Solid Phase Sandwich ELISA
96-well strip plate
7.81 - 500 pg/mL (Cell Culture Supernates, Serum, Heparin Plasma,)
Natural and recombinant mouse Shh-N. This kit also recognizes low levels of Shh-N in rat serum.
< 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 Sonic Hedgehog N-Terminus Immunoassay is a 4.5 hour solid phase ELISA designed to measure Shh-N in cell culture supernates, serum, and plasma. It contains recombinant mouse Shh-N and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate the recombinant factor accurately. Results obtained using natural mouse Shh-N showed dose-response curves that were parallel to the standard curves obtained using the recombinant Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural mouse Shh-N.
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested in separate assays to assess inter-assay precision.
Inter-Assay Precision (Precision between assays Three samples of known concentration were tested on one plate to assess intra-assay precision.Cell Culture Supernates, Serum, Heparin Plasma
|Standard Deviation||1.08||1.31||10.6||2.28||2.96||9.21 |
The recovery of mouse Shh-N spiked to levels throughout the range of the assay in various matrices was evaluated.
|Sample Type ||Average % Recovery ||Range % |
|Cell Culture Samples (n=6) ||101 ||96-109 |
|Heparin Plasma (n=4) ||97 ||86-108 |
|Serum (n=4) ||98 ||86-109 |
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of mouse Shh-N were serially diluted with Calibrator Diluent to produce samples with values within the dynamic range of the assay.
Human Shh cDNA encodes a 462 amino acid (aa) residue (45 kDa) precursor protein with a 23 aa signal peptide. An autocatalytic cleavage reaction yields a 19 kDa (residues 24 - 197) amino-terminal fragment (Shh-N), and a 25 kDa (residues 198 - 462) carboxy-terminal domain (Shh-C). The N-terminal domain retains all known signaling capabilities, while the C-terminal domain is responsible for the intramolecular processing, acting as a cholesterol transferase that covalently transfers the cholesterol molecule to the C-terminus of Shh-N. When Shh is expressed in insect or mammalian cells, a palmitoyl group is also attached to the N-terminal cysteine of Shh-N via an amide linkage. Although the binding affinity to their receptors is not changed, lipid-modified Shh-N proteins are more potent than the unmodified proteins in cell-based assays. Other hydrophobic modifications to unmodified Shh-N, including the substitution of the N-terminal cysteine residue with two hydrophobic isoleucine residues, can also increase Shh-N potency. At the cell surface, Shh-N activity is mediated by a multicomponent receptor complex involving the 12-pass transmembrane protein, Patched (Ptc) which binds Shh with high affinity and Smoothened (Smo), a signaling seven transmembrane G-protein coupled receptor. In the absence of Shh, Ptc represses Smo activity. The binding of Shh to Ptc releases the basal repression of Smo by Ptc. Shh is expressed in key embryonic tissues such as the Hensen’s node, the zone of polarizing activity in the posterior limb bud, the notochord, and the floor plate of the neural tube. Shh is involved in regulating the patterning of the developing central nervous system, somite, and limb. Shh plays an important role in the development of particular tissues such as whisker, hair, foregut, tooth and bone. Studies suggest that Shh is involved in regulating stem cell fates of neural and hematopoeitic lineages, and that aberrant Shh signaling is implicated in basal cell carcinomas and other diseases.
Related Research Areas
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