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Human NEP(Neprilysin) ELISA Kit

Uniprot : P08473

Cat.No.:E-EL-H0801
Reactivity: Human

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Product Details

Properties

Assay type	Sandwich-ELISA
Format	96T/48T
Assay time	3.5h
Detection range	0.16-10 ng/mL
Sensitivity	0.10 ng/mL
Sample type &Sample volume	serum, plasma and other biological fluids; 100μL
Specificity	This kit recognizes Human NEP in samples. No significant cross-reactivity or interference between Human NEP and analogues was observed.
Reproducibility	Both intra-CV and inter-CV are < 10%.
Application	This ELISA kit applies to the in vitro quantitative determination of Human NEP concentrations in serum, plasma and other biological fluids.

Test Principle

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human NEP. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human NEP and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human NEP, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human NEP. You can calculate the concentration of Human NEP in the samples by comparing the OD of the samples to the standard curve.

Kit components & Storage

An unopened kit can be stored at 2-8℃ for 1 month. If the kit is not supposed to be used within 1 month, store the components separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro ELISA Plate(Dismountable)	96T: 8 wells ×12 strips 48T: 8 wells ×6 strips	-20℃, 12 months
Reference Standard	96T: 2 vials 48T: 1 vial
Concentrated Biotinylated Detection Ab (100×)	96T: 1 vial, 120 μL 48T: 1 vial, 60 μL
Concentrated HRP Conjugate (100×)	96T: 1 vial, 120 μL 48T: 1 vial, 60 μL	-20℃(Protect from light), 12 months
Reference Standard & Sample Diluent	1 vial, 20 mL	2-8°C, 12 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	2-8℃(Protect from light)
Stop Solution	1 vial, 10 mL	2-8°C
Plate Sealer	5 pieces
Manual	1 copy
Certificate of Analysis	1 copy

Technical Data

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NEP were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NEP were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	0.49	1.16	3.64	0.53	1.07	3.70
Standard deviation	0.03	0.06	0.18	0.03	0.05	0.12
CV (%)	6.12	5.17	4.95	5.66	4.67	3.24

Recovery

The recovery of Human NEP spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum(n=8)	95-109	102
EDTA plasma (n=8)	96-112	102
Cell culture media (n=8)	94-110	101

Linearity

Samples were spiked with high concentrations of Human NEP and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Target Information

Database Links	SwissProt: P08473
Synonyms	CD10,MME,NEP,CALLA
Research Area	Cancer, Immunology, Stem cells

Assay Procedures

	1. Add 100μL standard or sample to the wells. Incubate for 90 min at 37°C
	2. Discard the liquid, immediately add 100μL Biotinylated Detection Ab working solution to each well. Incubate for 60 min at 37°C
	3. Aspirate and wash the plate for 3 times
	4. Add 100μL HRP conjugate working solution. Incubate for 30 min at 37°C. Aspirate and wash the plate for 5 times
	5. Add 90μL Substrate Reagent. Incubate for 15 min at 37°C
	6. Add 50μL Stop Solution
	7. Read the plate at 450nm immediately. Calculation of the results

Reviews/Q&A

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Reviews
Q&A

S***********nSubmitted [ Mar 06 2024 ]

Asked: Hello. Have you ever used this product to measure NEP in the cell culture supernatant of SH-SY5Y or other neuronal cells?

adminSubmitted [ Mar 06 2024 ]

Answered: We have validated E-EL-H0801 with SH-SY5Y cell culture supernatant, all the samples were undiluted and the reference value was ND-0.5ng/mL.

S******nSubmitted [ Mar 06 2024 ]

Asked: Hello, I am planning to perform an NEP ELISA using the pellet from cultured cells. Since NEP is a transmembrane protein, I am wondering if the protein can be sufficiently extracted using only the freezing-thawing or ultrasonic methods suggested in the enclosed protocol.

adminSubmitted [ Mar 06 2024 ]

Answered: Because NEP is a transmembrane protein, repeated freeze-thawing is recommended for cell lysate. If ultrasonic methods is selected, it is necessary to control the temperature and frequency of ultrasound.

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