CD28 Bioassay

Simple Bioassay, Consistent Results

The CD28 Bioassay is a bioluminescent cell-based assay used to measure the potency and stability of ligands or antibodies that activate CD28. CD28 binds to the B7 family members CD80 and CD86 (collectively referred to as B7 for this bioassay) on antigen-presenting cells (APCs). Co-stimulation of T cells by CD28 activation initiates signaling cascades that result in AP-1 and NFκB transcription factor activation and nuclear translocation. These pathways significantly enhance T cell cytokine production—specifically, interleukin-2 (IL-2)—which promotes T cell proliferation, differentiation and survival.

Antibodies that agonize CD28 have the potential to enhance immune responses against cancer and chronic infection. The CD28 Bioassay reflects the mechanism of action (MOA) of biologics designed to activate CD28.

CD28 Bioassay Components

The thaw-and-use format of the CD28 Bioassay Core Kit (Cat.# JA6701, JA6705) offers convenience and minimizes variability. The assay consists of the genetically engineered cell lines:

CD28 (TCR/CD3) Effector Cells: Jurkat T cells endogenously expressing TCR, CD3 and CD28 receptors and engineered with a luciferase reporter driven by a CD28 pathway-dependent promoter.

Based on the antibody properties to be tested, the CD28 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 CD28 in an FcγR-dependent manner. These cells must be purchased separately.

aAPC/CHO-K1 Cells (Cat.# JA9441, JA9445): CHO-K1 cells 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 a proprietary TCR-activating protein as well as human FcγRIIb. FcγRIIb serves as a crosslinking receptor that binds to antibody Fc domains and clusters agonist antibodies for maximal agonist activity.

The CD28 Effector Cells and FcγRIIb CHO-K1 Cells are also available in Cell Propagation Model format (CPM; Cat.# JA1072) 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 CD28 Bioassay measures the potency and stability of ligands or agonist antibodies and other biologics that bind and activate CD28.

**Representation of the CD28 Bioassay.** The CD28 Bioassay for FcγRIIb-dependent agonist antibodies consists of two cell lines, CD28 Effector Cells and FcγRIIb aAPC/CHO-K1 Cells. In the absence of agonist antibody, CD28 is not activated and the luminescence signal is low. In the presence of FcγRIIb aAPC/CHO-K1 Cells, anti-CD28 antibody can be crosslinked, thereby enhancing induction of CD28 pathway-activating luminescence in a dose-dependent manner by addition of Bio-Glo™ Reagent and quantitation with a luminometer. CD28 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 CD28 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 CD28 Bioassay Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	43.6
	70	67.2
	100	93.5
	140	139.9
	200	205.5
Repeatability (% CV)	100% (Reference)	4.9
Intermediate Precision (% CV)		6.3
Linearity (r²)		0.998
Linearity (y = mx + b)		y = 1.075x – 10.48
A 50–200% theoretical potency series of Control Ab, Anti-CD28, was analyzed in triplicate in three independent experiments performed on three days by two analysts using the CD28 Bioassay for FcγRIIb-dependent antibodies. Bio-Glo™ 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 CD28 Bioassay reflects the mechanism of action (MOA) and show specificity of biologics designed to activate CD28.

CD28 Bioassay Mechanism of Action — CD28 Effector Cells were incubated with FcγRIIb aAPC/CHO-K1 Cells in the presence of serial titrations of agonist antibodies as indicated. After a 5-hour induction, Bio-Glo™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were generated using thaw-and-use cells.

CD28 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-CD28 (Cat.# K1231), is available separately.

Protocols

Complete Protocol

CD28 Bioassay, Core Kit Technical ManualPDF (1895 KB) – English

CD28 Bioassay, Propagation Model Technical ManualPDF (1761 KB) – English

What's in the box?

Item	Part #	Size
TCR/CD3 Effector Cells (IL-2)	J129A	1 × 0.8ml
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

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.

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

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
TCR/CD3 Effector Cells (IL-2)	J129A	5 × 0.8ml
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.

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

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
TCR/CD3 Effector Cells (CPM) (IL-2)	J127A	2 × 1ml
FcγRIIb aAPC/CHO-K1 (CPM)	J325A	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.

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

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
Control Ab, Anti-CD28	K123A	1 × 1 each

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.

CD28 Bioassay

Measure Potency and Stability of Biologics Designed to Activate CD28