Fig 1: Antibody-dependent cell-mediated cytotoxicity (ADCC)

ADCC is a simple but important mechanism for the immune system to target diseased or infected cells. Antibodies bind to specific antigens on the surface of the target cell (see Fig 1).  PBMCs or natural killer (NK) cells, express Fc receptors on their cell surface and act as the effector cells. Interaction between the Fc region of the antibody and the Fc receptor induces the effector cell to degranulate, releasing IFN-γ, granzymes, and other cytotoxic compounds that lyse the target cell.

ADCC is not only a natural part of the adaptive immune response, but animal experiments have shown that it can also be seen as an important mechanism of action of therapeutic monoclonal antibodies (1), including the breast cancer drug trastuzumab, and rituximab, a drug used to treat diseases which show overactive, dysfunctional, or excessive numbers of B cells (e.g. lymphomas).

Cell lines to build up cellular ADCC screening assays

Fig 2: Principle of BPS’s ADCC cell lines

To enable researchers to build up a cellular ADCC screening system, BPS Biosciences have developed 2 reporter cell lines, which can replace NK cells or PBMCs in such a cellular assay (see Fig 2). The system is based on Jurkat cells that stably express human FcγRIIIa (CD16a), the receptor for the Fc region of human IgG. The FcγRIIIa on the Jurkat cells binds to the IgG on the surface of the target cell. This crosslinking causes the Jurkat cells to activate NFAT transcription, which induces the expression of luciferase and can be easily detected using the ONE-Step™ Luciferase Detection Reagents.

The effectiveness of ADCC depends on how well the effector cells are activated after the engagement of FcγRIIIa. Human FcγRIIIa displays dimorphism at amino acid 158 – one allele (V158) encodes a high Fc affinity receptor variant, while the other (F158) encodes a lower Fc affinity receptor variant.  BPS offers 2 different ADCC cell lines expressing either of these Fc receptors to allow selective antibody binding analyses using each type of receptor.

• ADCC Bioassay Effector Cell, F variant (Low Affinity)
• ADCC Bioassay Effector Cell V variant (High Affinity)

Get more information about our ADCC cell lines – just leave your questions or comments in the form below!

Reference:

(1) Clynes, RA, Towers, TL, Presta, LG, Ravetch, JV; Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets; Nat Med. 6 (4): 443-446 (2000)

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Detecting ubiquitylation

Ubitest Flow chart – LifeSensors at tebu-bio.com

Determining the linkage of polyubiquitin on target proteins is challenging. The traditional methods are either through Mass Spectrometry or immunoblot with linkage specific antibodies, which are cumbersome. LifeSensors has developed the UbiTest assay,  a more definitive method for demonstrating the ubiquitylation linkage of a protein, which is to couple immunoprecipitation of polyubiquitylated protein with digestion by a linkage specific deubiquitylase prior to immunoblot analysis. An increased signal for the unmodified substrate or a decreased signal of polyubiquitylated substrate at high molecular weight after K48/K63 specific DUB treatment is a clear indication that the protein was K48/K63 ubiquitylated.

Are you working on autophagy,? Or on GPCR de-orphaning? On the MAP kinase pathway or cellular metabolism? Maybe on pathways related to cellular metabolism?

Several compounds are known as modulators of these pathways and research fields and can be used as useful tools to study and characterize specific steps. Nonetheless, it is quite time consuming and expensive to order all these chemicals from different sources. Targeted compound libraries represent a convenient alternative.

Fig 1: CD39 and CD73 leading to elevated levels of extracellular adenosine.

Extracellular adenosine 5′-triphosphate (ATP) is released by dying and damaged cells, and it acts on many immune cells to promote inflammation. On the other hand, the unphosphorylated
metabolite, adenosine, functions as an anti-inflammatory molecule. Two extracellular ecto-5´-Nucleotidases, CD39, and CD73, convert extracellular ATP to ADP/AMP and AMP to adenosine, respectively, leading to elevated levels of extracellular adenosine (Fig 1).