Mouse Autotaxin ELISA
Autotaxin, also known as ENPP-2, is a secreted glycoprotein which belongs to the ectonucleotide pyrophosphatase/phosphodiesterase (NPP) family. Generally, NPPs can hydrolyze phosphates from nucleotides. Autotaxin exhibits the unique lysophospholipase D activity. The mature protein includes two somatomedin-B-like (SMB) cysteine knot domains, a catalytic domain, and an inactive C-terminal nuclease-like domain with an ef-hand-like motif that is important in cell motility, and a region involved in autotaxin secretion. There are three isoforms identified in mouse and human. Most circulating autotaxin is the β form which contains 863 amino acid. Autotaxin contributes to the predominant extracellular source of the phospholipid LPA (lysophosphatidic acid) from LPC (lysophosphatidylcholine). Autotaxin can also produce minor amounts of sphingosine 1-phosphate and cyclic phosphatidic acid which can antagonize many of the tumorigenic properties of LPA. Autotaxin stimulates tumor cell motility and enhances invasion and metastasis. It’s upregulated in melanoma, glioblastoma, breast and lung carcinoma, follicular lymphoma and other cancers. Autotaxin production by adipocytes enhances pre-adipocyte proliferation and may be
elevated in obesity. Autotaxin is present in blood, urine, saliva, seminal and cerebrospinal fluids. In addition, plasma autotaxin is cleared by the liver which is elevated in liver disease. Normal serum or plasma autotaxin concentration is reported to be slightly higher in females than in males and highest in pregnant females.
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.
Intra-assay Precision (Precision within an assay) CV%: 3.8%
Inter-assay Precision (Precision between assays) CV%: 4.9%
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.
Medium spike (2 ng/ml)
Cross reactivity with recombinant human PM20D1 protein.
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).