Human TRAIL R3 ELISA Assay

Additional Information

Summary

Human Trail R3, also called DcR1, CD263, LIT, or TRID is a glycosyl-phosphatidylinositol-linked membrane protein which binds TRAIL with high affinity. In the Trail receptor family, Trail R3 (DcR1) but also Trail R4 (DcR2) antagonize TRAIL-induced apoptosis whereas Trail R1 (DR4) and Trail R2 (DR5) transduce an apoptosis signal. Trail R3 has extracellular TRAIL-binding cystein-rich domain but lacks intracellular signalling domain. Expression of Trail R3 has been shown to protect cells bearing Trail R1 and/or Trail R2 from Trail-induced apoptosis. Many tumor cell lines don’t express the decoy receptors and are therefore sensitive to TRAIL if they express Trail R1 and/or Trail R2.

Assay Principle

A capture Antibody highly specific for TRAIL R3 has been coated to the wells of the microtiter strip plate provided during manufacture. Binding of TRAIL R3 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 R3 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 R3 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 R3 in any sample tested.

1. Add 100µl of Standard Diluent to zero wells and 100µl of Sample to sample wells
2. Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 2 hour(s)
3. Remove the cover and wash the plate
4. Add 50µl of diluted biotinylated anti TRAIL R3 to all wells
5. >Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 1 hour
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