Human Neprilysin DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Reagent Diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).
Blocking Buffer: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
Scientific Data
Product Datasheets
Preparation and Storage
Background: Neprilysin/CD10
The neprilysin (NEP) family of zinc metallopeptidases includes neprilysin, endothelin-converting enzyme-2 (ECE-2), PEX, damage induced neuronal endopeptidase (DINE), Kell and several neprilysin-like proteins. Neprilysin is expressed at the cell surface of a variety of cell types. Enzymatically, neprilysin functions both as an endopeptidase with a thermolysin-like specificity and as a dipeptidylcarboxypeptidase. Neprilysin has been shown to be involved in the degradation of enkephalins in the mammalian brain and the inactivation of circulating atrial natriuretic peptide. Neprilysin has also been identified as the common acute lymphoblastic leukemia antigen (CALLA), and to be expressed on the surface of lymphocytes in some disease states. These and other observations have resulted in considerable clinical interest in neprilysin as a potential target for analgesics and antihypertensive drugs. Neprilysin is also a major degrading enzyme of amyloid-beta peptide (A-beta) in the brain, indicating that down-regulation of neprilysin activity, which could be caused by aging, can contribute to the development of Alzheimers disease by promoting A-beta accumulation.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody (to the working concentration stated in the product datasheet ) in PBS without carrier protein. Immediately coat a 96-well microplate with 100 µL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 µL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block each well of the microplate as recommended in the product datasheet. Incubate at room temperature for a minimum of 1 hour.
Note: The recommended Reagent Diluent typically contains 1% BSA. Some DuoSet Development Kits require alternative blocking agents, or for plates to be blocked overnight with a higher percentage of BSA, please see the product datasheet for details.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
PRECAUTION
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.
Assay Procedure
- Add 100 µL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 µL of the Detection Antibody, diluted in Reagent Diluent (as recommended in the product datasheet), to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 µL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 µL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Human Neprilysin DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 8
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Plasma Neprilysin Displays No Relevant Association With Neurohumoral Activation in Chronic HFrEF
Authors: S Prausmülle, H Arfsten, G Spinka, C Freitag, PE Bartko, G Goliasch, G Strunk, N Pavo, M Hülsmann
J Am Heart Assoc, 2020-05-19;9(11):e015071.
Species: Human
Sample Types: Plasma
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The circulating form of neprilysin is not a general biomarker for overall survival in treatment-na�ve cancer patients
Authors: N Pavo, H Arfsten, A Cho, G Goliasch, PE Bartko, R Wurm, C Freitag, H Gisslinger, G Kornek, G Strunk, M Raderer, C Zielinski, M Hülsmann
Sci Rep, 2019-02-22;9(1):2554.
Species: Human
Sample Types: Plasma
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5-HIAA induces neprilysin to ameliorate pathophysiology and symptoms in a mouse model for Alzheimer's disease
Authors: C Klein, G Roussel, S Brun, C Rusu, C Patte-Mens, M Maitre, AG Mensah-Nya
Acta Neuropathol Commun, 2018-12-11;6(1):136.
Species: Human
Sample Types: Cell Lysates
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Neprilysin levels at the acute phase of ST-elevation myocardial infarction
Authors: H Bernelin, N Mewton, S Si-Mohamed, P Croisille, G Rioufol, E Bonnefoy-C, P Douek, N Dufay, C Amaz, C Jossan, M Ovize, T Bochaton
Clin Cardiol, 2018-12-10;0(0):.
Species: Human
Sample Types: Serum
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Possible Enzymatic Downregulation of the Natriuretic Peptide System in Patients with Reduced Systolic Function and Heart Failure: A Pilot Study
Authors: SS Zaidi, RD Ward, K Ramanathan, X Yu, IP Gladysheva, GL Reed
Biomed Res Int, 2018-07-26;2018(0):7279036.
Species: Human
Sample Types: Plasma
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Changes with age in the activities of beta-secretase and the Abeta-degrading enzymes neprilysin, insulin-degrading enzyme and angiotensin-converting enzyme.
Authors: Miners JS, van Helmond Z, Kehoe PG, Love S
Brain Pathol., 2010-01-12;20(4):794-802.
Species: Human
Sample Types: Tissue Homogenates
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CD10 enhances metastasis of colorectal cancer by abrogating the anti-tumoural effect of methionine-enkephalin in the liver.
Authors: Kuniyasu H, Luo Y, Fujii K, Sasahira T, Moriwaka Y, Tatsumoto N, Sasaki T, Yamashita Y, Ohmori H
Gut, 2009-10-14;59(3):348-56.
Species: Human
Sample Types: Tissue Homogenates
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Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.
Authors: Miners JS, Verbeek MM, Rikkert MO, Kehoe PG, Love S
J. Neurosci. Methods, 2007-08-25;167(2):229-36.
Species: Human
Sample Types: Tissue Homogenates
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Excellent ELISA.
We used it on human serum with a very good reproducibility.
At first, we used serum without dilution and then, outliers were assessed a second time with a 1:100 dilution
