ADCC Reporter Bioassay, F Variant
MOA-Based Bioassay to Measure FcγRIIIa (F158) Function
- No cell culture required
- Quantitate impact of F158 polymorphism on Fc effector function
- Supplied in convenient thaw-and-use format; also available in cell propagation model (CPM) format
Catalog Number:
Choose a Product
Size
Catalog Number: G9790
Catalog Number: G9798
Catalog Number: G9302
Specifically Quantitate the Effect of the FcγIIIa F158 Polymorphism
Fc receptor-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MOA) by which antibodies target disease cells for elimination.
The ADCC Reporter Bioassays, F Variant, use NFAT-mediated luciferase activity in effector cells as indicator of ADCC MOA, providing an alternative to traditional primary cell-based assays for measuring the effect of biologics on ADCC activity.
They are accurate, precise and stability-indicating and prequalified according to ICH guidelines, making them suitable for all stages of biologic research and development.
The Pathway
The human FcyRIIIa gene displays a polymorphism in the position coding for amino acid residue 158. This translates to a higher affinity FcyRIIIa variant having a valine at amino acid 158 (V158 and a lower affinity FcyRIIIa variant carrying phenylalanine F158). Killing of target cells is an endpoint of this pathway activation and is used in classic ADCC bioassays, which use donor peripheral blood mononuclear cells (PBMC) or the natural killer (NK) cell subpopulation as effector cells isolated from blood donors with FcyRIIIa VV, VF or FF alleles.
These primary cells can be highly variable in response, are tedious to prepare and can result in high background readings. It is highly challenging to quantitatively evaluate the impact of FcyRIIIa polymorphism by classic in vitro ADCC bioassays. The ADCC Reporter Bioassay, F Variant, overcomes many of these pain points.
The Workflow
Standardized Reagents Improve Results
- Thaw-and-use cell format provides significant time and labor savings over traditional methods while reducing variability.
- Kit includes all of the required reagents in standardized formats.
- Easily amenable to high-throughput workflows.
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 FcγRIIIa and NFAT-induced luciferase.
Luminescent Assay Monitors Fc Effector Activity
Following engagement with the Fc region of a relevant antibody bound to a Target Cell, ADCC Bioassay Effector Cells expressing FcγRIIIa F158 will transduce intracellular signals resulting in NFAT-mediated luciferase activity that can be easily quantified.
Assay Performance Data
Accuracy and Precision are critical to bioassays and development of biologics. The ADCC Reporter Bioassays are both accurate and precise. 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.
ADCC Reporter Bioassay, F Variant, Shows Precision, Accuracy and Linearity
WIL2-S Target Cells
| Parameter | Results | |
|---|---|---|
| Accuracy | % Expected Relative Potency | % Recovery |
| 50 | 87.7 | |
| 75 | 90.4 | |
| 150 | 95.1 | |
| 200 | 103.1 | |
| Repeatability (% CV) | 100% (Reference) | 3.4 |
| Intermediate Precision (% CV) | 6.2 | |
| Linearity (r2) | 0.997 (50–200% range) | |
| Linearity (y = mx + b) | y = 1.071x – 12.04 | |
| A 50–200% theoretical relative potency series was analyzed in triplicate in three independent experiments performed on three days. The effector-to-target cell ratio (E:T ratio) was 6:1. The ADCC Reporter Bioassay, F Variant, was characterized using ADCC Bioassay Target Cells (WIL2-S). Data were analyzed and relative potencies calculated after parallelism determination using PLA2.1 software from Stegmann Systems GmbH. Data were generated using thaw-and-use cells. | ||
Raji Target Cells
| Parameter | Results | |
|---|---|---|
| Accuracy | % Expected Relative Potency | % Recovery |
| 50 | 101.3 | |
| 75 | 99.2 | |
| 150 | 107.8 | |
| 200 | 99.3 | |
| Repeatability (% CV) | 100% (Reference) | 5.7 |
| Intermediate Precision (% CV) | 7.7 | |
| Linearity (r2) | 0.993 (50–200% range) | |
| Linearity (y = mx + b) | y = 1.071x + 0.596 | |
| A 50–200% theoretical relative potency series was analyzed in triplicate in three independent experiments performed on three days. The effector-to-target cell ratio (E:T ratio) was 6:1. The ADCC Reporter Bioassay, F Variant, was characterized using ADCC Bioassay Target Cells (Raji). Data were analyzed and relative potencies calculated after parallelism determination using PLA2.1 software from Stegmann Systems GmbH. Data were generated using thaw-and-use cells. | ||
The ADCC Reporter Bioassay is specific for FcγRIIIa-mediated (158F) ADCC
Use the ADCC Reporter Bioassay, F Variant, to:
- Demonstrate and/or confirm ADCC MOA (or lack thereof)
- Measure antibody potency and stability
- Determine the impact of antibody glycosylation and afucosylation on ADCC
- Screen antibodies
Product Format
- ADCC Bioassay, F Variant, is available in 1X and 5X sizes (Cat.# G9790, G9798).
- Includes ADCC Bioassay Effector Cells, Cell Culture Medium and Assay Reagents.
- Recommended for routine use with customer-defined antibody and target cells.
- ADCC Bioassay, F Variant, is also available in CPM format (Cat.# G9302).
- Includes ADCC Bioassay Effector Cells, F Variant, Propagation Model; 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 FcγRIIIa.
ADCC activity is a major mode of action of therapeutic monoclonal antibodies and is generally determined using a cell-based assay with primary PBMCs. ADCC Reporter Bioassay allows to measure the potency of an antibody without the constraint of PBMC donors’ variability and with a high level of performance. This assay was successfully validated and implemented in our company for batch release testing.
Dr. Fabian Vandermeers, Sr R&D Technical Leader, Quality Assistance S.A.
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
ADCC Bioassay Effector Cells, F Variant |
G979A | 1 × 1 vial |
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 SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage instructions.
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 propagate, modify, derivatize, genetically engineer or otherwise create variations of the Effector Cells or the luciferase gene stably transfected within the Effector Cells. 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.
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.
What's in the box?
| Item | Part # | Size |
|---|---|---|
ADCC Bioassay Effector Cells, F Variant |
G979A | 5 × 1 vial |
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 SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage instructions.
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 propagate, modify, derivatize, genetically engineer or otherwise create variations of the Effector Cells or the luciferase gene stably transfected within the Effector Cells. 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.
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.
What's in the box?
| Item | Part # | Size |
|---|---|---|
ADCC Bioassay Effector Cells, F Variant (CPM) |
G930A | 2 × 1ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage instructions.
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.
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.
Related Products
Similar Products
ADCC Reporter Bioassay, V Variant
Measure potency and stability of antibodies mediating ADCC through the high-affinity human FcγRIIIa-V receptor.
G7015, G7010, G7016, G7018, G7102, GA1130
FcγRIIa-H ADCP Bioassay
Characterize the Fc effector activity of antibodies that bind and activate the human FcγRIIa-H receptor.
G9901, G9991, G9995, G9871
Mouse FcγRIV ADCC Bioassay
Characterize the Fc effector activity of antibodies that bind and activate the mouse mFcγRIV receptor.
M1201, M1211, M1215, M1212
T Cell Activation Bioassay (NFAT/IL-2)
Choose NFAT or IL-2 kit to assess activity of biologics designed to bind and activate T cells via engagement of TCR/CD3.
J1621, J1625, J1601, J1651, J1655, J1631
Frequently Used With
Bio-Glo™ Luciferase Assay System
Bioluminescent detection reagent qualified for use with bioassays that utilize firefly luciferase.
G7940, G7941
GloMax® Discover System
High-performance microplate reader for detecting luminescence, fluorescence and absorbance.
GM3000
RealTime-Glo™ MT Cell Viability Assay
A bioluminescent method to kinetically monitor viability in cell culture up to 72 hours.
G9711, G9712, G9713
pHAb Reactive Dyes
Measure antibody internalization in real time with pH-sensitive fluorescent dyes.
G9841, G9831, G9835, G9845
