SIRPα/CD47 Blockade Bioassay

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.

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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.

Representative data for the SIRPα/CD47 Blockade Bioassay (Panel 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.

Protocols

Complete Protocol

SIRPα/CD47 Blockade Bioassay Technical ManualPDF (1851 KB) – English

SIRPα/CD47 Blockade Bioassay, Propagation Model Technical ManualPDF (1886 KB) – English

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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Certificate of Analysis

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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.

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.

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
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 SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

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.

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
CD47/FcγR-A CHO-K1 Target Cells (CPM)	GA120A	2 × 1ml
SIRPα Effector Cells (CPM)	GA129A	2 × 1ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

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. 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.

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
SIRPα Effector Cells (CPM)	GA129A	50 × 1ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

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
CD47/FcγR-A CHO-K1 Target Cells (CPM)	GA120A	50 × 1ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

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-SIRPα	K125A	1 × 50μg	0.5mg/ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Resources

Posters

Other Resources

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SIRPα/CD47 Blockade Bioassay

Measure Potency and Stability of Biologics that Bind and Block SIRPα/CD47 Interactions

Simple Bioassay, Quality Results

Bioassay Components and Product Formats

Workflow

Assay Principle

Assay Performance Data

A.

B.

Protocols

Complete Protocol

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Resources

Posters

Other Resources

Similar Products

PD-1/PD-L1 Blockade Bioassay

TLR Reporter Bioassay

ADCP Reporter Bioassay (THP-1)

SIRPα/CD47 Blockade Bioassay, Fc-Dependent

Frequently Used With

GloMax® Discover System

Lumit® FcγR Binding Immunoassays

ADCC Reporter Bioassay, V Variant

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