4-1BB Bioassay
A Simpler, More Consistent 4-1BB Bioassay
- Prequalified according to ICH guidelines
- Mechanism-of-action (MOA)-based assay
- No primary cell culture required
- Supplied in convenient thaw-and-use format; also available in cell propagation model (CPM) format
Catalog Number:
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Size
Catalog Number: JA2351
Catalog Number: JA2355
Catalog Number: J2332
Catalog Number: K1161
Simple Bioassay, Consistent Results
4-1BB (CD137/TNFRSF9), a member of the tumor necrosis factor receptor superfamily, is an inducible co-stimulatory receptor expressed on T cells, natural killer (NK) cells and innate immune cell populations. When present on the cell surface, 4-1BB interacts with 4-1BB ligand (4-1BBL) and induces subsequent cell proliferation and production of interferon gamma (IFNγ) and interleukin-2 (IL-2), particularly in T and NK cells.
Current methods used to measure the activity of biologic drugs targeting 4-1BB rely on assays that are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents and, as a result, are difficult to establish in a quality-controlled, drug-development setting.
The 4-1BB Bioassay is a biologically relevant, MOA-based assay that can be used to measure the potency and stability of ligands or agonist antibodies that can bind and activate 4-1BB.
Control Ab, Anti-4-1BB, is a mouse anti-human 4-1BB agonist antibody. It can be used as a positive control in the 4-1BB Bioassay in the presence of FcγRIIb CHO-K1 Cells (Cat.# JA2251, JA2255).
Assay Components
The thaw-and-use format of the assay (Cat.# JA2351, JA2355) offers convenience and minimizes variability. The assay consists of a genetically engineered Jurkat T cell line that expresses human 4-1BB and a luciferase reporter driven by a response element that can respond to 4-1BB ligand/agonist antibody stimulation. The 4-1BB Effector Cells included are provided as cryopreserved cells that can be thawed, plated and used in an assay without the need for cell propagation.
The assay is also available in CPM format (Cat.# J2332) containing cryopreserved cells that 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.
- Kit includes all of the required reagents in standardized formats.
- Easily amenable to high-throughput workflows.
Assay Principle
Panel A. Assay with FcγRIIb-dependent agonist antibody. The bioassay consists of two engineered cell lines, 4-1BB Effector Cells and FcγRIIb CHO-K1 Cells. In the presence of FcγRIIb CHO-K1 Cells, the anti-4-1BB antibody can be crosslinked, thereby inducing 4-1BB pathway-activated luminescence. Panel B. Assay with FcγRIIb-independent agonist antibody or ligand. The bioassay consists of one engineered cell line, 4-1BB Effector Cells. In the absence of agonist antibody or 4-1BB ligand, the 4-1BB receptor is not activated and luminescence signal is low. The addition of agonist antibody or 4-1BB ligand induces the 4-1BB pathway-activated luminescence, which can be detected in a dose-dependent manner.
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 4-1BB Bioassay Shows Precision, Accuracy and Linearity.
| Parameter | Results | |
|---|---|---|
| Accuracy | % Expected Relative Potency | % Recovery |
| 50 | 106.0 | |
| 70 | 96.2 | |
| 140 | 97.3 | |
| 200 | 103.2 | |
| Repeatability (% CV) | 100% (Reference) | 3.5 |
| Intermediate Precision (% CV) | 6.6 | |
| Linearity (r2) | 0.996 | |
| Linearity (y = mx + b) | y = 1.025x – 1.835 | |
| A 50–200% theoretical potency series of Control Ab, Anti-4-1BB, 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 4-1BB Bioassay reflects mechanism of action and shows specificity of biologics designed to activate 4-1BB. Panel A. 4-1BB Effector Cells were induced with a serial titration of ligands: GITRL, 4-1BBL and OX40L as indicated. Panel B. 4-1BB Effector Cells were induced with a serial titration of Control Ab, Anti-GITR (Cat.# K1171), Control Ab, Anti-4-1BB (Cat.# K1161), and Control Ab, Anti-OX40 (Cat.# K1191), as indicated, in the presence of FcγRIIb CHO-K1 Cells (Cat.# JA2251, JA2255). For both panels, Bio-Glo™ Reagent was added, and luminescence was quantified using the GloMax® Discover System.
Product Kit Formats and Related Products
- 4-1BB Bioassay is available in 1X and 5X sizes.
- 4-1BB Bioassay is also available in CPM format (Cat.# J2332).
- Control Ab, Anti-4-1BB (Cat.# K1161), is available separately.
- FcγRllb CHO-K1 Cells (Cat.# JA2251, JA2255) are available separately.
Protocols
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
4-1BB Effector Cells |
J235A | 1 × .5ml |
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
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Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
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.
What's in the box?
| Item | Part # | Size |
|---|---|---|
4-1BB Effector Cells |
J235A | 5 × .5ml |
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 SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
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.
What's in the box?
| Item | Part # | Size |
|---|---|---|
4-1BB Effector Cells (CPM) |
J233A | 2 × 1ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
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.
What's in the box?
| Item | Part # | Size | Concentration |
|---|---|---|---|
Control Ab, Anti-4-1BB |
K116A | 1 × 50μg | 1mg/ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage conditions.
Resources
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Frequently Used With
FcγRIIb CHO-K1 Cells
FcγRIIb CHO-K1 Cells function to crosslink co-stimulatory agonist antibodies for CD40, OX40, GITR or 4-1BB.
JA2251, JA2255, J2232
Bio-Gloâ„¢ 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
