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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

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Catalog number selected: JA1111

$ 1,060.00
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LAG-3/MHCII Blockade Bioassay
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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 CD8T 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.

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 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.

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. 

lag-3-mhcii-blockade-bioassay

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, Propagation Model 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 for Assay Performance Data

Accurate, but not precise


Accuracy
Measure of ability to replicated a reference or "target" value.

IntraLab Precision

Precise, but not accurate


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 Data Target 3

Accurate and precise


Intermediate Precision
Measure of repeatability of multiple assays of the same homogeneous sample with different analysts or equipment or on different days.

32454752-data-linear

Linear


Linearity
Ability of your assay to provide measured values that are proportional to the concentration of the sample.

Accuracy

Expected Relative Potency Recovery
50% 94.4%
75% 94.8%
100% 100.3%

140%

103.6%
 200% 99.8%

Repeatability (100% reference, %CV)

7.5%

Intermediate Precision (%CV)

9.7%

Linearity

r 2 = 0.998 (50-200% range)
y = 1.03x - 3.65


The LAG-3/MHCII Blockade Bioassay reflects the MOA and specificity of biologics designed to block the LAG-3/MHCII Interaction.

lag-3-mhcii-blockade-bioassay-mechanism-of-action

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.                                                                                 

lag-3-mhcii-blockade-bioassay-control-antibody-activity

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

  • Control Ab, Anti-LAG-3, is available separately.
  • TCR Activating Antigen is available separately.

Specifications

You are viewing: JA1111 Change Configuration

What's in the box?

Item Part # SizeAvailable 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

Choose language:

Download SDSPDF (380 KB) – English (United States)

Certificate of Analysis

See all certificates for JA1111

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.

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

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.

Specifications

You are viewing: JA1115 Change Configuration

What's in the box?

Item Part # SizeAvailable 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

Choose language:

Download SDSPDF (381 KB) – English (United States)

Certificate of Analysis

See all certificates for JA1115

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.

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

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.

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