Human/Cynomolgus Monkey PD-L1/B7-H1 Quantikine ELISA Kit Summary

Product Summary

The Quantikine^® Human/Cynomolgus Monkey B7-H1/PD-L1 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human/cynomolgus monkey B7-H1 in cell culture supernates, cell lysates, serum, plasma, and urine. It contains HEK293-expressed recombinant human B7-H1/PD-L1 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human/cynomolgus monkey B7-H1/PD-L1 showed linear curves that were parallel to the standard curves obtained using the Quantikine® kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human/cynomolgus monkey B7-H1/PD-L1.

Preparation and Storage

Background: PD-L1/B7-H1

PD-L1, also known as B7-H1 and CD274, is an approximately 65 kDa transmembrane glycoprotein in the B7 family of immune regulatory molecules. PD-L1 is expressed on inflammatory-activated immune cells including macrophages, T cells, and B cells, keratinocytes, enothelial and intestinal epithelial cells, as well as a variety of carcinomas and melanoma. PD-L1 binds to T cell B7-1/CD80 and PD-1. It suppresses T cell activation and proliferation and induces the apoptosis of activated T cells. It plays a role in the development of immune tolerance by promoting T cell anergy and enhancing regulatory T cell development. PD-L1 favors the development of anti-inflammatory IL-10 and IL-22 producing dendritic cells and inhibits the development of Th17 cells. In cancer, PD-L1 provides resistance to T cell mediated lysis, enhances EMT, and enhances the tumorigenic function of Th22 cells.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.