LAG-3/MHCII Blockade Bioassay
Measure Potency and Stability of Biologics Designed to Block LAG-3 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
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Catalog Number: JA1111
Catalog Number: JA1115
Catalog Number: JA1112
Catalog Number: K1150
Catalog Number: K1201
Measure the ability of biologics to block immune checkpoint signals
The LAG-3/MHCII Blockade Bioassay is a bioluminescent cell-based assay that measures potency and stability of antibodies and other biologics designed to block the interaction of LAG-3 with its best characterized ligand, major histocompatibility complex II (MHCII). LAG-3, also known as CD223, is an immune checkpoint receptor expressed on activated CD4+ and CD8+ T cells and natural killer (NK) cells. LAG-3 plays a critical role in regulating immune responses to tumor antigens and autoantigens. Engagement of LAG-3 by MHCII inhibits TCR signaling, cytokine production and proliferation of activated T cells. Therapeutic antibodies designed to block the LAG-3/MHCII interaction show promising results in clinical trials for the treatment of a variety of cancers.
The LAG-3/MHCII Blockade Bioassay reflects the mechanism of action (MOA) of biologics designed to block the interaction of LAG-3 with its ligand, MHCII.
The Control Ab, Anti-LAG-3 is a mouse anti-human LAG-3 antibody that shows blocking activity for LAG-3 with its ligand MHCII. It is intended for use as a control with the LAG-3/MHCII Blockade Bioassay, which can be used to measure the potency and stability of antibodies and other biologics designed to block the LAG-3/MHCII interaction.
TCR Activating Antigen (Cat.# K1201) is an antigen that is presented by MHCII on MHCII APC Cells to specifically activate the TCR on the LAG-3 Effector Cells, designed for use with the LAG-3/MHCII Blockade Bioassay.
Assay Components
The assay consists of two cell lines:
LAG-3 Effector Cells: Jurkat T cells stably expressing human LAG-3 and a luciferase reporter driven by T-cell activation pathway-dependent response elements.
MHC APC Cells: APC Cells presenting TCR Activating Antigen, a proprietary peptide designed to specifically activate the TCR on the LAG-3 Effector Cells.
When the two cell types are co-cultured in the presence of antigen, MHCII on the APC cells presents the Ag to the TCR on the LAG-3 Effector Cells to activate the Effector Cells. LAG-3 on the Effector Cells inhibits TCR signaling and promoter-mediated luciferase activity. Addition of the anti-LAG-3 antibody releases LAG-3 mediated inhibition, resulting in promoter-mediated luminescence.
The LAG-3/MHCII Blockade Bioassay is also available in CPM format, containing cryopreserved cells that can be thawed, propagated and banked for long-term use.
The 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.
The Assay Principle
The LAG-3/MHCII Blockade Bioassay is a bioluminescent cell-based assay that overcomes the limitations of existing assays and can be used to measure the potency and stability of antibodies and other biologics targeting LAG-3.
Representation of the LAG-3/MHCII Blockade Bioassay. The bioassay consists of two cell lines, LAG-3 Effector Cells and MHCII APC Cells. When co-cultured, the LAG-3 inhibits TCR pathway-activated luminescence. The addition of anti-LAG-3 antibody blocks LAG-3 binding to MHCII, resulting in full TCR pathway activation, 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. The LAG-3/MHCII 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 ability to replicated 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 your assay to provide measured values that are proportional to the concentration of the sample.
The CTLA-4 Blockade Bioassay Shows Precision, Accuracy and Linearity.
| Parameter | Results | |
|---|---|---|
| Accuracy | % Expected Relative Potency | % Recovery |
| 50 | 94.4 | |
| 75 | 94.8 | |
| 100 | 100.3 | |
| 100 | 100.3 | |
| 200 | 99.8 | |
| Repeatability (% CV) | 100% (Reference) | 7.5 |
| Intermediate Precision (% CV) | 9.7 | |
| Linearity (r2) | 0.998 | |
| Linearity (y = mx + b) | y = 1.03x – 3.65 | |
| A 50–200% theoretical potency series of Control Ab, Anti-LAG-3, 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 LAG-3/MHCII Blockade Bioassay reflects the MOA and specificity of biologics designed to block the LAG-3/MHCII Interaction.
LAG-3 Effector Cells were incubated with TCR Activating Antigen and MHCII APC cells in the presence of anti-LAG-3, anti-PD-1, anti-CTLA-4 or anti-TIGIT antibodies, as indicated.
The LAG-3/MHCII Blockade Bioassay measures the activity of Control Ab, Anti-LAG-3.
MHCII APC Cells and TCR Activating Antigen were plated overnight. The following day, a titration of Control Ab, Anti-LAG-3 was added to the plate, followed by LAG-3 Effector Cells, and then incubated for 5 hours.
Product Kit Formats and Related Products
- LAG-3/MHCII Blockade Bioassay is available in 1X and 5X sizes.
- LAG-3/MHCII Blockade Bioassay is also available in CPM format (Cat.# JA1112).
- Control Ab, Anti-LAG-3, is available separately (Cat.# K1150).
- TCR Activating Antigen is available separately (Cat.# K1201).
Specifications
Catalog Number:
Contenido
| Item | Part # | Presentación | Available Separately |
|---|---|---|---|
LAG-3 Effector Cells |
J249A | 1 × 0.5ml | |
MHCII APC Cells |
J250A | 1 × 0.5ml | |
TCR Activating Antigen Stock Solution |
K120A | 1 × 60μl | View Product |
DMEM with 4.5g/L Glucose, L-Glutamine, Sodium Pyruvate |
J236A | 1 × 60ml | |
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
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Use Restrictions
Not For Medical Diagnostic Use.Condiciones de Almacenaje
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™ 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.
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.
Contenido
| Item | Part # | Presentación | Available Separately |
|---|---|---|---|
LAG-3 Effector Cells |
J249A | 5 × 0.5ml | |
MHCII APC Cells |
J250A | 5 × 0.5ml | |
TCR Activating Antigen Stock Solution |
K120A | 5 × 60μl | View Product |
DMEM with 4.5g/L Glucose, L-Glutamine, Sodium Pyruvate |
J236A | 5 × 60ml | |
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 SDSCertificado de Análisis
Use Restrictions
Not For Medical Diagnostic Use.Condiciones de Almacenaje
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™ 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.
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.
Contenido
| Item | Part # | Presentación | Available Separately |
|---|---|---|---|
LAG-3 Effector Cells (CPM) |
J245A | 2 × 1ml | |
TCR Activating Antigen Stock Solution |
K120B | 1 × 500μl | View Product |
MHCII APC Cells (CPM) |
J246A | 2 × 1ml |
SDS
Search for SDSCertificado de Análisis
Use Restrictions
Not For Medical Diagnostic Use.Condiciones de Almacenaje
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™ 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.
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.
Contenido
| Item | Part # | Presentación | Concentración |
|---|---|---|---|
Control Ab, Anti-LAG-3 |
K115A | 1 × 100μg | 1mg/ml |
SDS
Search for SDSCertificado de Análisis
Use Restrictions
Not For Medical Diagnostic Use.Condiciones de Almacenaje
See the Protocol for detailed storage conditions.
Contenido
| Item | Part # | Presentación | Available Separately |
|---|---|---|---|
TCR Activating Antigen Stock Solution |
K120B | 1 × 500μl | View Product |
SDS
Search for SDSCertificado de Análisis
Use Restrictions
Not For Medical Diagnostic Use.Condiciones de Almacenaje
See the Protocol for detailed storage conditions.
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