SIRPα/CD47 Blockade Bioassay
Measure Potency and Stability of Biologics that Bind and Block SIRPα/CD47 Interactions
- Prequalified according to ICH guidelines
- 96- and 384-well plate formats
- No primary cell culture required
- Supplied in convenient thaw-and-use format; also available in cell propagation model (CPM) format
Catalog Number:
Choose a Product Format
Size
Catalog Number: JA6011
Catalog Number: JA6015
Catalog Number: JA6012
Catalog Number: GA6000
Catalog Number: GA6040
Catalog Number: K1251
Simple Bioassay, Quality Results
Antibody-dependent cellular phagocytosis (ADCP) is an important mechanism-of-action (MOA) for antibody-based immunotherapies. ADCP is regulated by a complex network of checkpoints, including SIRPα and CD47, that facilitate removal of aberrant or infected cells while preserving healthy tissues. Despite its important physiological role, the SIRPα/CD47 checkpoint contributes to immune escape by tumor cells, many of which overexpress CD47 and thereby evade phagocytosis. Biologics that inhibit SIRPα/CD47 interaction enhance phagocytosis of tumor cells in vitro and have shown promising therapeutic efficacy.
Current methods for assessing the activity of SIRPα/CD47 checkpoint inhibitors rely on primary monocyte-derived macrophages and direct measurement of phagocytosis. These assays are laborious and highly variable due to their reliance on donor cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in a quality-controlled setting.
The SIRPα/CD47 Blockade Bioassay is a bioluminescent reporter cell-based assay used to measure the potency and stability of Fc-silent or Fc-null ligands and antibodies that bind and block SIRPα and CD47 interactions. For Fc-functional inhibitors, we recommend using the SIRPα/CD47 Blockade Bioassay, Fc-Dependent.
Bioassay Components and Product Formats
The blockade bioassay consists of two genetically engineered cell lines:
SIRPα Effector Cells: A monocytic cell line with a stably integrated NanoLuc® (NL) luciferase reporter driven by FcγR and SIRPα/CD47 pathway-dependent response elements.
CD47/FcγR-A CHO-K1 Target Cells: CHO-K1 cells engineered to express human CD47 and an engineered cell-surface protein designed to activate FcγRs.
Thaw-and-Use Kits (1X, 5X):
- Include SIRPα Effector Cells, CD47 CHO-K1 Target Cells, cell culture media and assay reagents
- Recommended for routine use with customer-defined antibody
- Available in 1X and 5X sizes
Cell Propagation Model format (CPM; Cat.# JA6012):
- Includes the SIRPα Effector Cells and CD47 CHO-K1 Target Cells (CPM); these are cryopreserved cells that can be thawed, propagated and banked for long-term use.
- Use with Bio-Glo-NL™ Luciferase Assay System
Cell Bank format (Cat.# GA6000, GA6040):
- Available for customers who are planning to expand and bank their CPM purchase, but they would like to use the cell expansion service offered by Promega.
When the two cell types are co-cultured, SIRPα/CD47 interaction inhibits FcγR signaling and promoter-driven luminescence. Addition of Fc-silent anti-SIRPα or anti-CD47 antibody that blocks SIRPα/CD47 interaction releases the inhibitory signal and results in FcγR activation and promoter-driven luminescence.
Workflow
Standardized Reagents Improve Results
- Thaw-and-use format provides significant time and labor savings over traditional methods while reducing variability.
- Easily amenable to high-throughput workflows.
Assay Principle
The SIRPα/CD47 Blockade Bioassay measures the potency and stability of biologics designed to block the SIRPα/CD47 interaction.
Representation of the SIRPα/CD47 Blockade Bioassay. The SIRPα/CD47 Blockade Bioassay consists of two cell lines, SIRPα Effector Cells and CD47/FcγR-A CHO-K1 Target Cells. When co-cultured, the SIRPα/CD47 interaction inhibits FcγR-mediated luminescence. When the SIRPα/CD47 interaction is disrupted, FcγR activation triggers promoter-driven luminescence that can be detected and quantified using Bio-Glo-NL™ Reagent.
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 SIRPα/CD47 Blockade Bioassay Shows Precision, Accuracy and Linearity.
| Parameter | Results | |
|---|---|---|
| Accuracy | % Expected Relative Potency | % Recovery |
| 50 | 95.6 | |
| 70 | 93.9 | |
| 100 | 97.9 | |
| 130 | 95.8 | |
| 150 | 99.5 | |
| Repeatability (% CV) | 100% (Reference) | 5.0 |
| Intermediate Precision (% CV) | 4.7 | |
| Linearity (r2) | 0.998 | |
| Linearity (y = mx + b) | y = 1.006x – 3.570 | |
|
A 50–150% theoretical potency series of Control Ab, Anti-SIRPα, was analyzed in triplicate in three independent experiments performed on three days by two analysts using the SIRPα/CD47 Blockade Bioassay. Bio-Glo-NL™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were analyzed and relative potencies calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells. |
||
A.
B.
Representative data for the SIRPα/CD47 Blockade Bioassay. Panel A. SIRPα Effector Cells were incubated with CD47/FcγR-A CHO-K1 Target Cells in the presence of serial titrations of blocking antibodies as indicated. Panel B. The SIRPα/CD47 Blockade Bioassay was performed in 96-well plates as described in the technical manual with a titration of Control Ab, Anti- SIRPα. Data were generated using thaw-and-use cells. Luciferase activity was quantified using Bio-Glo-NL™ Reagent.
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
SIRPα Effector Cells |
GA131A | 1 × 0.5ml |
CD47/FcγR-A CHO-K1 Target Cells |
GA125A | 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
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Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.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 |
|---|---|---|
SIRPα Effector Cells |
GA131A | 5 × 0.5ml |
CD47/FcγR-A CHO-K1 Target Cells |
GA125A | 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 SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.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 |
|---|---|---|
CD47/FcγR-A CHO-K1 Target Cells (CPM) |
GA120A | 2 × 1ml |
SIRPα Effector Cells (CPM) |
GA129A | 2 × 1ml |
SDS
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Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.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 |
|---|---|---|
SIRPα Effector Cells (CPM) |
GA129A | 50 × 1ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.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 |
|---|---|---|
CD47/FcγR-A CHO-K1 Target Cells (CPM) |
GA120A | 50 × 1ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.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-SIRPα |
K125A | 1 × 50μg | 0.5mg/ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
Please see the Protocol for detailed storage conditions.
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