Mouse CCL3/MIP-1 alpha Quantikine ELISA Kit Summary

Product Summary

The Quantikine Mouse MIP-1 alpha Immunoassay is a 4.5 hour solid phase ELISA designed to measure mouse MIP-1 alpha levels in cell culture supernates and serum. It contains E. coli-expressed recombinant mouse MIP-1 alpha and antibodies raised against the recombinant protein. Results obtained for naturally occurring mouse MIP-1 alpha 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 mouse MIP-1 alpha.

Preparation and Storage

Background: CCL3/MIP-1 alpha

MIP-1 alpha (macrophage inflammatory protein 1 alpha) is a member of the CC or beta chemokine subfamily that was originally purified from the conditioned media of an LPS-stimulated murine macrophage cell line. MIP-1 alpha acts as a chemoattractant to a variety of cells including monocytes, T cells, B cells and eosinophils.

The two human MIP-1 alpha genes arise by duplication/mutation. They code for MIP-1 alpha isoforms CCL3/LD78a and CCL3L1/LD78b, which share 94% amino acid sequence homology. Whereas the human CCL3/LD78a is a single-copy gene, the human CCL3L1/LD78b gene copy number varies within the population. Human CCL3L1/LD78b binds and signals through chemokine receptors CCR1, CCR5. When compared to CCL3/LD78a, CCL3L1/LD78b has higher binding affinity to CCR5, which also functions as a coreceptor for HIV-1 entry. The copy number of CCL3L1 is one of several genetic determinants of HIV-1 susceptibility.

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.