Human TRAIL R2 ELISA Assay

Additional Information

Assay Background

Human Trail R2, also called DR5 (Death Receptor 5), CD262, TRICK 2 or Apo2 is a TNF receptor, which is a receptor for Trail (APO2 ligand). Trail R2 is involved in apoptosis. In the Trail receptor family, Trail R2, but also Trail R1 (DR4) transducer an apoptosis signal, whereas Trail R3 (DcR1) and Trail R4 (DcR2) antagonize TRAIL-induced apoptosis. Binding of trimeric Trail to Trail R1 induces apoptosis by oligomerization (likely to be trimerization) of the receptor. Trail R2 cDNA encodes a 440 amino acid residue precursor protein containing extracellular cysteine-rich domains, a transmembrane domain and a cytoplasmic death domain. Trail R2 shares 55% of its amino acid sequence with Trail R1.Like DR4, DR5 transcript is widely expressed in normal tissues and in many types of tumor cells.

Assay Principle

A capture Antibody highly specific for the Eagle Biosciences TRAIL R2 has been coated to the wells of the microtitre strip plate provided during manufacture. Binding of TRAIL R2 in samples and known standards to the capture antibodies is completed and then any excess unbound analyte is removed. During the next incubation period the binding of the biotinylated anti- TRAIL R2 secondary antibody to the analyte occurs. Any excess unbound secondary antibody is then removed. The HRP conjugate solution is then added to every well including the zero wells, following incubation excess conjugate is removed by careful washing. A chromogen substrate is added to the wells resulting in the progressive development of a blue coloured complex with the conjugate. The colour development is then stopped by the addition of acid turning the resultant final product yellow. The intensity of the produced coloured complex is directly proportional to the concentration of TRAIL R2 present in the samples and standards. The absorbance of the colour complex is then measured and the generated OD values for each standard are plotted against expected concentration forming a standard curve. This standard curve can then be used to accurately determine the concentration of TRAIL R2 in any sample tested.

Assay Procedure

1. Add 100µl of standard diluent to zero wells and 100µl of Samples to sample wells.
2. Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 1 hour(s).
3. Remove the cover and wash the plate.
4. Add 50µl of diluted biotinylated anti TRAIL R2 to all wells.
5. Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 1 hour(s).
6. Repeat wash step 4.
7. Add 100μl of Streptavidin-HRP solution into all wells.
8. Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 30 min.
9. Repeat wash step 4.
10. Add 100μl of ready-to-use TMB Substrate Solution into all wells.
11. Incubate in the dark for 10-20 minutes* at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil.
12. Add 100μl of H2SO4:Stop Reagent into all wells

Typical Standard Curve