OX40 Bioassay

Simple Bioassay, Consistent Results

The OX40 Bioassay is a bioluminescent cell-based assay that measures the potency and stability of ligands or agonist antibodies that can bind and activate OX40. OX40 (CD134/TNFRSF4), a member of the tumor necrosis factor (TNF) receptor superfamily, is a costimulatory receptor expressed primarily on activated T cells, and on neutrophils and natural killer (NK) cells to a lesser extent. When present on the cell surface, OX40 interacts with OX40 ligand (OX40L) and induces subsequent cell proliferation, survival and production of cytokines, particularly in T cells.

The OX40 Bioassay reflects the mechanism of action (MOA) of biologics designed to activate OX40 following the addition of OX40 ligand or OX40 agonist antibodies.

Control Ab, Anti-OX40, is a mouse anti-human OX40 agonist antibody. It can be used as a positive control in the OX40 Bioassays in the presence of FcγRIIb CHO-K1 Cells (Cat.# J2232, JA2251, JA2255).

Assay Components

The thaw-and-use format of the OX40 Bioassay (Cat.# JA2191, JA2195) offers convenience and minimizes variability. The assay consists of a genetically engineered cell line, OX40 Effector Cells, that expresses human OX40 and a luciferase reporter driven by a response element that can respond to OX40 ligand/agonist antibody stimulation.

Based on the antibody properties to be tested, the OX40 Bioassay can be conducted in either single-cell systems or with FcγRIIb CHO-K1 Cells (Cat.# JA2251, JA2255) to determine the agonist antibodies that can activate OX40 in a FcγR-dependent manner.

The OX40 Effector Cells are also available in Cell Propagation Model format (CPM; Cat.# J2172), as 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

The OX40 Bioassays are designed to measure the potency and stability of antibodies and other biologics targeting OX40.

OX40 schematic showing FcgRIIb-dependent agonist antibody. 15616ma-wa — **Panel A. Assay with FcγRIIb-dependent agonist antibody.** The bioassay consists of two engineered cell lines, OX40 Effector Cells and and FcγRIIb CHO-K1 Cells. In the presence of FcγRIIb CHO-K1 Cells, the anti-OX40 antibody can be crosslinked, thereby inducing OX40 pathway-activated luminescence.

OX40 Bioassay schematic showing FcgRIIb-independent agonist antibody or ligand. 15616ma-wb — **Panel B. Assay with FcγRIIb-independent agonist antibody or ligand.** The OX40 Bioassay consists of one engineered cell line, OX40 Effector Cells. In the absence of agonist antibody or OX40 ligand, the OX40 receptor is not activated and luminescence signal is low. The addition of agonist antibody or OX40 ligand induces the OX40 pathway-activated luminescence that 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 OX40 Bioassays Show Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	99.4
	70	100.8
	140	101.2
	200	109.9
Repeatability (% CV)	100% (Reference)	5.4
Intermediate Precision (% CV)		9.0
Linearity (r²)		0.996
Linearity (y = mx + b)		y = 1.123x – 8.895
A 50–200% theoretical potency series of Control Ab, Anti-OX40 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 OX40 Bioassays reflect the mechanism of action and show specificity of biologics designed to activate OX40.

Showing specificity and MOA, OX40 Effector Cells were induced with ligands for GITRL, 4-1BBL, OX40L in the OX40 Bioassay. 15617ma-wa — **Panel A.** OX40 Effector Cells were induced with a serial titration of the following ligands: GITRL crosslinked by anti-HA Ab; 4-1BBL crosslinked by anti-His Ab; and OX40L. After a 5-hour induction, Bio-Glo™ Reagent was added, and luminescence was quantified using the GloMax® Discover System.

OX40 Effector Cells were induced with Anti-4-1BB, Anti-OX40 and Anti-GITR Abs. 15617ma-wb — **Panel B.** OX40 Effector Cells were induced with a titration of Control Ab, Anti-GITR (Cat.# K1171), Control Ab, Anti-4-1BB (Cat.# K1161) and Control Ab, Anti-OX40 (Cat.# K1191), in the presence of FcγRIIb CHO-K1 Cells (Cat.# JA2232). After a 5-hour induction, Bio-Glo™ Reagent was added, and luminescence was quantified using the GloMax® Discover System.

OX40 Bioassay is available in 1X and 5X sizes.
OX40 Bioassay is also available in CPM format (Cat.# J2172).
Control Ab, Anti-OX40 (Cat.# K1191) is available separately.
FcγRllb CHO-K1 Cells (Cat.# JA2251, JA2255) are available separately.

Protocols

Complete Protocol

OX40 Bioassay Technical ManualPDF (2154 KB) – English

OX40 Bioassay, Propagation Model, Technical ManualPDF (2244 KB) – English

What's in the box?

Item	Part #	Size
OX40 Effector Cells	J219A	1 × 0.5ml
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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Certificate of Analysis

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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.

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
OX40 Effector Cells	J219A	5 × 0.5ml
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 SDS

Certificate of Analysis

Search by lot number

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.

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
OX40 Effector Cells (CPM)	J217A	2 × 1ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

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 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-OX40	K119A	1 × 50μg	1mg/ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

Please see the Protocol for detailed storage conditions.

OX40 Bioassay

Part Numbers: JA2191, JA2195, J2172, K1191

Measure Potency and Stability of Biologics Designed to Activate OX40