iCrea™ ADCC mTNF-alpha (+) Target Cell Line, Human HEK293 Cells

Description

The iCrea™ ADCC Target mTNFα (+) Assay Cell Line are human cells engineered to overexpress mTNFα and optimized to give high sensitivity and specificity when used together with iLite ADCC Effector (V) Assay Cell Line. iLite ADCC Target mTNF-alpha (+) Assay Cell Line are based on a human embryonic kidney cell line, HEK293 ( CRL-1573), and have been genetically engineered and optimized to express high and constant levels of the surface antigen mTNF-alpha. The cells are to be use as target cells together with iCrea™ ADCC Effector (V) Assay Cell Line for measuring the ADCC activity of anti-TNF-alpha antibodies.

Features

Read-out of results: Quantitative
Detection system: Luminescence
Regulatory status: Research Use Only

Applications

The iCrea™ mTNF-alpha (+) Target Assay Cell Line can be used together with iCrea™ ADCC Effector (V) and iCrea™ mTNF-alpha (-) Target Assay Cell Line for the quantification ADCC activity of anti-mTNF-alpha antibodies.

Principle

The iCrea™ ADCC Assay Cell Line are engineered cells that enable antibody-dependent cell-mediated cytotoxicity (ADCC) to be examined through the specific expression of Firefly luciferase. When the antibodies of interest bind to the antigens on the surface of the target cell, the target-bound antibodies will be presented to the Fc receptors (FcγRIIIa) on the effector cell. When the Fc-portion of the targetbound antibodies binds to the receptor, multiple cross-linking of the two cell types occurs. This will initiate a signaling cascade which triggers the expression of Firefly luciferase (FL) in the effector cell. In this application note, we describe the use of an effector cell line (iCrea™ ADCC Effector (V) Assay Cell Line) that over-express FcγRIIIa and contain the FL reporter gene that responds to the principal transcription factors that mediate signaling from the FcγRIIIa receptor, together with a positive target cell line which over-expresses membrane bound TNF-alpha (iCrea™ mTNF-alpha (+) Target Assay Cell Line). iCrea™ ADCC Effector (V) Assay Cell Line also contain the Renilla Luciferase (RL) reporter gene, under the control of a constitutive promoter, that allows anti-TNF drug-induced FL activity to be normalized with respect to the constitutive expression of RL. This renders assay results independent of variations in cell number, serum matrix effects, or lysis of the effector cells by the target cells. In addition, we also describe the use of a negative control in the form of a target cell line without expression of membrane bound TNF-alpha (iCrea™ mTNF-alpha (-) Target Assay Cell Line). The Firefly luciferase signal can be measured in a luminometer following addition and incubation of luciferase substrate. The Firefly luciferase signal is proportional to the functional activity of Infliximab in the sample (Fig.1).

Size

1 vial corresponding to one 96 well plate

Species Reactivity

Human

Cell Background

HEK293 Cells

Sample

Works with both adherent and suspension cells.

Growth Medium

Gibco (cat no 12648-010)

Cell Purity

>95%

Cell Viability

>90%

Sterility Testing

Creative Biolabs provides sterility testing in accordance with USP and EP regulations. All of our sterility testing is performed in an isolator or clean room environments. The cell line has been screened using the membrane filtration testing methods to confirm the absence of aerobic, anaerobic and fungi microorganisms.

Identity Testing

Identity testing is required for newly established cell lines. Isoenzyme analysis is used to confirm the identity of the species of a cell line. Alternative methods for identity testing include DNA fingerprinting, STR analysis and karyology.

Virological Safety Testing

A broad range of viruses is susceptible to affecting human cell lines. We can provide in vivo/vitro virus saftey assays by utilizing various animal systems. These viruses include: adventitious viruses, bovine viruses, human and simian viruses, porcine viruses, retrovirus and rodent viruses.

Genetic Stability Testing

We perform cell genetic stability studies under ICH guidelines. We can provide guidance on the appropriate testing program upon your requirements.

Mycoplasma Testing

Guaranteed mycoplasma-free

Shipping

Dry ice

Storage

Upon receipt confirm that adequate dry-ice is present, and the cells are
frozen. Immediately transfer to -80°C storage. Cells should be stored at
-80°C (do not store at any other temperature) and are stable as
supplied until the expiry date shown. Cells should be used within 30 min
of thawing.

Warnings

Avoid multiple freeze/thaw cycles

Research Use Only

Our recombinant cell are for research use only, not for diagnostic or therapeutic use.

Quality Control

The expression of hVISTA has been verified by flow cytometry.
The induction of antibody-dependent cellular cytotoxicity (ADCC) has been verified using anti-hVISTA-hIgG1 antibody and Jurkat-NFAT Lucia™ CD16 reporter cell line.
The stability of the 20 channels after thawing has been verified.
Raji-hVISTA cells are guaranteed to be free of mycoplasma.

Figure 1 Example of Infliximab calibration curve using the firefly luciferase substrate luciferase reagent.

Figure 2
A: Measurement of the specific Firefly (FL) signal. Cells were stimulated with increasing concentrations of Trastuzumab.
B: Measurement of the Renilla (RL) signal for normalization, from the identical assay as in A.

Figure 3 Dose response curve after normalization of the specific Firefly signal with the according Renilla signal.

iCrea™ ADCC mTNF-alpha (+) Target Cell Line, Human HEK293 Cells (CAT#: AD-P019-C) Datasheet

Overview

Specification

Images

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