TIM-3 Bioassay
Measure Potency and Stability of Biologics Designed to Block TIM-3 Interactions
- Uses Bio-Glo-NLâ„¢ Luciferase Assay System
- 96- and 384-well plate formats
- No primary cell culture required
- Supplied in convenient thaw-and-use format and cell propagation model (CPM) format
Catalog Number:
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Catalog Number: JA2211
Catalog Number: JA2215
Catalog Number: JA2222
Catalog Number: K1210
Measure the Ability of Biologics to Block Immune Checkpoint Signals
The TIM-3 Bioassay is a bioluminescent reporter 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 TIM-3. TIM-3 (CD366, HAVCR2) is an immune checkpoint receptor expressed on a subset of activated T cells, regulatory T cells (Tregs), macrophages and dendritic cells. TIM-3 has the capacity to inhibit or costimulate T-cell receptor (TCR) signaling in different in vitro systems.
The TIM-3 Bioassay consists of two cell lines:
- TIM-3 Effector Cells: Human T cells genetically engineered to express human TIM-3 and a NanoLuc® luciferase reporter driven by T cell activation pathway-dependent response elements
- TIM-3 Target Cells: MHCII-positive human cell line
When the two cell types are co-cultured, TIM-3 Target Cells provide low-level stimulation to the TIM-3 Effector Cells through the TCR. The combined stimulation of the TCR and TIM-3 induces promoter-mediated luciferase activation and luminescence. Adding a TIM-3 blocking antibody prevents TIM-3 signaling and reduces promoter-mediated luminescence.
The Control Ab, Anti-TIM-3, is a mouse anti-human TIM-3 antibody that shows blocking activity for TIM-3 with its ligands. It is intended for use as a control with the TIM-3 Bioassay, which can be used to measure the potency and stability of antibodies and other biologics designed to block the TIM-3 signaling.
Assay Components
The thaw-and-use format of the TIM-3 Bioassay (Cat.# JA2211, JA2215) offers convenience and minimizes variability. The assay consists of two cell lines: A genetically engineered cell line, TIM-3 Effector Cells, that expresses human TIM-3 and a NanoLuc® luciferase reporter driven by T cell activation pathway-dependent response elements; and TIM-3 Target MHCII-positive human cell line.
The assay is also available in CPM format (Cat.# JA2222) containing cryopreserved cells that can be thawed, propagated and banked for long-term use.
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.
Assay Principle
The TIM-3 Bioassay measures the potency and stability of antibodies and other biologics that block TIM-3 signaling.
Representation of the TIM-3 Bioassay. The bioassay consists of two cell lines, TIM-3 Effector Cells and TIM-3 Target Cells. When cocultured, TIM-3 augments TCR pathway-activated luminescence of NanoLuc® luciferase. Adding anti-TIM-3 antibody blocks TIM-3 binding to ligands, including phosphatidylserine (PS), resulting in reduced TCR pathway activation, which can be detected in a dose-dependent manner by addition of Bio-Glo-NL™ 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.
The TIM-3 Bioassay Shows Precision, Accuracy and Linearity.
| Parameter | Results | |
|---|---|---|
| Accuracy | Percent Expected Relative Potency | Percent Recovery |
| 50 | 102.6 | |
| 70 | 100.2 | |
| 100 | 97.7 | |
| 130 | 99.8 | |
| 150 | 107.7 | |
| Repeatability (% CV) | 100% (Reference) | 12.9 |
| Intermediate Precision (% CV) | 13.4 | |
| Linearity (r2) | 0.904 | |
| Linearity (y = mx + b) | y = 1.078x – 3.277 | |
| A 50–150% theoretical potency series of Control Ab, Anti-TIM-3, was analyzed in triplicate in three independent experiments performed on three days by two analysts. 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. | ||
Product Kit Formats and Related Products
- TIM-3 Bioassay is available in 1X and 5X sizes.
- Control Ab, Anti-TIM-3, is available separately (Cat.# K1210).
- TIM-3 Bioassay is also available in CPM format (Cat.# JA2222).
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
TIM-3 Effector Cells |
J313A | 1 × 0.5ml |
TIM-3 Target Cells |
J314A | 1 × 0.9ml |
Fetal Bovine Serum |
J121A | 1 × 4ml |
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
Not For Medical Diagnostic Use.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. 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 |
|---|---|---|
TIM-3 Effector Cells |
J313A | 5 × 0.5ml |
TIM-3 Target Cells |
J314A | 5 × 0.9ml |
Fetal Bovine Serum |
J121A | 5 × 4ml |
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
Not For Medical Diagnostic Use.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. 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 |
|---|---|---|
TIM-3 Effector Cells (CPM) |
J306A | 2 × 1ml |
TIM-3 Target Cells (CPM) |
J307A | 2 × 1ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.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 |
|---|---|---|
Control Ab, Anti-TIM-3 |
K121A | 1 × 100μg |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
Not For Medical Diagnostic Use.Storage Conditions
Please see the Protocol for detailed storage conditions.
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