Sample
Cell culture supernates, tissue lysates, serum, plasma, saliva, urine, human milk, fecal extract
Intended Use
For the quantitative determination of human S100A8/S100A9 Heterodimer concentrations in cell culture supernates, tissue lysates, serum, plasma, saliva, urine, human milk, and fecal extract.
Contents of Kit
1.Human S100A8/S100A9 Heterodimer Microplate, 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody specific for human S100A8/S100A9 Heterodimer. Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 1 month at 2-8 °C.*
2.Human S100A8/S100A9 Heterodimer Standard, 2 vials of recombinant human S100A8/S100A9 Heterodimer in a buffered protein base with preservatives; lyophilized. Refer to the vial label for reconstitution volume. Discard after use. Use a new standard for each assay.
Once opened, the folowing Reagents may be stored for up to 1 month at 2-8 °C.*
3.Human S100A8/S100A9 Heterodimer Conjugate, 21 mL of a monoclonal antibody specific for human S100A8/S100A9 Heterodimer conjugated to horseradish peroxidase with preservatives.
4.Assay Diluent RD1-34, 11 mL of a buffered protein base with preservatives.
5.Calibrator Diluent RD5-10, 3 vials (21 mL/vial) of a buffered protein base with preservatives.
6.Wash Buffer Concentrate, 21 mL of a 25-fold concentrated solution of buffered surfactant with preservative. May turn yellow over time.
7.Color Reagent A, 12 mL of stabilized hydrogen peroxide.
8.Color Reagent B, 12 mL of stabilized chromogen (tetramethylbenzidine).
9.Stop Solution, 6 mL of 2 N sulfuric acid.
10.Plate Sealers, 4 adhesive strips.
* Provided this is within the expiration date of the kit.
Storage
Store the unopened kit at 2-8 °C. Do not use past kit expiration date.
Precision
Intra-assay Precision (Precision within an assay)
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay Precision (Precision between assays)
Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.
Sensitivity
Twenty-one assays were evaluated and the minimum detectable dose (MDD) of human S100A8/S100A9 Heterodimer ranged from 0.005-0.215 ng/mL. The mean MDD was 0.086 ng/mL.
The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
General Description
S100A8 (also known as MRP8, Calgranulin A, and CP-10) and S100A9 (also known as MRP14 and Calgranulin B) are pro-inflammatory members of the S100 family of secreted calcium binding proteins (1, 2). The 10 kDa human S100A8 and 14 kDa S100A9 each contain two EF-hand calcium binding motifs. Human S100A8 shares 57% and 61% amino acid (aa) sequence identity with mouse and rat S100A8, respectively. Human S100A9 shares 57% and 62% amino acid sequence identity with mouse and rat S100A9, respectively (3, 4). In the presence of calcium or zinc, S100A8 and S100A9 can associate into disulfide-linked homodimers and 34-35 kDa heterodimers known as S100A8/S100A9 Heterodimer or Calprotectin (5-8).
S100A8 and S100A9 are upregulated in neutrophils, monocytes, Schwann cells, and keratinocytes at sites of inflammation (9-15). The S100A8/S100A9 Heterodimer is elevated in rheumatoid arthritis synovial fluid and in the serum of cardiovascular disease, atopic dermatitis, and psoriatic arthritis patients (1, 11, 15-17). Its levels correlate with body adiposity and leukocyte count (18). S100A8/S100A9 Heterodimer exerts its effects through the receptors RAGE and TLR4 (13, 19). The heterodimer promotes neutrophil infiltration into sites of inflammation and inflammatory cytokine production by monocytes (6, 10, 11, 14, 20). It promotes bone spur formation in osteoarthritis (21), upregulation of Complement Component C3 (20), astrocyte proliferation (19), and the suppression of tumor growth by promoting the influx and activation of NK cells (12, 13). The S100A8/S100A9 Heterodimer additionally binds to fatty acids such as arachidonic acid (9), and it sequesters manganese, thereby restricting the growth of Mn-dependent bacteria (22).
The Human S100A8/S100A9 Heterodimer ELISA Kit 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 kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human S100A8/S100A9 Heterodimer.
Standard Curve
This standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed.
Citations
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