Human CXCL5/ENA-78 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human ENA-78 Immunoassay is a 4.5 hour solid phase ELISA designed to measure human ENA-78 in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human ENA-78 and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate the recombinant human ENA-78 accurately. Results obtained using natural human ENA-78 showed dose curves that were parallel to the standard curves obtained using the recombinant Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural human ENA-78.

Preparation and Storage

Background: CXCL5/ENA-78

CXCL5/Epithelial Cell-derived Neutrophil-activating Peptide (ENA-78), is a member of the CXC subfamily of chemokines. Full-length CXCL5/ENA-78 is 114 amino acids (aa) in length with a predicted molecular weight of 12 kDa. Following the removal of the signal peptide, bioactive CXCL5/ENA-78 is 78 aa in length. CXCL5/ENA-78 can be N-terminally cleaved by Cathepsin G and Chymotrypsin to CXCL5/ENA-74 (74 aa) and CXCL5/ENA-70 (70 aa), which show increased potency relative to CXCL5/ENA-78. While murine LIX was thought to be an ortholog to human CXCL5/ENA-78, genome-wide analysis and a consensus in the field suggests that human CXCL5/ENA-78 does not have a true murine ortholog. CXCL5/ENA-78 is upregulated at sites of inflammation and is expressed by multiple hematopoietic cell types, fibroblasts, endothelial cells, vascular smooth muscle cells, and adipocytes. It acts as a chemoattractant for neutrophils, has angiogenic properties, and contributes to cancer progression.

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.