5-Nucleotidase ELISA Kit (5-NT)

Full Name: 5-Nucleotidase ELISA Kit (5-NT)
Reactivity: Human
Sample Type: Serum, Plasma, Biological Fluids, Tissue Homogenates
Sensitivity: 0.188 ng/ml

INTRODUCTION

5-Nucleotidase (5-NT) is an enzyme that hydrolysis nucleoside 5′-monophosphates into their respective nucleosides and free phosphate molecules. It can be classed into either Type I or Type II, this classification is primarily based on their substrate specificity. It is mainly present in the brain, endocrine pancreas, liver heart and blood vessels. It is associated with the canalicular and sinusoidal cell membranes, where elevated serum levels are hepatobiliary in origin. Deoxyribonucleoside monophosphates (dNMPs) are broken down into their respective dNMPs in 5-NT Type I, but in 5-NT Type II the enzyme catalyses both the hydrolysis and phosphorylation activities on nucleosides. This enzyme’s initial application was to recognise cobalamins that undergo 5-phosphate metabolism. This is a mitochondrial enzyme that exists naturally.

5-Nucleotidase is composed of a single polypeptide chain. It contains an active site that is made up of a catalytic triad of amino acid residues (namely histidine, aspartate and a serine). It is a metalloenzyme which catalyses the transfer of a phosphate group from ATP to nucleosides, and the subsequent release of ADP. This enzyme is usually ubiquitous in nature and therefore can be found in almost any cells, organs and tissues. The enzyme is located predominantly in the cytosol and nuclei. Active sites of this enzyme are also found on the surfaces of many plasma membrane bound proteins. In humans, the 5-NT gene has been mapped to chromosome 2p23.3 and is composed of 26 exons that encode a protein of 366 amino acids with a predicted molecular weight of 36,773 Daltons.

INTENDED USE

Human 5-nucleotidase ELISA kit is designed for determining levels of 5-NT (5-nucleotidase, CD73, NTE) present in human in serum, plasma, biological fluids and tissue homogenates.

CONTENT

All reagents supplied need to be stored at 2 °C – 8 °C, unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.

• One 96-Well Plate: Pre-coated with 5-NT.
• Standards: Lyophilized recombinant.
• Sample/Standard Dilution Buffer.
• Biotinylated-labelled Antibody.
• Antibody Dilution Buffer.
• HRP-Streptavidin Conjugate (SABC).
• SABC Dilution Buffer.
• TMB Substrate.
• Wash Buffer (25x).
• Plate Sealer.
• Product Instructions.

TYPICAL RESULTS

The following 5-nucleotidase ELISA kit it is recommended that a standard curve is generated for each assay carried out.

Standard Curve: 0, 0.312, 0.625, 1.25, 2.5, 5, 10, 20 ng/ml.
Reactivity: Human
Sensitivity: 0.188 ng/ml
Range: 0.313 – 20 ng/ml
Principle: Sandwich
Application: Research Use Only.

ASSAY CHARACTERISTICS

– Specificity: Highly specific for 5-NT, no cross reactivity or interference between 5-NT and analogues was detected.
– Recovery: Serum (94 – 100%), EDTA Plasma (91 – 100%), Heparin Plasma (86 – 99%).
– Linearity: Serum (92 – 97%), EDTA Plasma (85 – 101%), Heparin Plasma (80 – 93%).
– Precison Intra-Assay: CV < 8%.
– Precison Inter-Assay: CV < 10%.
– Stability: Less than 10%.

REFERENCES

1. 5′-Nucleotidase: molecular structure and functional aspects. Biochem J. (1992) 1285 (Pt 2)(Pt 2): 345-65. Zimmermann H.
2. Mammalian deoxyribonucleoside kinases. Pharmacol Ther. (1995) 67 (2): 155-86. Arnér E.S and Eriksson S.
3. The clinical biochemistry of 5′-nucleotidase. Ann Clin Lab Sci. (1990) 20 (2): 123-39. Sunderman F.W.

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