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4-1BB Bioassay

A Simpler, More Consistent 4-1BB Bioassay

  • Prequalified according to ICH guidelines
  • Mechanism-of-action-based assay
  • No primary cell culture required

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Catalog number selected: JA2351

$ 760.00
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4-1BB Bioassay
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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 bioluminescent cell-based assay that overcomes the limitations of existing assays and can be used to measure the potency and stability of ligands or agonist antibodies that can bind and activate 4-1BB. 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 are provided in thaw-and-use format as cryopreserved cells that can be thawed, plated and used in an assay without the need for cell culture and propagation.

Assay Components

The 4-1BB Effector Cells included are provided in a thaw-and-use format as cryopreserved cells that can be thawed, plated and used in an assay without the need for cell propagation. 

The 4-1BB Effector Cells have been engineered to express the response element (RE) upstream of luc2P, as well as exogenous 4-1BB receptor. When 4-1BB binds to 4-1BB Effector Cells, the receptor transduces intracellular signals resulting in luminescence.The bioluminescent signal is detected and quantified using Bio-Glo™ Luciferase Assay System (Cat.# G7940) and a standard luminometer such as the GloMax® Discover System.

The Workflow

Traditional Method

Standard bioassay workflow with cultured cells

'Cells as reagents' Method

Improved bioassay workflow with thaw-and-use cells

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

 
4-1BB Bioassay schematic showing assay with FcγRIIb-dependent agonist antibody.
4-1BB Bioassay schematic showing assay with FcγRIIb-independent agonist antibody or ligand.

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. The 4-1BB Bioassay is 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. 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 for Assay Performance Data
Accurate, but not precise

Accuracy
Measure of ability to replicate a reference or "target" value.
IntraLab Precision
Precise, but not accurate

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 Data Target 3
Accurate and precise

Intermediate Precision

Measure of repeatability of multiple assays of the same homogeneous sample with different analysts or equipment or on different days.

32454752-data-linear
Linear

Linearity

Ability of your assay to provide measured values that are proportional to the concentration of the sample.

Accuracy

Expected Relative Potency Recovery
50% 106.0%
70% 96.2%
140% 97.3%
200% 103.2%

Repeatability (100% reference, %CV)

100%

Intermediate Precision (%CV)

6.6%

Linearity

r 2 = 0.996 (50–200% range)
y = 1.025x–1.835


The 4-1BB Bioassay reflects mechanism of action and shows specificity of biologics designed to activate 4-1BB.

4-1BB Bioassay Sensitivity and MOA Panel A 15367ma-a
4-1BB Bioassay sensitivity and MOA schematic Panel B 15367ma-b

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 anti-GITR antibody, Control Ab, Anti-4-1BB (Cat.# K1161) and anti-OX40 antibody, as indicated, in the presence of FcγRIIb CHO-K1 Cells (Cat.# JA2251). For both panels, Bio-Glo™ Reagent was added, and luminescence was quantified using the GloMax® Discover System.

Product Kit Formats and Related Products

  • Kits are available in 1X and 5X sizes.
  • Control Ab, Anti-4-1BB (Cat.# K1161), is available separately.

Protocols

Specifications

You are viewing: JA2351 Change Configuration

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

Choose language:

Download SDSPDF (303 KB) – English (United States)

Certificate of Analysis

See all certificates for JA2351

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

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.

U.S. Pat. No. 8,008,006 and European Pat. No. 1341808.

Patent Pending.

U.S. Pat. No. 10,077,244 and other patents.

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.

Product cannot be used for proficiency testing.

Specifications

You are viewing: JA2355 Change Configuration

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

Choose language:

Download SDSPDF (304 KB) – English (United States)

Certificate of Analysis

See all certificates for JA2355

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

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.

Patent Pending.

U.S. Pat. No. 8,008,006 and European Pat. No. 1341808.

U.S. Pat. No. 10,077,244 and other patents.

Product cannot be used for proficiency testing.

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.

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