Membrane TNFα Target Cells

Measure Effector Functions and Antibody Binding Affinity

Tumor necrosis factor alpha (TNFα) is a key cytokine involved in immune and inflammatory responses. TNFα is produced primarily by macrophages, but it can be generated by other leukocytes as well as endothelial cells, cardiomyocytes and other cell types. TNFα binding to its receptors, TNF receptor type 1 (TNFR1) and TNFR2, induces a myriad of cellular responses that are cell-context dependent. Typically, TNFα binding to TNFR1 and/or TNFR2 induces pro-inflammatory responses, such as immune cell activation, promotion of effector functions and cytokine production, as well as the acute phase response. TNFα exists in both a trimeric membrane-bound form (mTNFα) and as a soluble protein. Binding of TNFα-targeted antibodies to mTNFα-expressing cells can induce effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) to destroy the mTNFα-expressing inflammatory cells. However, such studies are hampered by the lack of model cell lines naturally expressing mTNFα.

mTNFα Target Cells are a genetically engineered cell line stably expressing TNFα on the cell surface. They are designed to be used as target cells in assays that measure the effector functions, such as ADCC and CDC, of anti-TNFα blockers. In addition, they can be used to measure antibody binding affinity to mTNFα.

Assay Components

Membrane TNFα Target Cells are provided in thaw-and-use format (Cat.# J3331, J3335). These cryopreserved cells are ready to be thawed for use in assays that measure the effector functions of anti-TNFα blockers.

Membrane TNFα Target Cells are also available in Cell Propagation Model (CPM) format (Cat.# J3322). These cryopreserved cells 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.
Easily amenable to high-throughput workflows.

Assay Principle

17870ma — **mTNFα Target Cells used to measure ADCC with the ADCC Reporter Bioassay.** Following engagement with the Fc region of a relevant antibody bound to a Target Cell, ADCC Bioassay Effector Cells expressing the FcγR will transduce intracellular signals, resulting in NFAT-mediated luciferase activity that can be easily quantified.

17871ma — **mTNFα Target Cells used to measure CDC with the CytoTox-Glo™ Assay.** The assay measures dead-cell protease activity, and the amount of luminescence directly correlates with the percentage of cells undergoing cytotoxic stress.

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 ADCC Reporter Bioassay using mTNFα Target Cells Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	49.1
	70	72.0
	100	100.9
	140	150.1
	200	213.4
Repeatability (% CV)	100% (Reference)	2.1
Intermediate Precision (% CV)		9.4
Linearity (r²)		0.9991
Linearity (y = mx + b)		y = 1.099x – 6.024
A 50–200% simulated potency series of infliximab was analyzed in triplicate in three independent experiments performed on three days by two analysts using mTNFα Target Cells in the ADCC Reporter Bioassay. Bio-Glo™ 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.

17855ma — **The ADCC Reporter Bioassay with mTNFα Target Cells measures the activity of the anti-TNFα antibodies infliximab (Panel A) and adalimumab (Panel B).** mTNFα Target Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of the assay, ADCC Effector Cells and a titration of infliximab or adalimumab were added. After a 24-hour induction at 37°C, Bio-Glo™ Luciferase Reagent was added and luminescence measured using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. The EC₅₀ was 148ng/ml for infliximab and 10.7ng/ml for adalimumab. Fold induction was 4.9 and 2.9 for infliximab and adalimumab, respectively.

17856ma — **A CDC assay measures the activity of the anti-TNFα antibodies infliximab (Panel A) and adalimumab (Panel B).** mTNFα Target Cells were added to a 96-well assay plate 18 hours prior to the assay. On the day of assay, 10% CDC-Qualified Human Serum and a titration of infliximab or adalimumab were added. After a 6-hour induction at 37°C, CytoTox-Glo™ Cytotoxicity Reagent was added and luminescence measured using the GloMax® Discover System. Data were fitted to a four-parameter logistic curve using GraphPad Prism® software. Infliximab gave an EC₅₀ of 0.73µg/ml, fold induction of 7.7 and specific lysis of 40.2%. With adalimumab, the EC₅₀ was 0.19µg/ml, the fold induction was 15.7 and the specific lysis was 37.1%.

Protocolos

Complete Protocol

Membrane TNFα Target Cells Technical ManualPDF (2159 KB) – English

Membrane TNFα Target Cells, Propagation Model Technical ManualPDF (2234 KB) – English

Contenido

Item	Part #	Presentación
Membrane TNFα Target Cells	J333A	1 × 0.5ml

SDS

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Certificado de Análisis

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Use Restrictions

Not For Medical Diagnostic Use.

Condiciones de Almacenaje

Please see the Protocol for detailed storage conditions.

Patentes y Exclusiones

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 luminescent assay reagents purchased from Promega for all luminescence-based 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.

Product cannot be used for proficiency testing.

Contenido

Item	Part #	Presentación
Membrane TNFα Target Cells	J333A	5 × 0.5ml

SDS

Search for SDS

Certificado de Análisis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Condiciones de Almacenaje

Please see the Protocol for detailed storage conditions.

Patentes y Exclusiones

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 luminescent assay reagents purchased from Promega for all luminescence-based 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.

Product cannot be used for proficiency testing.

Contenido

Item	Part #	Presentación
Membrane TNFα Target Cells (CPM)	J332A	2 × 1ml

SDS

Search for SDS

Certificado de Análisis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Condiciones de Almacenaje

Please see the Protocol for detailed storage conditions.

Patentes y Exclusiones

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 luminescent assay reagents purchased from Promega for all luminescence-based 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.

Product cannot be used for proficiency testing.

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