ADCC activity detection

As a component of the immune system, antibodies play an important role in fighting diseases. Antibody-dependent cell-mediated cytotoxicity ( ADCC ) is one of the ways in which antibodies work, when IgG antibodies interact with target cells (virus-infected cells and tumor cells) surface antigenic determinants through the Fab fragment After specific binding, its Fc segment can bind to effector cells such as FcγR killer cells ( NK cells, monocyte-macrophages, neutrophils), trigger the killing activity of effector cells, and directly kill target cells (virus infection Cells and tumor cells).

ADCC is an ideal mechanism for antibody drugs to kill target cells, and the anti-tumor activity of monoclonal antibodies can be verified by ADCC test. Early ADCC verification methods are mostly based on freshly prepared peripheral blood mononuclear cells or NK cells as effector cells. However, these methods have disadvantages such as difficulty in cell classification and culture, cumbersome operation, high background value, and being affected by individual differences. 

Icartab is committed to the research and development of tumor antibody drugs. In order to solve the common problems in the traditional ADCC test, we used the luciferase reporter gene detection system as the platform to construct stable transfection of CD16 receptor and NFAT (Nuclear Factor of Activated T -cells) Jurkat-NFAT-Luc-CD16 cell line of the original reaction When an antibody Fab after antigen-binding segment to the target cell, antibody Fc segments effector cells Jurkat-NFAT-Luciferase-CD16 cell surface (FcyRIIIA) binding, causing Jurkat-NFAT-Luciferase(New)-CD16 associated The signal pathway is activated, which in turn leads to an increase in luciferase expression. The system has the following advantages:

  • The data result is stable and the detection process is fast;

  • Higher sensitivity and excellent repeatability;

  • Can be applied to various target cells suspended or attached;

  • It can be used to quality control the efficacy and stability of therapeutic antibody drugs.

Illustration: When the Fab fragment of the antibody binds to the antigen on the target cell, the Fc fragment of the antibody binds to the effector cell Jurkat-NFAT-Luciferase (New)-CD16 cell membrane surface CD16 (FcγRIIIA), Causing the activation of the intracellular signal domain of CD16 (FcγRIIIA) and activates the Syk protein,The activation of the Syk protein leads to Ca+ efflux in the endoplasmic reticulum, which increases the concentration of Ca2+ in the cytoplasm. After Ca2+ binds to Calmodulin, it can further transmit signals downstream, making it in a state of inhibition The phosphorylated NFAT is dephosphorylated and the inhibition is released, and NFAT is transferred to the nucleus, resulting in the activation of NAFT-related promoter genes and the activation of Luciferase gene expression. By detecting the expression level of Luciferase at different antibody concentrations, the ADCC activity of related antibodies can be evaluated. The ADCC effect is closely related to the affinity of the antibody antigen, the type of the Fc segment of the antibody, and the concentration of the antibody.

Case show: 

  • Prepare target cells and incubate with the antibody to be tested

  • Prepare effector cells

  • ADCC activity detection

  • Cycle 1-2 weeks

  • Basic information about antibodies

  • Target cell

  • Project Report