Human CCL26/Eotaxin-3 Quantikine ELISA Kit Summary

Product Summary

The Quantikine^® Human CCL26/Eotaxin-3 Immunoassay is a 4.5 hour solid phase ELISA designed to measure human Eotaxin-3 levels in cell culture supernates, cell lysates, serum, and plasma. It contains E. coli-expressed, recombinant human Eotaxin-3 and antibodies raised against the recombinant factor. Results obtained for naturally occurring human Eotaxin-3 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 natural human Eotaxin-3.

Preparation and Storage

Background: CCL26/Eotaxin-3

Eotaxin-3 (also known as CCL26 or SCYA26) is a CC chemokine with potent chemotactic activity for eosinophils (1-4). It was originally cloned from the GenBank human expressed sequence tag (EST) database (1). Eotaxin-3, along with Eotaxin-1 and Eotaxin-2, selectively activates the CC chemokine receptor 3 (CCR3). The Eotaxin-3-CCR3 interaction may play an important role in allergic diseases such as atopic dermatitis and bronchial asthma (5). The full-length cDNA for Eotaxin-3 encodes a protein of 94 amino acids (aa) with a putative signal peptide of either 23 or 26 aa residues (1, 2). Both the 71 and 68 aa residue variants of recombinant Eotaxin-3 demonstrate equal potency in inducing chemotaxis of a human CCR3-transfected cell line (2). The mature Eotaxin-3 protein demonstrates 44% aa identity with MIP-1β and 40% aa identity with MIP-1α, RANTES and MCP-4 (1). Unlike most other CC chemokines, Eotaxin-3 maps to human chromosome 7q11.2, within 40 kilobases of the Eotaxin-2 loci (1, 2). Eotaxin-3 and Eotaxin-2 are unique in that they are the only chemokines identified to date that map to chromosome 7 (2, 6).

Like Eotaxin-1 and Eotaxin-2, Eotaxin-3 is a ligand for CCR3. The potency of Eotaxin-3 as a CCR3 ligand, however, is ten-fold less than that of Eotaxin-1 (2). CCR3 is a seven-transmembranespanning G-protein-linked receptor expressed on eosinophils, basophils, subpopulations of Th2 lymphocytes, and keratinocytes (5). Signal transduction via CCR3 is characterized by actin polymerization, a transient rise in cytosolic calcium concentration, and release of reactive oxygen species. Other chemokines capable of signaling through CCR3 include Eotaxin-1, Eotaxin-2, MCP-2, MCP-3, MCP-4, MIP-1δ, and RANTES. Studies indicate that Eotaxin-3 is capable of cross-desensitizing cells to other CCR3 ligands. Northern blot analysis demonstrates that Eotaxin-3 mRNA is constitutively expressed within the heart, liver, and ovary (1, 2). Low levels of Eotaxin-3 expression can also be detected in a variety of other tissues (1). Expression of Eotaxin-3 mRNA in vascular endothelial cells can be up-regulated by the cytokines IL-13 and IL-4 in a STAT6-dependent fashion (3, 7). In contrast to other potent eosinophil chemoattractants (i.e. Eotaxin-1, RANTES and MCP-4) that are induced by proinflammatory cytokines, neither TNF-α, IL-1β, IFN-γ, nor TNF-α plus IFN-γ are effective at up-regulating Eotaxin-3 mRNA expression. Eotaxin-3 appears to be important for contributing to eosinophil accumulation in atopic disease. The mRNA expression of Eotaxin-3, unlike Eotaxin-1 or Eotaxin-2, is up-regulated in asthmatics in the stages following allergen challenge (8). These data suggest the possibility that Eotaxin-3 may be responsible for the continuing recruitment of eosinophils to asthmatic airways during this period. The Quantikine® Human CCL26/Eotaxin-3 Immunoassay is a 4.5 hour solid phase ELISA designed to measure human Eotaxin-3 levels in cell culture supernates, cell lysates, serum, and plasma. It contains E. coli-expressed, recombinant human Eotaxin-3 and antibodies raised against the recombinant factor. Results obtained for naturally occurring human Eotaxin-3 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 natural human Eotaxin-3.

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.