Mouse PM20D1 immunoassay kit
PM20D1 is a bidirectional N-fatty-acyl amino acid synthase/hydrolase that regulates the production of N-fatty-acyl amino acids. These metabolites are endogenous chemical uncouplers of mitochondrial respiration. In an UCP1-independent manner, maybe through interaction with mitochondrial transporters, they promote proton leakage into the mitochondrial matrix. PM20D1 may indirectly regulate the bodily dissipation of chemical energy as heat through thermogenic respiration.
PRINCIPLE OF THE ASSAY
This assay is a quantitative sandwich ELISA. The microplate is pre-coated with a polyclonal antibody specific for mouse PM20D1. Standards and samples are pipetted into the wells and any human PM20D1 present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase biotin-labelled polyclonal antibody specific for mouse PM20D1 is added to the wells. After wash step to remove any unbound reagents, streptavidin-HRP conjugate (STP-HRP) is added. After the last wash step, an HRP-substrate solution is added and colour develops in proportion to the amount of mouse PM20D1 bound initially. The assay is stopped and the optical density of the wells determined using a microplate reader. Since the increases in absorbance are directly proportional to the amount of captured mouse PM20D1, the unknown sample concentration can be interpolated from a reference curve included in each assay.
A. Typical representation of standard curve
The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample assay.
The lowest level of mouse PM20D1 that can be measured by this assay is 0.156 ng/ml.
Cross reactivity with recombinant human PM20D1 protein.
Intra-assay Precision (Precision within an assay)
Two samples of known concentration were tested 8 times on one plate.
Inter-assay Precision (Precision between assays)
Two samples of known concentration were tested in 8 separate assays.
Serum samples were assayed by adding 90 µl of sample and 10 µl of spike stock.
Solution calculated to yield the intended 0, 1, 2 or 4 ng/ml spike concentration.
Low spike (1 ng/ml)
Medium spike (2 ng/ml)
High spike (4 ng/ml)
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of mouse PM20D1 were serially diluted with the 1×Assay buffer to produce samples with values within the dynamic range of the assay.
Mouse serum from 18 weeks after AAV-PM20D1 overexpressed fed with High-fat diet or Standard chow and control group (AAV-luciferase).