Human CCL4/MIP-1 beta Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human MIP-1 beta Immunoassay is a 3.0 or 4.5 hour solid phase ELISA designed to measure MIP-1 beta in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human MIP-1 beta (Act-2 variant) and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant human MIP-1 beta. Results obtained using natural human MIP-1 beta 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 MIP-1 beta.

Preparation and Storage

Background: CCL4/MIP-1 beta

CCL4/MIP-1 beta is a beta chemokine that is secreted at sites of inflammation by activated leukocytes, lymphocytes, vascular endothelial cells, and pulmonary smooth muscle cells. It attracts a variety of immune cells to sites of microbial infection as well as to other pathologic inflammation such as allergic asthma and ischemic myocardium. CCL4 is secreted from activated monocytes as a heterodimer with CCL3/MIP-1 alpha. It signals through CCR5, and an N-terminally trimmed form additionally interacts with CCR1 and CCR2. In humans, the ability of CCL4 to bind CCR5 inhibits the cellular entry of M-tropic HIV-1 which utilizes CCR5 as a coreceptor.

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.