays of the same homogeneous sample with different analysts or equipment or on different days.

Linearity: Ability of the assay to provide measured values that are proportional to the concentration of the sample.

The ADCC Reporter Bioassay using mRANKL Target Cells Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	46.0
	70	68.9
	100	100.3
	150	144.9
	200	214.7
Repeatability (% CV)	100% (Reference)	10.4
Intermediate Precision (% CV)		8.3
Linearity (r²)		0.9928
Linearity (y = mx + b)		y = 1.093x – 9.838
A 50–200% simulated potency series of denosumab-IgG1 was analyzed in triplicate in three independent experiments performed on three days by two analysts using mRANKL Target Cells in the ADCC Reporter Bioassay. Bio-Glo™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were analyzed and relative potencies calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells.

17859ma — **The ADCC Reporter Bioassay with mRANKL Target Cells measures the activity of the anti-RANKL antibody denosumab-IgG1.** mRANKL Target Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of the assay, ADCC Effector Cells and a titration of denosumab-IgG1 were added. After a 24-hour induction at 37°C, Bio-Glo™ Luciferase Reagent was added and luminescence measured using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. The EC₅₀ was 69ng/ml for denosumab-IgG1 and fold induction was 10.2.

17860ma — **A CDC assay measures the activity of the anti-RANKL antibody denosumab-IgG1.** mRANKL Target Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of assay, 10% Normal Human Serum Complement and a titration of denosumab-IgG1 were added. After a 6-hour induction at 37°C, CytoTox-Glo™ Cytotoxicity Reagent was added and luminescence measured using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. For denosumab-IgG1, the EC₅₀ was 261ng/ml, fold induction was 11.0 and specific lysis was 53%.

Protocols

Complete Protocol

Membrane RANKL Target Cells Technical ManualPDF (2251 KB) – English

Membrane RANKL Target Cells, Propagation Model Technical ManualPDF (2397 KB) – English

What's in the box?

Item	Part #	Size
Membrane RANKL Target Cells	J338A	1 × 0.5ml

SDS

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Certificate of Analysis

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Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

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) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. 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 propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use luminescent assay reagents purchased from Promega for all luminescence-based assays using 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.

Product cannot be used for proficiency testing.

What's in the box?

Item	Part #	Size
Membrane RANKL Target Cells	J338A	5 × 0.5ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

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) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. 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 propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use luminescent assay reagents purchased from Promega for all luminescence-based assays using 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.

Product cannot be used for proficiency testing.

What's in the box?

Item	Part #	Size
Membrane RANKL Target Cells (CPM)	J336A	2 × 1ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

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) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. 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 cells or genes stably transfected within the cells except that recipient may propagate and store the cells for long-term use. In addition, recipient must use luminescent assay reagents purchased from Promega for all luminescence-based assays using 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.

Product cannot be used for proficiency testing.

Membrane RANKL Target Cells

Assess the Mechanism of Action of Biologics Designed to Affect RANKL Signaling