Mouse IFN gamma Assay KIT For mIFNg detection in cell supernatants DELPHINE JAGA CATHY DREXLER December 2012 –V0 April 2013 –V0 Sales training

Mouse IFN gamma assay (V0) IFNg background IFNg (type II interferon) is a glycoprotein homodimer (20-25kDa) produced by a variety of cells : NK, monocytes/macrophage, B cells, mast cells, dendritic cells. 38-44% identity between mouse and human, canine, equine, cotton rat. IFNg plays a key role in host defense by exerting anti viral, anti proliferative and immunoregulatory activities. On many cell types, IFNg induces the production of cytokines and upregulates the expression of various membrane proteins (MHC class I&II, Fc receptors, leucocyte adhesion molecules…) IFNg key functions: • Influences T helper cells phenotype by stimulating Th1 and inhibiting Th2. • Activates NK, Ag presentation & lysosomes activity of macrophages • Activates IFN g receptor (heterodimer1&2) and JAK-STAT signaling pathway. • It is used to treat chronic granulomatous diseases (=Bridges-Good Syndrome) and osteopetrosis (marble bone disease). Mouse IFN gamma assay (V0)

Target Customer Who Cares High probability users: • Biotechs and Pharmas working on inflammation and measuring the concentration of mouse IFNg in cell supernatants • This kit can be not sensitive enough to quantify in serum or plasma • Therapeutic area of interest: auto-immunity, oncology, infectious disease, allergy. Identify customers / Key questions to ask: • Do you quantify cytokine biomarkers using established mouse or human cell lines? • What is your reference technology (ELISA, AlphaLISA, MSD)? • What is the panel of cytokines which fits to your needs? Mouse IFN gamma assay (V0)

Store the kit at -20°C upon reception Mouse IFN gamma assay (V0) Mouse IFNg assay Included in the kit: - 1 vial of mIFNg standard - 1 vial of anti-mIFNg -d2 - 1 vial of anti-mIFNg -K - 1 vial of diluent - 1 vial of conjugate buffer 1000 tests (20µL basis) Ref 6FMIFPEB 10 000 tests (20µL basis) Ref 6FMIFPEH Store the kit at -20°C upon reception Mouse IFN gamma assay (V0)

Mouse IFN gamma assay (V0) Assay Principle Mouse IFNg Anti-Mouse IFNg Cryptate conjugate d2 conjugate This assay is designed for the quantitative determination of Mouse IFNg. Mouse IFNg is measured in a sandwich assay format using 2 different specific monoclonal antibodies, one labelled with cryptate (donor) and the second with d2 (acceptor). The intensity of the signal obtained is proportional to the number of antigen-antibody complexes formed and therefore the fluorescence is proportional to the concentration of the antigen. Mouse IFN gamma assay (V0)

Mouse IFN gamma assay (V0) Assay protocol The reagents are dispensed as per the order shown below in a white plate (384 wells small volume) Incubate 2h at 18-22°C Read on an HTRF® compatible reader 16 µL Sample or standard 2 µL Anti-Mouse IFNg-d2 conjugate 2 µL Anti-Mouse IFNg-K conjugate (Or 4 µL pre-mixed) Mouse IFN gamma assay (V0)

Mouse IFN-g Standard curve The experiment was run in diluent and read on a PHERAstar FS Laser. Mouse IFN gamma assay (V0)

Analytical characteristics Detection limit = 30 pg/mL Linear range study 30 to 2000 pg/mL Incubation time 2h at 18-22°C Mouse IFN gamma assay (V0)

Linearity Limit - Hook effect Mouse IFN gamma assay (V0)

Validation on cell based assay at NIH (USA) (384 LV well plate) Customer data - CONFIDENTIAL for internal use only Mouse IFN gamma assay (V0)

Customer data - CONFIDENTIAL for internal use only Validation on cell based assay at NIH (USA) (1536 well plate) Customer data - CONFIDENTIAL for internal use only Mouse IFN gamma assay (V0)

Mouse IFN gamma assay (V0) Value proposition Easy to run and faster process: • 1 single incubation step • cell-based (culture media compatible) Cost effective: • cheaper compared to our competitors High robustness: • signal stability • low interferences • low CVs Mouse IFN gamma assay (V0)

Associated HTRF® products hIL1β kit (62IL1PEB /C) hIL2 kit (64IL2PEB/C) hIL4 kit (63ADKPEBU006/CU006) hIL5 kit (63ADKPEBU007/PECU007) mouse & hIL6 kit (63ADKPEBU043/CU043 & 62IL6PEB /C) hIL8 kit (62IL8PEB /C) hIL10 kit (6FH10PEB /C) mouse & hIL17 kits (64M17PEB /C & 64H17PEB/C) hIL12 (6FH12PEB/H) mouse & hTNFα kit (6FMTNPEB/H & 62TNFPEB /C) mouse & hIFNγ kit (6FMIFPEB /H & 62IFNPEB /C) hGROa (CXCL1) (6FGROPEG/K) Mouse IFN gamma assay (V0)

Mouse IFN gamma assay (V0) At launch Website update (Dec 16) Biomarker section page New page for m & hIFNg Cytokine flyer updated  pakage insert and MSDS are on request Mouse IFN gamma assay (V0)

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