ICOS Blockade Bioassay

Simple Bioassay, Consistent Results

The ICOS Blockade Bioassay is a bioluminescent cell-based assay used to measure the potency and stability of of ligands or antibodies that bind and block ICOS/ICOSL. 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 Blockade Bioassay reflects the mechanism of action (MOA) of biologics designed to block the interaction of ICOS with its ligand, ICOSL.

ICOS Blockade Bioassay Components

The assay consists of two 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.

ICOSL aAPC/CHO-K1 Cells: CHO-K1 cells expressing an engineered cell surface protein designed to activate TCR/CD3 in an antigen-independent manner, and ICOSL.

The ICOS Effector Cells and ICOSL aAPC/CHO-K1 Cells are also available in Cell Propagation Model format (CPM; Cat.# JA6072) as cryopreserved cells that can be thawed, propagated and banked for long-term use.

When the two cell types are co-cultured, the ICOSL aAPC/CHO-K1 Cells activate TCR/CD3 and ICOS on the ICOS Effector Cells to induce maximum promoter-mediated luminescence. Adding a biologic that blocks ICOS/ICOSL inhibits co-stimulation by ICOS and results in decreased promoter-mediated luminescence.

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 Blockade Bioassay measures the potency and stability of biologics designed to block the ICOS/ICOSL interaction.

**Representation of the ICOS Blockade Bioassay.** The ICOS Blockade Bioassay consists of two cell lines, ICOS Effector Cells and ICOSL aAPC/CHO-K1 Cells. When co-cultured, ICOSL aAPC/CHO-K1 Cells activate TCR/CD3 and ICOS on the ICOS Effector Cells to induce maximum promoter-mediated luminescence. Adding a biologic that blocks ICOS/ICOSL inhibits T cell co-stimulation by ICOS and results in decreased promoter-mediated luminescence. This decrease in luminescence can be detected in a dose-dependent manner by adding Bio-Glo-NL™ Reagent and quantitating with a luminometer.

Assay Performance Data

Accuracy and precision are critical to bioassays and the development of biologics. The ICOS Blockade 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.

Illustration of bioassay accuracy and precision

The ICOS Blockade Bioassay Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	53.0
	70	71.0
	100	101.5
	140	145.1
	200	205.2
Repeatability (% CV)	100% (Reference)	5.6
Intermediate Precision (% CV)		6.5
Linearity (r²)		0.9996
Linearity (y = mx + b)		y = 1.025x – 0.3804
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 Blockade Bioassay. 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 Blockade Bioassay reflects the mechanism of action (MOA) and specificity of biologics designed to block the ICOS/ICOSL interaction.

ICOS/ICOSL Blockade Bioassay reflects mechanism of action and specificity of biologics designed to block the ICOS/ICOSL interaction — ICOS Effector Cells were incubated with ICOSL aAPC/CHO-K1 Cells in the presence of serial titrations of blocking 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.

ICOSL aAPC/CHO-K1 Cells were added to a 96-well assay overnight. On the day of 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.

ICOS Blockade Bioassay is available in 1X and 5X sizes.
Control Ab, Anti-ICOS (Cat.# K1241), is available separately.

Protocols

Complete Protocol

ICOS Blockade Bioassay Technical ManualPDF (1771 KB) – English

ICOS Blockade Bioassay, Propagation Model Technical ManualPDF (1287 KB) – English

What's in the box?

Item	Part #	Size
ICOS Effector Cells	J328A	1 × 1 vial
ICOSL aAPC/CHO-K1 Cells	J330A	1 × 0.5ml
Fetal Bovine Serum	J121A	1 × 4ml
Ham's F-12 Medium	J123A	1 × 25ml
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

Search for SDS

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-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.

Patent 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.

Product cannot be used for proficiency testing.

What's in the box?

Item	Part #	Size
ICOS Effector Cells	J328A	5 × 1 vial
ICOSL aAPC/CHO-K1 Cells	J330A	5 × 0.5ml
Fetal Bovine Serum	J121A	5 × 4ml
Ham's F-12 Medium	J123A	5 × 25ml
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 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-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.

Patent 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.

Product cannot be used for proficiency testing.

What's in the box?

Item	Part #	Size
ICOS Effector Cells (CPM)	J327A	2 × 1ml
ICOSL aAPC/CHO-K1 Cells (CPM)	J329A	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-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.

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-ICOS	K124A	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.

ICOS Blockade Bioassay

Measure Potency and Stability of Biologics Designed to Block ICOS Interactions