FcγRIIa-H ADCP Bioassay

Measure Potency and Stability of Biologics that Bind and Activate FcyRIIa-H

抗体依存性細胞貪食 (ADCP) は治療用抗体の重要な作用機序 (MOA: mechanism of action) であり、In vivoではADCPはFcγRIIa、FcγRIおよびFcγRIIIa を介して単球、マクロファージ、好中球、樹状細胞により媒介されます。3つすべての受容体がADCPに関与しますが、FcγRIIaはこのプロセスに最も重要なFcγ受容体であると考えられています。

FcγRIIa-H ADCP Reporter Bioassay は生物学的に関連性のあるMOAベースのアッセイで、FcγRIIaに特異的に結合し、活性化する抗体やその他のバイオロジックスの力価や安定性測定に使用することができます。本アッセイに含まれるJurkat細胞はヒト FcγRIIa-H (高親和性 H131 バリアント) を安定に発現し、NFAT-誘導性ルシフェラーゼが含まれます。

このバイオアッセイは、ICHガイドラインに準じた事前承認がなされており、力価および安定性の試験におけるルーチンな使用に要求される精度 (precision) と正確度 (accuracy) および直線性を示します。また、このバイオアッセイのワークフローは従来のプライマリー細胞ベースのアッセイとは異なり、シンプルかつ頑健で96/384 ウェルプレートフォーマットに適合し、品質管理された薬剤開発セッティングにそのまま利用することができます。

キットの構成

FcγRIIa-H ADCP Reporter Bioassayは複数の製品キットからお選びいただけます:

Complete Kit （スターターキット）

構成品： FcγRIIa-H Effector Cells, Target Cells (Raji), Control Antibody, Cell Culture Medium および Assay Reagent

Core Kits （自前の抗体および標的細胞を用いたルーチンでの使用に推奨）

構成品： FcγRIIa-H Effector Cells, Cell Culture Medium および Assay Reagent
1X と 5X の2サイズを用意

FcγRIIa を介したADCP作用機序の証明と確認
抗体の力価および安定性の測定
抗体のグリコシル化およびアフコシル化の機能への影響を評価
抗体のスクリーニング

FcγRIIIa-H ADCP Bioassay Effector Cells, Propagation Modelは FcγRIIa-H Effector Cellsの増殖、バンキングを行うことができます。 Bio-Glo™ Luciferase Assay Systemは FcγRIIa-H ADCP Bioassay Effector Cells, Propagation Modelを使用する場合に必要な試薬です。

【ライセンス】ADCP Bioassay

Richards, J.O. et al. (2008) Optimization of antibody binding to FcγRIIa enhances macrophage phagocytosis of tumor cells. Mol. Cancer Ther. 7, 2517–27.
Dugast, A.S. et al. (2011) Decreased Fc-receptor expression on innate immune cells is associated with impaired antibody mediated cellular phagocytic activity in chronically HIV-1 infected individuals. Virology 415, 160–7.
Ackerman, M.E. et al. (2013) Enhanced phagocytic activity of HIV-specific antibodies correlates with natural production of immunoglobulins with skewed affinity for FcγR2a and FcγR2b. J. Virol. 87, 5468–76.
Tebo, A.E. et al. (2002) Fcγ receptor-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes in vitro. Clin. Exp. Immunology 130, 300–6.

ワークフロー

Standard bioassay workflow with cultured cells

Improved bioassay workflow with thaw-and-use cells

標準化された試薬による改善点

製品に含まれる細胞を融解して使用するだけなので、従来法よりも飛躍的に時間と労力が低減し、バラつきも減少
キットは標準化フォーマットとして必要な全ての試薬を供給
ハイスループットなワークフローにも容易に適応

The Assay Principle

The Fc Effector Reporter Bioassays use an alternative readout from traditional primary cell based assays: the FcγR and NFAT-mediated activation of luciferase activity in the effector cells. Primary cells are replaced with a Jurkat cell line stably expressing human FcγRIIa H-131and NFAT-induced luciferase.

Luminescent Assay Monitors Fc Effector Activity

Following engagement with the Fc region of a relevant antibody bound to a Target Cell, ADCP Bioassay Effector Cells expressing FcγRIIa-H131 with transduce intracellular signals resulting in NFAT-RE-mediated luciferase activity that can be easily quantified.

Assay Performance Data

Accuracy and Precision are critical to bioassays and the development of new biologics. The ADCP Reporter Bioassays are both accurate and precise and have been prequalified according to ICH guidelines and show the precision, accuracy and linearity required for routine use in potency and stability studies. Accuracy is a measure of how close results are to a target or reference value. Precision is a measure of repeatability.

Accuracy: Measure of ability to replicate 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.

ADCP Reporter Bioassay Shows Precision, Accuracy and Linearity

Raji Target Cells

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	99.1
	75	102.7
	150	105.0
	200	99.1
Repeatability (% CV)	100% (Reference)	6.5
Intermediate Precision (% CV)		3.5
Linearity (r²)		0.997 (50–200% range)
Linearity (y = mx + b)		y = 0.997x + 1.95
A 50–200% theoretical relative potency series of the ADCP Control Ab, anti-CD20 (IgG1), was analyzed in triplicate in three independent experiments performed on three days. Luciferase activity was quantified using the Bio-Glo™ Reagent. Data were analyzed and relative potencies calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells.

The ADCP Bioassay reflects the MOA and specificity of antibodies designed to bind and activate FcγRIIa-H

13449MA-W-a — Increasing concentrations of trastuzumab (anti-HER2) or rituximab (anti-CD20) were incubated with SKBR3 (HER2+) target cells and FcγRIIa-H Effector Cells.

13449MA-W-b — Increasing concentrations of trastuzumab (anti-HER2) or rituximab (anti-CD20) were incubated with Raji (CD20+) target cells and FcγRIIa-H Effector Cells.

13449MA-W-c — Increasing concentrations of rituximab were incubated with Raji target cells and NFAT-RE effector cells either with or without FcγRIIa-H expression. Bio-Glo™ Reagent was added, and luminescence was measured. Data were fitted to a 4PL curve using GraphPad Prism® software.

Product Format

Complete Kit

Includes FcγRIIa-H Effector Cells, Target Cells (Raji), Control Antibody, Cell Culture Medium and Assay Reagents.
Recommended for use as a starter kit.

Core Kit

Includes FcγRIIa-H Effector Cells, Cell Culture Medium and Assay Reagents.
Recommended for routine use with customer-defined antibody and target cells.
Available in 1X and 5X sizes.

Cell Propagation Model

Includes FcγRIIa-H Effector Cells (CPM); these are cryopreserved cells that can be thawed, propagated and banked for long-term use.
Use to measure the potency and stability of antibodies and other biologics with Fc domains that specifically bind and activate FcγRIIa.

プロトコール

Complete Protocol

Fc(gamma)RIIa-H ADCP Reporter Bioassay, Complete Kit PDF (1362 KB) – English

Fc(gamma)RIIa-H ADCP Reporter Bioassay, Core Kit Technical ManualPDF (1358 KB) – English

FcγRIIa-H ADCP Bioassay Effector Cells, Propagation Model Technical ManualPDF (1752 KB) – English

選択製品の構成品内容

Item	Part #	Size
FcγRIIa-H Effector Cells	G988A	1 × 0.62ml
Target Cells (Raji)	G960A	1 × 0.55ml
ADCP Control Ab, Anti-CD20	G989A	1 × 2.5μg
Low IgG Serum	G711A	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

Search by lot number

使用制限

Not For Medical Diagnostic Use.

保存条件

Please see the Protocol for detailed storage conditions.

パテントおよび免責条項

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
FcγRIIa-H Effector Cells	G988A	1 × 0.62ml
Low IgG Serum	G711A	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

Search by lot number

使用制限

Not For Medical Diagnostic Use.

保存条件

Please see the Protocol for detailed storage conditions.

パテントおよび免責条項

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
FcγRIIa-H Effector Cells	G988A	5 × 0.62ml
Low IgG Serum	G711A	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

Search by lot number

使用制限

Not For Medical Diagnostic Use.

保存条件

Please see the Protocol for detailed storage conditions.

パテントおよび免責条項

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
FcγRIIa-H Effector Cells (CPM)	G987A	2 × 1ml

SDS

Search for SDS

分析証明書

Search by lot number

使用制限

Not For Medical Diagnostic Use.

保存条件

Please see the Protocol for detailed storage conditions.

パテントおよび免責条項

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.

FcγRIIa-H ADCP Bioassay

Part Numbers: G9901, G9991, G9995, G9871

FcγRIIa-H への結合ならびにそれを介してADCPを活性化するバイオロジックスの力価と安定性を測定

Measure Potency and Stability of Biologics that Bind and Activate FcyRIIa-H

ワークフロー

The Assay Principle

Assay Performance Data

ADCP Reporter Bioassay Shows Precision, Accuracy and Linearity

Raji Target Cells

The ADCP Bioassay reflects the MOA and specificity of antibodies designed to bind and activate FcγRIIa-H

Product Format

プロトコール

Complete Protocol

製品仕様

選択製品の構成品内容

SDS

分析証明書

使用制限

保存条件

パテントおよび免責条項

製品仕様

選択製品の構成品内容

SDS

分析証明書

使用制限

保存条件

パテントおよび免責条項

製品仕様

選択製品の構成品内容

SDS

分析証明書

使用制限

保存条件

パテントおよび免責条項

製品仕様

選択製品の構成品内容

SDS

分析証明書

使用制限

保存条件

パテントおよび免責条項

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