Human CD14 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human soluble CD14 Immunoassay is a 4.5 hour solid phase ELISA designed to measure human soluble CD14 in cell culture supernates, serum, and plasma. It contains recombinant human CD14 expressed from CHO cells and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant factor. Results obtained measuring natural human sCD14 showed dose-response curves that were parallel to the standard curves obtained using the kit standards. These results indicate that this kit can be used to determine relative mass values for natural human sCD14.

Preparation and Storage

Background: CD14

CD14 is a glycoprotein that mediates the interaction of lipopolysaccharide (LPS, endotoxin)with cells, thereby signaling the presence of gram-negative bacteria (1-3). CD14 is eithersoluble (CD14) (4, 5) or membrane-bound (mCD14) by a glycosylphosphatidylinositol (GPI)anchor (6, 7). mCD14 is a 55 kDa glycoprotein (1), while CD14 varies from about 43 to 53 kDa,depending on the degree of glycosylation and whether it was synthesized without the anchoror was shed by phospholipase cleavage of the anchor or by proteolysis (12-14). There is noevidence for different mRNAs for m- and CD14. There is no apparent sequence homology withother proteins. The sequence of human CD14 is 63-73% identical to that of mouse, rat, or rabbitCD14 (15).

mCD14 is expressed primarily on myeloid cells, such as monocytes, macrophages andneutrophils (1-3), the cells most sensitive to LPS, and to a lesser extent on other cells, such as Bcells (8) and a circulating dendritic cell progenitor (9). CD14 appears to mediate LPS stimulationof cells that do not express mCD14 (10, 11), such as endothelial, epithelial and smooth-musclecells. CD14 is found in both serum and urine (5).

The binding of LPS to CD14 requires an acute phase protein, LPS-binding protein (LBP) (16).The relationship of mCD14, CD14, LPS and LBP is complicated. At low concentrations of LPS,LBP is essential for the binding of LPS to CD14, but at high concentrations, LBP may actuallyinhibit binding of LPS to CD14. In addition, CD14 may compete with mCD14 for LPS (17) andmay serve to help clear LPS (18). These four factors thus appear to participate in a complexfeedback mechanism of immune regulation involving both up-regulation and down-regulationof the inflammatory process triggered by LPS. It is loss of control of this mechanism thatappears to lead to septic shock. LPS-bound CD14 signals production of inflammatory cytokinesand other inflammatory proteins, but the mechanism of signal transduction is unclear. Sincea GPI anchor is not transmembrane, there presumably is another transmembrane protein oncells through which LPS-bound CD14 transmits a signal (19).

In addition to its well known role in gram-negative infections, CD14 likely serves otherfunctions as well. It recognizes soluble peptidylglycan from gram-positive cell walls (20), and ithas been reported to bind apoptotic cells and induce their phagocytosis (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.