Mouse/Rat CCL5/RANTES Quantikine ELISA Kit Summary

Product Summary

The Quantikine Mouse/Rat CCL5/RANTES Immunoassay is a 4.5 hour solid phase ELISA designed to measure mouse or rat RANTES levels in cell culture supernates, serum, and platelet-poor plasma. It contains E. coli-expressed recombinant mouse RANTES as well as antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate recombinant RANTES. Results obtained using natural mouse or rat RANTES showed linear curves that were parallel to the standard curves obtained using the recombinant kit standards. These results indicate that this kit can be used to determine relative mass values for natural mouse and rat RANTES.

Preparation and Storage

Background: CCL5/RANTES

RANTES (Regulated upon Activation, Normal T cell-expressed, and presumably Secreted), also known as CCL5, a member of the CC chemokine family of inflammatory and immunoregulatory cytokines, initially discovered by subtractive hybridization as a T cell-specific molecule (1, 2). Mouse RANTES cDNA encodes a 91 amino acid (aa) residue precursor protein with a presumed signal peptide of 23 aa residues that is cleaved to generate the 68 aa residue mature protein (3). At the amino acid sequence level, mouse RANTES is 84% identical to human RANTES. Both human and mouse RANTES exhibit cross species activity. Naturally occurring human RANTES has been found to be a mixture of the 68 aa residue mature protein and a 66 aa residue amino-terminally truncated form (4, 5). Cells known to express RANTES include keratinocytes, eosinophils, platelets, endothelium, fibroblasts, respiratory epithelium, astrocytes, vascular smooth muscle, and CD4⁺ and CD8⁺ T cells (6-14). As suggested by its acronym, RANTES production by the various cell types is induced in response to cytokine stimulation (8-13). 

Like other CC chemokines, RANTES is a monocyte chemoattractant (2). RANTES can also chemoattract unstimulated CD4⁺/CD45RO⁺ memory T cells and stimulated CD4⁺ and CD8⁺ T cells with the naive and memory phenotypes. In addition, RANTES can chemoattract and degranulate eosinophils, as well as chemoattract and induce histamine release from basophils. Human RANTES has been shown to be an inhibitor of HIV infection of human mononuclear cells (15, 16). Several CC chemokine receptors, including CCR-1, CCR-3, CCR-4, and CCR-5, have been shown to bind RANTES and subsequently to transduce a signal by increasing the intracellular calcium ion level (2, 17-21).

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.