Human Vitamin D BP Quantikine ELISA Kit Summary

Product Summary

The Quantikine^® Human Vitamin D BP immunoassay is a 3.5 hour sandwich-type solid phase ELISA designed to measure human Vitamin D BP in cell culture supernates, serum, plasma, and urine. It contains HEK293-expressed recombinant human Vitamin D BP and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human Vitamin D BP 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 Vitamin D BP.

Preparation and Storage

Background: Vitamin D BP

Vitamin D binding protein (VitD BP), also known as DBP and Gc-globulin, is a 58 kDa glycoprotein that circulates at high concentration in the serum and serves as a carrier protein for vitamin D. The transport of vitamin D by VitD BP is important for the function of a wide variety of tissues. VitD BP binds both the 25(OH) and the hormonally active 1,25(OH)2 forms of vitamin D. VitD BP is primarily expressed in hepatocytes and to a lesser extent in the kidney. It delivers vitamin D into cells by Megalin-mediated endocytosis. A selectively deglycosylated form of VitD BP known as macrophage activating factor (MAF) blocks the angiogenic effects of FGF basic, VEGF, and Angiopoietin 2. VitD BP enhances the chemotaxis of monocytes and neutrophils to the activated complement component C5a or C5a des Arg (a C-terminally processed form of C5a). The chemotactic cofactor property of VitD BP is eliminated by binding to 1,25(OH)2 vitamin D, but it is not altered by binding to 25(OH) vitamin D or actin.

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.