Cat No.: 32700 Other Names: Peptidase M20 domain containing 1, Cps1
Introduction
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 microtiter plate is pre coated with affinity purified antibody against 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-horseradish peroxidase (STP-HRP)
conjugate is added. After the last wash step, an HRP-substrate(TMB) 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.
Assay Performance
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.
Mouse PM20D1 (ng/mL) | Absorbance (450 nm) | Blanked Absorbance |
0 | 0.096 | 0 |
0.312 | 0.128 | 0.032 |
0.625 | 0.161 | 0.065 |
1.25 | 0.23 | 0.134 |
2.5 | 0.371 | 0.275 |
5 | 0.609 | 0.513 |
10 | 1.116 | 1.07 |
20 | 2.191 | 2.095 |
B. Sensitivity
The lowest level of mouse PM20D1 that can be measured by this assay is 0.156 ng/mL.
C. Specificity
No cross reactivity or interference was observed when test with recombinant human PM20D1 protein.
D. Precision
Intra-assay Precision (Precision within an assay)
Two samples of known concentration were tested 8 times on one plate.
CV%: 3.8%
Inter-assay Precision (Precision between assays)
Two samples of known concentration were tested in 8 separate assays.
CV%: 4.9%
E. Spiking and recovery
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.
Spike level | Expected (ng/mL) | Observed (ng/mL) | Recovery (%) |
Low spike (1 ng/mL) | 0.94 | 1.04 | 110.7 |
Medium spike (2 ng/mL) | 1.91 | 2.21 | 115.8 |
High spike (4 ng/mL) | 4.10 | 3.83 | 93.4 |
F. Linearity and recovery
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.
Sample 1:
Dilution | Measured (ng/mL) | Expected (ng/mL) | Recovery (%) |
1:2 | 13.69 | 14.37 | 95.3 |
1:4 | 8.39 | 7.76 | 92.5 |
1:8 | 3.86 | 3.29 | 117.2 |