5'-nucleotidase (5'-NT), also known as ecto-5'-nucleotidase or CD73 (Cluster of Differentiation 73), is an enzyme that in humans is encoded by the NT5E gene. Ecto-5-prime-nucleotidase catalyzes the conversion at neutral pH of purine 5-prime mononucleotides to nucleosides, the preferred substrate being AMP. The enzyme consists of a dimer of 2 identical 70-kD subunits bound by a glycosyl phosphatidyl inositol linkage to the external face of the plasma membrane. And the enzyme is used as a marker of lymphocyte differentiation. Consequently, a deficiency of NT5 occurs in a variety of immunodeficiency diseases. Other forms of 5-prime nucleotidase exist in the cytoplasm and lysosomes and can be distinguished from ecto-NT5 by their substrate affinities, requirement for divalent magnesium ion, activation by ATP, and inhibition by inorganic phosphate. Rareallelic variants are associated with a syndrome of adult-onset calcification of joints and arteries (CALJA) affecting the iliac, femoral, and tibial arteries reducing circulation in the legs and the joints of the hands and feet causing pain.
Sample Type
Cell culture supernates, serum and plasma (heparin, EDTA)
Intended Use
For quantitative detection of human CD73 in cell culture supernates, serum and plasma (heparin, EDTA).
Specificity
Natural and recombinant human CD73
Test Principle
This human CD73 BioAssay™ ELISA Kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. A monoclonal antibody from mouse specific for CD73 has been precoated onto 96-well plates. Standards(CHO, W27-K547) and test samples are added to the wells, a biotinylated detection polyclonal antibody from goat specific for CD73 is added subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the human CD73 amount of sample captured in plate.
Kit Components
96-well plate precoated with human CD73 antibody, 1 Lyophilized recombinant human CD73 standard, 10ng/tube×2 Biotinylated human CD73 antibody, 130ul (dilution 1:100) Avidin-Biotin-Peroxidase Complex (ABC), 130ul (dilution 1:100) Sample diluent buffer, 30ml Antibody diluent buffer, 12ml ABC diluent buffer, 12ml TMB color developing agent, 10ml TMB stop solution, 10ml
Storage and Stability
Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Important Note
This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
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