Human PM20D1 ELISA Kit

Human PM20D1 ELISA Kit

SKU: 31700

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Assay range: 31.2-2000 pg/mL

Kit Size: 96 wells/Kit

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 a 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 human 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 human 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 human 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 human 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. 

    Human PM20D1(pg/mL)

    Absorbance

    (450 nm)

    Blanked Absorbance

    0

    0.103

    0

    31.2

    0.126

    0.023

    62.5

    0.161

    0.058

    125

    0.237

    0.134

    250

    0.421

    0.318

    500

    0.747

    0.644

    1000

    1.134

    1.031

    2000

    2.12

    2.017

    B. Sensitivity:

    The lowest level of human PM20D1 that can be measured by this assay is 7.8 pg/mL.

     C. Specificity:

    No cross reactivity or interference was observed when test with recombinant mouse PM20D1 protein.

    D. Precision:

    Intra-assay Precision (Precision within an assay)

    Two samples of known concentration were tested 8 times on one plate.

    Sample

    Mean (ng/mL)

    SD 

    CV (%)

    1

    19.98

    0.74

    3.7

    2

    10.74

    0.37

    3.4

     Inter-assay Precision (Precision between assays)

    Two samples of known concentration were tested in 8 separate assays.

    Sample

    Mean (ng/mL)

    SD 

    CV (%)

    1

    19.84

    0.76

    3.8

    2

    10.58

    0.3

    2.8

    E. Spike 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, 50, 400 or 800 pg/ml spike concentration.

    Spike level

    Expected

    (pg/mL)

    Observed

    (pg/mL)

    Recovery (%)

    Low spike (50 pg/mL)

    49.83

    47.83

    95

    Medium spike
    (400 pg/mL)

    396.5

    357.33

    90.1

    High spike (800 pg/mL)

    679.83

    724

    106.5

    F. Linearity and Recovery:

    To assess the linearity of the assay, samples containing high concentrations of human PM20D1 were serially diluted with the 1×Assay buffer to produce samples with values within the dynamic range of the assay.

    Dilution

    Measured (pg/mL)

    Expected (pg/mL)

    Recovery (%)

    1:100

    3072.46

    27850.9

    110.3

    1:200

    30770

    27850.9

    110.5

     

     

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