Human CCL5/RANTES Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human RANTES Immunoassay is a 3.5 hour solid phase ELISA designed to measure human RANTES levels in cell culture supernates, serum, plasma, and urine. It contains E. coli-derived recombinant human RANTES and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant factor. Results obtained using natural human RANTES 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 of natural human RANTES.

Preparation and Storage

Background: CCL5/RANTES

RANTES (Regulated upon Activation, Normal T cell Expressed and presumably Secreted), also known as CCL5, is a member of the "CC" subfamily of chemokines. It plays a primary role in the inflammatory immune response via its ability to chemoattract leukocytes and modulate their function. The cDNA for RANTES was initially discovered by subtractive hybridization as a T cell specific sequence (1, 2). Human RANTES cDNA encodes a highly basic 91 amino acid (aa) residue precursor polypeptide with a 23 aa hydrophobic signal peptide that is cleaved to generate the 68 aa mature protein (1, 2). Human RANTES exhibits approximately 85% homology with mouse RANTES at the deduced aa level (3, 4). 

RANTES is a potent chemoattractant for a number of different cell types including unstimulated CD4⁺/CD45RO⁺ memory T cells and stimulated CD4⁺ and CD8⁺ T cells with naive and memory phenotypes, NK cells, basophils, eosinophils, dendritic cells, mast cells, monocytes, and microglia (5-13). In addition to its effects on migration, RANTES can activate a number of cell types including T cells (14-16), monocytes (17), neutrophils (17), NK cells (7), dendritic cells (18), and astrocytes (19). T cell activation generally requires relatively high RANTES concentrations (~ 1 μM) and is dependent upon aggregation of the molecule and association with cell surface glycosaminoglycans (GAGs) (15-17). Whether this activity occurs in vivo remains unclear although in mice, intraperitoneally injected RANTES mutants that are unable to aggregate and/or bind GAG, are not capable of attracting leukocytes when compared to wild-type controls (20). Other in vivo studies show that RANTES knockout mice exhibit deficient recruitment of leukocytes to sites of acute inflammation (21). 

RANTES, is known to interact with four identified seven transmembrane G-protein coupled receptors: CCR1, CCR3, CCR4, and CCR5 (22-25). RANTES stimulation can initiate a variety of signaling cascades that are cell context dependent. For instance, in T-cells, RANTES can stimulate elevations of intracellular Ca²⁺ (26), and activation of focal adhesion kinase (FAK) (27), protein kinase A (28), PI3-kinase (14), Rho GTPase (29), and JAK/STAT signaling pathways (30). The cytomegalovirus protein US28 exhibits significant homology with CC chemokine receptors and is capable of binding RANTES (31). Membrane-spanning US28 can, depending on the context, signal in a constitutive manner (32), bind RANTES and initiate G-protein-mediated signaling cascades (33), or sequester RANTES and potentially alter inflammatory responses (34-36). 

The RANTES receptor CCR5 is also the primary co-receptor for R5 (M-tropic) variants of HIV-1 (37, 38). It has been demonstrated that RANTES, as well as the other CCR5 ligands, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, can competitively inhibit CCR5/HIV-1 interaction and suppress viral infection in vitro (39, 40). These effects apparently do not require fully intact signaling from the CCR5 receptor (41). Consequently, modified forms of RANTES and non-peptide compounds that block the interaction of HIV-1 with CCR5 show promise for future therapies (41-44). In contrast, several reports show that RANTES can enhance in vitro replication of X4 (T-tropic) variants of HIV-1 that use CXCR4 as a co-receptor rather than CCR5 (45, 46). This activity usually requires relatively high RANTES concentrations (~μM) and is dependent upon interaction with cell surface GAGs, oligomerization, and activation of tyrosine kinase and MAP kinase signaling cascades (46, 47).

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.