ICOS Bioassay
Measure Potency and Stability of Biologics Designed to Activate ICOS
- Use with Bio-Glo-NLâ„¢ Luciferase Assay System
- Prequalified according to ICH guidelines
- 96- and 384-well plate compatible
- Available in convenient thaw-and-use format
Catalog Number:
Choose an option
Catalog Number: JA6801
Catalog Number: JA6805
Catalog Number: JA3072
Catalog Number: K1241
Simple Bioassay, Consistent Results
The ICOS Bioassay is a bioluminescent cell-based assay used to measure the potency and stability of ligands or agonist antibodies that activate ICOS. ICOS (CD278) binds to its ligand ICOSL (B7-H2, CD275), which is constitutively expressed on B cells, monocytes and dendritic cells, and can be induced on endothelial and epithelial cells during inflammation. ICOS co-stimulation induces the production of effector T cell cytokines such as interferon (IFN)-γ, interleukin (IL)-4 and IL-10.
The ICOS Bioassay reflects the mechanism of action (MOA) of biologics designed to activate ICOS.
ICOS Bioassay Components
The thaw-and-use format of the ICOS Bioassay, Core Kit (Cat.# JA6801, JA6805) offers convenience and minimizes variability. The assay consists of the genetically engineered cell lines:
ICOS Effector Cells: Jurkat T cells that endogenously express TCR/CD3 and are engineered to express human ICOS and a NanoLuc® (NL) luciferase reporter driven by ICOS and TCR/CD3 pathway-dependent response elements.
Based on the antibody properties to be tested, the ICOS Bioassay can be conducted with either aAPC/CHO-K1 Cells (Cat.# JA9441, JA9445) or FcγRIIb aAPC/CHO-K1 Cells (Cat.# JA9331, JA9335) to determine if the agonist antibodies can activate ICOS in an FcγR-dependent manner. These cells must be purchased separately.
aAPC/CHO-K1 Cells (Cat.# JA9441, JA9445): CHO-K1 cell engineered to express a cell-surface protein designed to activate the TCR complex in an antigen-independent manner. Intended for use with FcγRIIb-independent antibodies or other biologics that do not require crosslinking for optimal activity.
FcγRIIb aAPC/CHO-K1 Cells (Cat.# JA9331, JA9335): CHO-K1 cells engineered to express human FcγRIIb and an engineered cell-surface protein designed to activate the TCR complex in an antigen-independent manner. FcγRIIb serves as a crosslinking receptor that binds to antibody Fc domains and clusters agonist antibodies for maximal agonist activity.
The ICOS Effector Cells and FcγRIIb aAPC/CHO-K1 Cells are also available in Cell Propagation Model format (CPM; Cat.# JA3072) 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 ICOS Bioassay measures the potency and stability of ligands or agonist antibodies and other biologics that bind and activate ICOS.
Representation of the ICOS Bioassay. The ICOS Bioassay for FcγRIIb-dependent agonist antibodies consists of two cell lines, ICOS Effector Cells and FcγRIIb aAPC/CHO-K1 Cells. In the absence of agonist antibody, ICOS is not activated and the luminescence signal is low. In the presence of FcγRIIb aAPC/CHO-K1 Cells, anti-ICOS antibody can be crosslinked, thereby inducing ICOS pathway-activating luminescence in a dose-dependent manner by adding Bio-Glo-NL™ Reagent and quantitating with a luminometer. ICOS Effector Cells can also be used with antibodies that are FcγRIIb-independent by substituting aAPC/CHO-K1 Cells.
Assay Performance Data
Accuracy and precision are critical to bioassays and the development of biologics. The ICOS 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: 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 ICOS Bioassay Shows Precision, Accuracy and Linearity.
| Parameter | Results | |
|---|---|---|
| Accuracy | % Expected Relative Potency | % Recovery |
| 50 | 46.6 | |
| 70 | 69.0 | |
| 100 | 102.8 | |
| 140 | 143.1 | |
| 200 | 211.7 | |
| Repeatability (% CV) | 100% (Reference) | 2.7 |
| Intermediate Precision (% CV) | 5.8 | |
| Linearity (r2) | 0.9996 | |
| Linearity (y = mx + b) | y = 1.094x – 7.835 | |
| A 50–200% theoretical potency series of Control Ab, Anti-ICOS, was analyzed in triplicate in three independent experiments performed on three days by two analysts using the ICOS Bioassay for FcγRIIb-dependent antibodies. Bio-Glo-NL™ Luciferase Reagent was added, and luminescence was quantified using the GloMax® Discover System. Data were analyzed, and relative potencies were calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells. | ||
The ICOS Bioassay reflects the mechanism of action (MOA) and show specificity of biologics designed to activate ICOS.
ICOS Effector Cells were incubated with FcγRIIb aAPC/CHO-K1 Cells in the presence of serial titrations of agonist antibodies as indicated. After a 6-hour induction, Bio-Glo-NL™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were generated using thaw-and-use cells.
FcγRIIb aAPC/CHO-K1 Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of the assay, ICOS Effector Cells and a titration of Control Ab, Anti-ICOS (Cat.# K1241), were added. After a 6-hour induction at 37°C, Bio-Glo-NL™ Luciferase Reagent was added and luminescence measured using the GloMax® Discover System.
Product Kit Formats and Related Products
- ICOS Bioassay, Core Kit, is available in 1X and 5X sizes.
- FcγRIIb aAPC/CHO-K1 Cells (Cat.# JA9331, JA9335) are available separately.
- aAPC/CHO-K1 Cells (Cat.# JA9441, JA9445) are available separately.
- Control Ab, Anti-ICOS (Cat.# K1241), is available separately.
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
ICOS Effector Cells |
J328A | 1 × 1 vial |
Fetal Bovine Serum |
J121A | 1 × 4ml |
RPMI 1640 Medium |
G708A | 1 × 36ml |
Bio-Glo-NL™ Luciferase Assay Buffer |
J308A | 1 × 10ml |
Bio-Glo-NL™ Luciferase Assay Substrate |
J309A | 1 × 200μl |
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-NL™ 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. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.
U.S. Pat. No. 8,809,529, European Pat. No. 2635582, Japanese Pat. No. 5889910 and other patents and patents pending.
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 |
|---|---|---|
ICOS Effector Cells |
J328A | 5 × 1 vial |
Fetal Bovine Serum |
J121A | 5 × 4ml |
RPMI 1640 Medium |
G708A | 5 × 36ml |
Bio-Glo-NL™ Luciferase Assay Buffer |
J308A | 5 × 10ml |
Bio-Glo-NL™ Luciferase Assay Substrate |
J309A | 5 × 200μl |
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-NL™ 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. 8,809,529, European Pat. No. 2635582, Japanese Pat. No. 5889910 and other patents and patents pending.
U.S. Pat. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.
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 |
|---|---|---|
FcγRIIb aAPC/CHO-K1 (CPM) |
J325A | 2 × 1ml |
ICOS Effector Cells (CPM) |
J327A | 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-NL™ 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. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.
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-ICOS |
K124A | 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.
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