GITR Bioassay

Simple Bioassay, Consistent Results

The GITR Bioassays are bioluminescent cell-based assays used to measure the potency and stability of ligands or agonist antibodies that bind and activate GITR. GITR (CD357/TNFRSF18), a member of the tumor necrosis factor (TNF) receptor superfamily, is a costimulatory receptor widely expressed on most immune cells and further upregulated on activated T cells. When engaged with GITR ligand (GITRL) on the cell surface, GITR enhances subsequent T cell expansion and cytokine production including interleukin-2 (IL-2) and IL-9.

The GITR Bioassay reflects the mechanism of action (MOA) of biologics designed to activate GITR.

Control Ab, Anti-GITR, is a rabbit anti-human GITR agonist antibody. It can be used as a positive control in the GITR Bioassays (Cat.# JA2291, JA2295, J2272) in the presence of FcγRIIb CHO-K1 Cells (Cat.# J2232, JA2251, JA2255).

Assay Components

The thaw-and-use format of the GITR Bioassay (Cat.# JA2291, JA2295) offers convenience and minimizes variability. The assay consists of a genetically engineered cell line, GITR Effector Cells, that expresses human GITR and a luciferase reporter driven by a response element that can respond to GITR ligand/agonist antibody stimulation.

Based on the antibody properties to be tested, the GITR 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 GITR in a FcγR-dependent manner.

The GITR Effector Cells are also available in Cell Propagation Model format (CPM; Cat.# J2272), 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 GITR Bioassays measure the potency and stability of ligands or agonist antibodies and other biologics that bind and activate GITR.

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

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

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	99.3
	75	98.9
	125	102.5
	150	102.9
Repeatability (% CV)	100% (Reference)	1.3
Intermediate Precision (% CV)		6.6
Linearity (r²)		0.997
Linearity (y = mx + b)		y = 1.053x – 3.742
A 50–150% theoretical potency series of Control Ab, Anti-GITR 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.

GITR Bioassays reflect the mechanism of action (MOA) and show specificity of biologics designed to activate GITR.

Panel A. GITR Effector Cells induced with serial titrations of ligands GITRL, 4-1BBL or OX40L. — **Panel A.** GITR Effector Cells were induced with a serial titration of ligands GITRL, 4-1BBL or OX40L. After a 6-hour induction at 37°C, Bio-Glo™ Reagent was added, and luminescence was quantified using the GloMax® Discover System. Data were generated using thaw-and-use cells.

GITR Effector Cells induced with titrations of anti-GITR, anti-4-1BB or anti-OX40 antibodies in the presence of FcγRIIb CHO-K1 Cells. — **Panel B.** GITR Effector Cells were induced with a serial titration of anti-GITR, anti-4-1BB or anti-OX40 antibodies in the presence of FcγRIIb CHO-K1 Cells (Cat.# JA2251, JA2255). After a 6-hour induction at 37°C, Bio-Glo™ Reagent was added, and luminescence was quantified using the GloMax® Discover System. Data were generated using thaw-and-use cells.

GITR Bioassay is available in 1X and 5X sizes.
Control Ab, Anti-GITR (Cat.# K1171) is available separately.
FcγRllb CHO-K1 Cells (Cat.# JA2251, JA2255) are available separately.

Protocols

Complete Protocol

GITR Bioassay Technical ManualPDF (1400 KB) – English

GITR Bioassay, Propagation Model, Technical ManualPDF (2193 KB) – English

What's in the box?

Item	Part #	Size
GITR Effector Cells	J229A	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.

Patent Pending.

U.S. Pat. No. 8,008,006.

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.

What's in the box?

Item	Part #	Size
GITR Effector Cells	J229A	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.

Patent Pending.

U.S. Pat. No. 8,008,006.

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.

What's in the box?

Item	Part #	Size
GITR Effector Cells (CPM)	J227A	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.

Patent Pending.

U.S. Pat. No. 8,008,006.

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.

What's in the box?

Item	Part #	Size	Concentration
Control Ab, Anti-GITR	K117A	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.

GITR Bioassay

Measure Potency and Stability of Biologics Designed to Activate GITR