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CD40 Bioassays

Measure Potency and Stability of Biologics Designed to Activate CD40

  • Prequalified according to ICH guidelines
  • 96- and 384-well plate formats
  • Supplied in convenient thaw-and-use format; also available in cell propagation model (CPM) format

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Catalog number selected: J2132

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CD40 Bioassays
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Simple Bioassay, Consistent Results

The CD40 Bioassay is a bioluminescent cell-based assay that measures potency and stability of ligands or agonist antibodies and other biologics that can bind and activate CD40. CD40 is expressed on the surface of B cells, dendritic cells and monocytes and is a member of the tumor necrosis factor receptor superfamily. CD40 ligand (CD154) is the primary ligand for CD40 and is expressed by activated T cells, critical regulators of cellular and humoral immunity. Signaling via CD40 triggers activation of antigen-presenting cells (APC).

Agonist CD40 antibodies can mimic the CD40 ligand and are capable of substituting for CD4+ helper T cells in murine models of T cell-mediated immunity. Therefore, agonist CD40 antibodies can rescue the function of APC in tumor-bearing hosts and restore effective immune responses against tumor antigens. Subsequent data from multiple preclinical models has demonstrated synergistic enhancement from combining CD40 agonists with cytotoxics, especially in chemotherapy.

The CD40 Bioassay reflects the mechanism of action (MOA) of biologics designed to activate the CD40 receptor. 

Assay Components

The thaw-and-use format of the CD40 Bioassay (Cat.# JA2151, JA2155) offers convenience and minimizes variability. The assay consists of a genetically engineered cell line, CD40 Effector Cells,  that expresses human CD40 and a luciferase reporter driven by a response element that can respond to CD40 ligand/agonist antibody stimulation. 

Based on the antibody properties to be tested, the CD40 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 CD40 in a FcγR-dependent manner.

The CD40 Effector Cells are also available as a Cell Propagation Model (CPM; Cat.# J2132), cryopreserved cells that can be thawed, propagated and banked for long-term use.

Workflow

Standard bioassay workflow with cultured cells
Improved bioassay workflow with thaw-and-use cells

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 CD40 Bioassay measures the potency and stability of ligands or agonist antibodies and other biologics that bind and activate CD40.

CD40 Bioassay Schematic, Antibody-dependent. Panel A. 15232mb-a

Panel A. Assay with FcγRIIb-dependent antibody. In the presence of FcγRIIb CHO-K1 Cells, the anti-CD40 antibody is cross-linked, thereby inducing CD40 pathway-activated luminescence, which can be detected in a dose-dependent manner by addition of Bio-Glo™ Reagent and quantitation with a luminometer. 

CD40 Bioassay Schematic, Antibody/Ligand Independent. Panel B. 15232mb-b
Panel B. Assay with FcγRIIb-independent antibody or ligand. In the absence of agonist antibody or CD40 ligand, the CD40 receptor is not activated and luminescence signal is low. The addition of CD40 ligand or antibody induces the CD40 pathway-activated luminescence, which can be detected in a dose-dependent manner by addition of Bio-Glo™ Reagent and quantitation with a luminometer.

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.

32454752-data-target-combined

The CD40 Bioassay Shows Precision, Accuracy and Linearity.

Parameter Results
Accuracy % Expected Relative Potency % Recovery
50 98.7
70 99.8
140 104.0
200 100.8
Repeatability (% CV) 100% (Reference) 5.1
Intermediate Precision (% CV)   7.0
Linearity (r2)   0.997
Linearity (y = mx + b)   y = 1.028x – 0.0284
A 0–200% theoretical potency series of Control Ab, Anti-CD40 (Cat.# K1181) 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 CD40 Bioassay reflects the mechanism of action (MOA) and specificity of biologics designed to activate the CD40 receptor.

CD40 Bioassay data. Panel A. 15186ma-a
Panel A. CD40 Effector Cells were induced with a serial titration of anti-CD40 antibody, anti-4-1BB antibody, anti-OX40 antibody or anti-GITR antibody, as indicated, in the presence of FcγRllb CHO-K1 Cells. Bio-Glo™ Reagent was added, and luminescence was quantified using the GloMax® Discover Detection System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. Data were generated using thaw-and-use cells.
15186ma-b
Panel B. CD40 Effector Cells were induced with a serial titration of CD40 Ligand, 4-1BB Ligand, OX40 Ligand or GITR Ligand, as indicated. Bio-Glo™ Reagent was added, and luminescence was quantified using the GloMax® Discover Detection System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. Data were generated using thaw-and-use cells.
  • CD40 Bioassay is available in 1X and 5X sizes.
  • Control Ab, Anti-CD40, is available separately (Cat.# K1181).
  • FcγRllb CHO-K1 Cells are available separately (Cat.# JA2251, JA2255).

Specifications

You are viewing: J2132 Change Configuration

What's in the box?

Item Part # Size

CD40 Effector Cells (CPM)

J213A 2 × 1ml

SDS

Choose language:

Download SDSPDF (198 KB) – English (United States)

Certificate of Analysis

See all certificates for J2132

Use Restrictions

Not For Medical Diagnostic Use.

Storage Conditions

XX

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 and European Pat. No. 1341808.

Patent 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.

Specifications

You are viewing: JA2151 Change Configuration

What's in the box?

Item Part # Size

CD40 Effector Cells

J215A 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

Choose language:

Download SDSPDF (302 KB) – English (United States)

Certificate of Analysis

See all certificates for JA2151

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. 8,008,006 and European Pat. No. 1341808.

Patent Pending.

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.

Specifications

You are viewing: JA2155 Change Configuration

What's in the box?

Item Part # Size

CD40 Effector Cells

J215A 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

Choose language:

Download SDSPDF (302 KB) – English (United States)

Certificate of Analysis

See all certificates for JA2155

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. 8,008,006 and European Pat. No. 1341808.

Patent Pending.

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.

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