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Analysis Kinetics of anti-CD39 Clone A1 mAb by BLI (CAT#: STEM-MB-0290-CJ)

Introduction

CD39, encoded by the gene ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1), is an integral membrane protein that metabolizes extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to adenosine monophosphate (AMP). CD39 appears to be the major extracellular enzyme catabolizing ATP in the tumor microenvironment (TME). CD39 is constitutively expressed on immune cell populations, such as B cells, natural killer cells, dendritic cells, Langerhans cells, monocytes, macrophages, mesangial cells, neutrophils, activated T cells, and regulatory T cells (Tregs), as well as endothelial cells. During chronic viral infection in humans and in a mouse model, CD39 is a CD8+ T cell exhaustion marker. In an oncology setting, CD39 is highly expressed by tumor-infiltrating lymphocytes, especially Tregs, exhausted CD8+ T cells, and myeloid-derived cells such as myeloid-derived suppressor cells. In addition, CD39 also is the rate-limiting enzymatic step in the production of immunosuppressive adenosine, and should therefore be viewed as an immunological switch that shifts ATP-driven pro-inflammatory immune cell activity toward an anti-inflammatory, immunosuppressed state mediated by adenosine.




Principle

Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time.

Applications

Immunology/Inflammation

Procedure

1. Detect Buffers and prepare samples. BLI experiments are set up with one molecule immobilised on the surface of the biosensor (load sample) and a second molecule in solution (the analytical sample).
2. Fix the load sample on the biocompatible biosensor while the analytical sample is in solution.
3. The biosensor tip is immersed in the solution so that the target molecule begins to bind to the analysis sample.
4. Set up and run the BLI experiment. Molecules bound to or dissociated from the biosensor can generate response curves on the BLI system; unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interferogram pattern.
5. Collect and analyse data on the BLI's system.

Materials

• Equipment: Fortebio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: Mouse anti-CD39, Guinea pig anti-human CD39, Polyclonal rabbit anti-ALPL (TNAP) antibody
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