< 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.
No significant interference observed with available related molecules.
The Quantikine Mouse Vitamin D BP Immunoassay is a 2.5 hour solid phase ELISA designed to measure mouse Vitamin D BP levels in cell culture supernates, tissue lysates, serum, plasma, and urine. It contains NS0-expressed recombinant mouse Vitamin D BP and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate the recombinant mouse Vitamin D BP accurately. Results obtained using natural mouse Vitamin D BP showed dose-response curves that were parallel to the standard curves obtained using the recombinant kit standards. These results indicate that this kit can be used to determine relative mass values for natural mouse Vitamin D BP.
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.
The recovery of mouse Vitamin D BP spiked to three levels throughout the range of the assay was evaluated.
Average % Recovery
Cell Culture Media (n=4)
To assess the linearity of the assay, samples containing high concentrations of mouse Vitamin D BP were serially diluted with Calibrator Diluent to produce samples with values within the dynamic range of the assay. Samples were diluted prior to assay.
Preparation and Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
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.
Refer to the product for 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.
Prepare all reagents, standard dilutions, and samples as directed in the product insert.
Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
100 µL Standard, Control, or Sample
Add 100 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 1 hour on a horizontal orbital microplate shaker.
Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.
100 µL Conjugate
Add 100 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 1 hour on the shaker.
Aspirate and wash 4 times.
100 µL Substrate Solution
Add 100 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes on the benchtop. PROTECT FROM LIGHT.
100 µL Stop Solution
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.
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