Human PD-L2/B7-DC DuoSet ELISA Summary

Product Summary

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human PD-L2 / B7-DC. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet ELISA.

Preparation and Storage

Background:  PD-L2/B7-DC

PD-L2 (Programmed Death Ligand 2), also known as B7-DC and butyrophilin-like protein, is a transmembrane protein expressed on dendritic cells, subsets of activated CD4+ and CD8+ T cells, and memory B cells that differentiate into plasma cells. At inflammatory sites, PD-L2 is upregulated on synoviocytes, infiltrating macrophages, dendritic cells, and airway epithelial cells. PD-L2, along with B7-H1/PD-L1, binds to T cell PD-1 where it promotes IFN-gamma production and CD40 Ligand upregulation while inhibiting IL-4 production. In addition, PD-L2 binds to RGM-B on macrophages and alveolar epithelial cells, supporting respiratory immune tolerance. In asthma, PD-L2 suppresses IL-5 and IL-13 production, promotes IL-12 production by dendritic cells, and supports allergen-induced airway hyper-responsiveness and mucus production.

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.