Membrane RANKL Target Cells
Assess the Mechanism of Action of Biologics Designed to Affect RANKL Signaling
- Prequalified according to ICH guidelines
- 96- and 384-well plate formats
- No primary cell culture required
- Supplied in convenient thaw-and-use format; also available in cell propagation model (CPM) format
Catalog Number:
Choose a Product Format
Size
Catalog Number: J3381
Catalog Number: J3385
Catalog Number: J3362
エフェクター機能と抗体結合親和性の測定
Receptor activator of nuclear factor κB (RANK/TRANCE receptor, TNFRSF11A) は、腫瘍壊死因子受容体 (TNFR) ファミリーの一員である。この受容体にリガンドである RANKL が結合すると、骨のモデリングやリモデリングにおける破骨細胞の形成、活性化、生存が制御されるほか、骨のターンオーバーの増加を特徴とするいくつかの病的な状態も制御しています。RANKLは、最初はII型膜貫通タンパク質（mRANKL）として産生され、プロテアーゼにより切断されて可溶性の RANKL となる。RANKL を標的とした抗体が mRANKL 発現細胞に結合すると、抗体依存性細胞媒介性細胞毒性（ADCC）や補体依存性細胞毒性（CDC）などのエフェクター機能が誘導され、mRANKL発現細胞が破壊されます。
mRANKL Target Cells は、遺伝子操作により、切断抵抗性の mRANKL を安定的に発現させた細胞株であり、mRANKL の表面発現を強化しています。mRANKL Target Cells は、抗RANKLブロッカーのADCCやCDCなどのエフェクター機能を測定するアッセイにおいて、標的細胞として使用できるように設計されています。また、mRANKLに対する抗体の結合親和性を測定するためにも使用することができます。
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
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.
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. |
||
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.
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%.
Specifications
Catalog Number:
選択製品の構成品内容
| Item | Part # | Size |
|---|---|---|
Membrane RANKL Target Cells |
J338A | 1 × 0.5ml |
SDS
Search for SDS分析証明書
使用制限
Not For Medical Diagnostic Use.保存条件
Please see the Protocol for detailed storage conditions.
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.
選択製品の構成品内容
| Item | Part # | Size |
|---|---|---|
Membrane RANKL Target Cells |
J338A | 5 × 0.5ml |
SDS
Search for SDS分析証明書
使用制限
Not For Medical Diagnostic Use.保存条件
Please see the Protocol for detailed storage conditions.
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.
選択製品の構成品内容
| Item | Part # | Size |
|---|---|---|
Membrane RANKL Target Cells (CPM) |
J336A | 2 × 1ml |
SDS
Search for SDS分析証明書
使用制限
Not For Medical Diagnostic Use.保存条件
Please see the Protocol for detailed storage conditions.
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.
Resources
No related resources available
Related Products
類似製品
RANKL Bioassay
RANK/RANKL パスウェイへの刺激、阻害するバイオロジックスの効果および安定性を測定
J3102, JA2701, JA2705
Membrane TNFα Target Cells
Assess the mechanism of action of biologics designed to affect TNFα signaling.
J3331, J3335, J3322
Membrane VEGF Target Cells
Assess the mechanism of action of biologics designed to affect VEGF signaling.
J3351, J3355, J3342
関連製品
ADCC Reporter Bioassay, V Variant
高親和性ヒトFcγRIIIa-V受容体を介したADCC活性を持つ抗体の力価および安定性を評価
G7015, G7014, G7010, G7016, G7013, G7018, G7102, GA1130
CytoTox-Glo™ Cytotoxicity Assay
細胞集団中の死細胞数を発光で測定する細胞毒性アッセイ
G9290, G9291, G9292
GloMax® Discover System
発光・蛍光・吸光度測定に対応した高性能マイクロプレートリーダー
GM3000
Bio-Glo™ Luciferase Assay System
レポーターバイオアッセイに理想的な安定性の高いルシフェラーゼ試薬
G7940, G7941
