iLite anti-HER2 ADCC Activity Set

$1,850.00

The Eagle Biosciences iLite Assay Ready Cells are designed for the specific detection of drug potency in serum/plasma as well as neutralizing antibodies. They utilize an assay technique called reporter gene assay ( RPG ) to analyze serum/plasma samples. The reporter genes utilized are encoded with a bioluminescent luciferase (Firefly luciferase). Different levels of luminescence detected from each reporter gene cell indicates different levels of expression. The iLite anti-HER2 ADCC Activity Set are for research use only.

iLite anti-HER2 ADCC Activity Set

For Research Use Only

The Eagle Biosciences iLite® anti-HER2 ADCC Activity Set includes (components are also sold separately):


Key benefits of iLite® ADCC Activity Assays

  • Unparalleled sensitivity
  • High serum tolerance
  • Normalization read-out included
  • Negative control available for screening of unspecific activity
  • Easy to use – no culturing required on target or effector cells

Product Developed and Manufactured by Svar Life Science

Additional Information

Summary

The products included in the iLite® anti-HER2 ADCC Activity Set can be used for the quantification ADCC activity of anti-HER2 antibodies.

Background

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism whereby pathogenic cells are lysed by lymphocytes, most often Natural Killer (NK) cells. The mechanism involves binding of antibodies to surface antigens on the pathogen. Crosslinking of these antibodies to NK cells through the binding of the Fc-portion to Fc receptors on the NK cells leads to activation of the NK cell and formation of an immune synapse with the pathogenic cell. The NK cell releases cytotoxic granules containing granzymes and perforin into the synapse, leading to apoptosis of the targeted cell.

Breast cancer is the most common cancer in women worldwide, and the second most common cancer overall. Survival rates have improved in the recent years, and stratification of patients into subgroups has vastly improved treatment options for many patients. As an example, patients with HER2 positive breast cancer generally have a poor prognosis, but treatment with trastuzumab, a monoclonal antibody targeting the HER2 receptor, has shown to increase both overall survival and disease free survival when given together with chemotherapy. Trastuzumab’s mechanism of action is mediated in part by inducing ADCC when crosslinking HER2 positive cells with the patient’s immune cells. The drug was FDA approved for treatment of breast cancer patients in 1998, and many biosimilars are currently in development.

Assay Principle

The iLite® ADCC Assay Ready Cells 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 target-bound 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 (iLite® ADCC Effector (V) Assay Ready Cells) 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 the surface antigen HER2 (iLite® ADCC Target HER2 (+) Assay Ready Cells). iLite® ADCC Effector (V) Assay Ready Cells also contain the Nano Luciferase (NL) reporter gene, under the control of a constitutive promoter, that allows drug-induced FL activity to be normalized with respect to the constitutive expression of NL. 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 depleted of HER2 expression (iLite® Target HER2 (-) Assay Ready Cells). 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 trastuzumab in the sample (Fig.1)

Assay Quick Guide

Click here for the Assay Summary: anti-HER2 ADCC Activity Protocol


FIGURE 1: The interaction between the reporter gene Effector cell and the Target cell, generated by the crosslinking via a specific drug antibody. The resulting luminescence originates exclusively from this crosslinking and the signaling from the CD16 receptor to the Firefly Luciferase.


FIGURE 2: When the Target cell have been depleted for the specific surface target molecule, the crosslinking between the Effector and the Target cell cannot be established and hence no luminescence from Firefly Luciferase is generated. But luminescence from NanoLuc, which is expressed under a constitutive promotor, is still present.

Frequently Asked Questions


Manual

Product Manual