CTLA-4 Blockade Bioassay
Measure Potency and Stability of Biologics Designed to Block CTLA-4 Interactions
- 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
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Catalog Number: JA3001
Catalog Number: JA3005
Catalog Number: JA1400
Catalog Number: JA1020
免疫チェックポイントシグナルを遮断するバイオロジックスの力価測定法
CTLA-4 （CD152としても知られる）は 制御性T細胞 (Tregs) 上で構成的に発現する免疫抑制性受容体でT細胞を下方制御します。CTLA-4 は癌抗原や自己抗原に対する免疫応答の調節に重要は役割を果たしています。T細胞表面でのCTLA-4発現が上方制御されると、T細胞はB7と親和性高く結合し、CD28からの正の共刺激シグナルが抑えられます。さらに CTLA-4 と免疫提示細胞 (APC) 上のリガンド CD80 (B7-1) または CD86 (B7-2) が結合すると CD28のT細胞活性化の共刺激や細胞増殖やサイトカイン産生が阻害されます。
CTLA-4 Blockade Bioassay はCTLA-4とそのリガンドCD80 および CD86 との相互作用を遮断するためにデザインされたバイオロジックスの作用機序 (MOA) を反映しています。
アッセイの構成品
本アッセイに含まれる２種類の遺伝子改変細胞株：
CTLA-4 Effector Cells: ヒトCTLA-4 および TCR/CD28の活性化に応答するネイティブプロモーター駆動のルシフェラーゼを安定発現する Jurkat T 細胞
aAPC/Raji Cells: 抗原非依存的に相応のTCR（T細胞受容体）を活性化するよう設計された遺伝子組み換えタンパク質を細胞表面に発現し、またCTLA-4のリガンドであるCD80とCD86も内在的に発現するRaji細胞
この２種類の細胞を共培養した場合、CTLA-4はそのリガンドであるCD80とCD86を共有するCD28と競合し、CD28経路の活性化およびプロモーターによる発光が抑制されます。一方、抗CTLA-4抗体を添加することで、CTLA-4とCD80とCD86との相互作用がブロックされ、プロモーターによる発光が生じます。
CTLA-4 Blockade Bioassayは、既存のアッセイの制限を克服し、CTLA-4を標的とする抗体やその他のバイオロジックの効力と安定性を測定するために使用される、生物発光ベースの細胞アッセイです。
ワークフロー
従来法 （プライマリー細胞を使用）
標準化された試薬による最適な結果
- ”解凍 ＆ アッセイ” だけのフォーマットは従来法に比べ飛躍的に時間と労力を削減し、バラつきも低減
- キットは標準化されたフォーマットとして必要なすべての試薬を供給
- ハイスループットワークフローにも容易に適応
アッセイの原理
CTLA-4 Blockade Bioassayは、既存のアッセイの制限を克服し、CTLA-4を標的とする抗体やその他のバイオロジックの効力と安定性を測定するために使用される、生物発光ベースの細胞アッセイです。
Representation of the CTLA-4 Blockade Bioassay. The bioassay consists of two genetically engineered cell lines, CTLA-4 Effector Cells and aAPC/Raji Cells. Panel A. When co-cultured, the CTLA-4/CD80 and CD86 interaction inhibits CD28 pathway-activated luminescence. The addition of anti-CTLA-4 antibody blocks the CTLA-4/CD80 and CD86 interaction, thereby re-establishing CD28 pathway-activated luminescence, which can be detected in a dose-dependent manner by addition of Bio-Glo™ Reagent and quantitation with a luminometer. Panel B. When co-cultured with non-CTLA-4-expressing Effector Cells (Cat.# J1631), activation also induces luminescence by activation of the CD28 pathway but in a manner independent of anti-CTLA-4 antibody.
アッセイパフォーマンスデータ
正確度 (Accuracy) と 精度 (Precision) はバイオアッセイやバイオロジックス開発において非常に重要です。CTLA-4 Blockade Bioassayは正確度と精度を兼ね備えたアッセイです（正確度とは標的または参照値に対してどれほど近かったかという度合いであり、精度は再現性の度合いです）。本アッセイは正確度 と 精度 のためのICHガイドラインに従い開発、事前承認されており、抗体医薬の研究開発に使用するバイオアッセイの様々なアプリケーションに必要とされるパフォーマンス特性を備えています。
正確度 ：参照値あるいは標的値との近さの度合い
ラボ内再現精度：同一試験室内で試験者、装置、試験日時などの測定条件を変えずに繰り返し測定するときの再現性の度合い
室内再現精度：同一試験室内で試験者、装置、試験日時などの一部又はすべての条件を変えて，繰り返し測定するときの再現性の度合い
直線性：測定値とサンプル濃度が比例する度合
The CTLA-4 Blockade Bioassay Shows Precision, Accuracy and Linearity.
| Parameter | Results | |
|---|---|---|
| Accuracy | % Expected Relative Potency | % Recovery |
| 50 | 105.3 | |
| 70 | 96.7 | |
| 130 | 108.4 | |
| 150 | 112.3 | |
| Repeatability (% CV) | 100% (Reference) | 6.7 |
| Intermediate Precision (% CV) | 10.2 | |
| Linearity (r2) | 0.991 | |
| Linearity (y = mx + b) | y = 1.175x – 11.45 | |
| A 50–150% theoretical potency series of ipilimumab (CTLA-4 blocking antibody) was analyzed in triplicate in three independent experiments performed on three days by two analysts. Bio-Glo™ Reagent was added and luminescence quantified using the GloMax® Discover Detection System. Data were analyzed and relative potencies calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells. | ||
The CTLA-4 Blockade Bioassay reflects the mechanism of action (MOA) and specificity of biologics designed to block the CTLA-4/CD80 and CD86 interaction.
The CTLA-4 Blockade Bioassay measures the inhibitory activity of clinical grade anti-CTLA-4 blocking antibodies.
CTLA-4 Effector Cells were incubated with aAPC/Raji Cells in the absence or presence of anti-CTLA-4, anti-PD-1, anti-TIGIT or anti-HER2 blocking antibodies, as indicated.
CTLA-4 Effector Cells and aAPC/Raji Cells were incubated with increasing concentrations of anti-CTLA-4 antibody, ipilimumab, for 6 hours.
キットのフォーマットと関連製品
-
"Thaw-and-Use" フォーマットで1X および 5X サイズより選択可能
-
Cell Propagation Model format (Cat.# JA1400)
- Control Ab、Anti-CTLA-4P の別売品 (Cat.# JA1020)
Specifications
Catalog Number:
選択製品の構成品内容
| Item | Part # | Size |
|---|---|---|
CTLA-4 Effector Cells |
J158A | 1 × 0.8ml |
aAPC/Raji Cells |
J159A | 1 × 0.8ml |
Fetal Bovine Serum |
J121A | 1 × 4ml |
RPMI 1640 Medium |
G708A | 1 × 36ml |
Bio-Glo™ Luciferase Assay Buffer |
G719A | 1 × 10ml |
Bio-Glo™ Luciferase Assay Substrate |
G720A | 1 × 1 vial |
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 Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence 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.
Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.
選択製品の構成品内容
| Item | Part # | Size |
|---|---|---|
CTLA-4 Effector Cells |
J158A | 5 × 0.8ml |
aAPC/Raji Cells |
J159A | 5 × 0.8ml |
Fetal Bovine Serum |
J121A | 5 × 4ml |
RPMI 1640 Medium |
G708A | 5 × 36ml |
Bio-Glo™ Luciferase Assay Buffer |
G719A | 5 × 10ml |
Bio-Glo™ Luciferase Assay Substrate |
G720A | 5 × 1 vial |
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 Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence 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.
Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.
選択製品の構成品内容
| Item | Part # | Size |
|---|---|---|
CTLA-4 Effector Cells (CPM) |
J146A | 2 × 1ml |
aAPC/Raji Cells (CPM) |
J148A | 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 Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence 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.
Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.
選択製品の構成品内容
| Item | Part # | Size | Concentration |
|---|---|---|---|
Control Ab, Anti-CTLA-4 |
JA102A | 1 × 100μg | 1mg/ml |
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.
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