FAS ELISA Kit (APO-1, TNFRSF6 and CD95)

INTENDED USE

Human FAS ELISA kit can be used for analysing in vitro quantitative concentration of FAS (APO-1, TNFRSF6 and CD95) in human serum, cell culture supernatant and plasma. This assay has a minimum sensitivity limit of 18.75 pg/ml.

BACKGROUND

Fas can also be called CD95, TNFRSF6 and APO-1 and it belongs to the NGF/TNF receptor superfamily where it can mediate apoptosis. The mature protein is made up of 319 amino acids and has predicted molecular weight of 48 kDa. The extracellular domain is found to be rich in cysteine residue and it displays high similarity to human nerve growth factor receptor, B cell antigen CD40 and tumor necrosis factor receptor.

Fas has an important role in the formation of the death-inducing signalling complex (DISC) upon ligand binding. This results in a membrane anchored ligand trimer on the surface of an adjacent cell causing oligomerisation. Following the process of death domain (DD) aggregation, the receptor complex is then internalised through the cellular endosomal machinery. This permits the adaptor molecule (FADD) to bind the death domain via its own death domain. Recently, there are evidence to indicate that it has the ability to promote tumor growth, since during tumor progression, it is frequently downregulated or cells become rendered apoptosis resistant. There are also some reports which indicate that the extrinsic Fas-pathway can be sufficient to induce complete apoptosis in certain cell types through DISC assembly and subsequent caspase-8 activation.

CONTENT

All reagents supplied need to be stored at 2 °C – 8 °C, unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.

• One 96-Well Plate: Pre-coated with anti-TNFRSF6 antibody.
• Standards: Concentrations 0, 31.25, 62.5, 125, 250, 500, 1000, 2000 pg/ml, made up from lyophilized recombinant human FAS.
• Sample/Standard Dilution Buffer.
• Biotinylated-labelled TNFRSF6 Antibody.
• Antibody Dilution Buffer.
• HRP-Streptavidin Conjugate (SABC).
• SABC Dilution Buffer.
• TMB Substrate.
• Wash Buffer (25x).
• Plate Sealer.
• Product Instructions.

SENSITIVITY

The minimum detection sensitivity level of fas (CD95, APO-1, TNFRSF6) using this APO-1 ELISA kit was 18.75 pg/ml. The dynamic range for this assay is 31.25 – 2,000 pg/ml.

ASSAY CHARACTERISTICS

– Specificity: Highly specific for TNFRSF6, no cross reactivity or interference between TNFRSF6 and analogues was detected.
– Recovery: Serum (86 – 105%), EDTA Plasma (85 – 102%), Heparin Plasma (87 – 105%).
– Linearity: Serum (90 – 104%), EDTA Plasma (83 – 96%), Heparin Plasma (81 – 97%).
– Precison Intra-Assay: CV < 8%.
– Precison Inter-Assay: CV < 10%.
– Stability: Less than 10%.

REFERENCES

1. Lipid raft-mediated Fas/CD95 apoptotic signaling in leukemic cells and normal leukocytes and therapeutic implications. J Leukoc Biol. (2015) 98 (5): 739-59. Review. Gajate C. and Mollinedo F.
2. Fas and TRAIL ‘death receptors’ as initiators of inflammation: Implications for cancer. Semin Cell Dev Biol. (2015) 39: 26-34. Review. Cullen S.P. and Martin S.J.
3. Regulation of CD95/Fas signaling at the DISC. Cell Death Differ. (2012) 19 (1): 36-41. Review. Lavrik I.N. and Krammer P.H.
4. Fas-Fas Ligand: Checkpoint of T Cell Functions in Multiple Sclerosis. Front Immunol. (2016) 7: 382. Review. Volpe E., et al.
5. The many roles of FAS receptor signaling in the immune system. Immunity. (2009) 30 (2): 180-92. Review. Strasser A., et al.
6. Role of Fas/Fas-L in vascular cell apoptosis. J Cardiovasc Pharmacol. (2009) 53 (2): 100-8. Review. Stoneman V.E. and Bennett M.R.

ADDITIONAL INFORMATION

• Full Name: FAS ELISA Kit (APO-1, TNFRSF6 and CD95)
• Reactivity: Human
• Sample Type: Plasma, Biological Fluids, Serum, Tissue Homogenates
• Sensitivity: 18.75 pg/ml

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