Human S100A8/S100A9 Heterodimer Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human S100A8/S100A9 Heterodimer Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human S100A8/S100A9 Heterodimer in cell culture supernates, tissue lysastes, serum, plasma, saliva, urine, human milk, and fecal extract. It contains E. coli-expressed recombinant human S100A8/S100A9 Heterodimer and antibodies raised against the recombinant factor. Results obtained using natural human S100A8/S100A9 Heterodimer 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 S100A8/S100A9 Heterodimer.

Preparation and Storage

Background: S100A8/S100A9 Heterodimer

S100A8 (also MRP8 and calgranulin A) is a 10 kDa member of the S100 family, EF-hand superfamily of Ca-binding proteins. It is produced by neutrophils and monocytes, and forms Ca²⁺-dependent heterodimer/heterotetramer complexes (termed calprotectin) with S100A9. It functions both intracellularly and extracellularly, where it binds to RAGE and CD36. Human S100A8 is 93 amino acids (aa) in length. It contains two EF-hand motifs (aa 12 - 47 and 46 - 81) and one high-affinity Ca²⁺-binding site (aa 59 - 70). There may be one splice form that shows a 15 aa substitution for the C-terminal 14 amino acids. Although mouse S100A8 is cleaved by MMP-2 after Asn21, it is unclear if human S100A8 is susceptible. Full-length human S100A8 is 57% and 74% aa identical to mouse and canine S100A8, respectively.

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.