ELISA Kit for Neprilysin (CD10)

NEP; MME; CALLA; SFE; Atriopeptidase; Enkephalinase; Skin fibroblast elastase; Common Acute Lymphoblastic Leukemia Antigen; Membrane Metallo-Endopeptidase

Specificity

This assay has high sensitivity and excellent specificity for detection of Neprilysin (CD10).
No significant cross-reactivity or interference between Neprilysin (CD10) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Neprilysin (CD10) and the recovery rates were calculated by comparing the measured value to the expected amount of Neprilysin (CD10) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 80-98 89
EDTA plasma(n=5) 85-104 97
heparin plasma(n=5) 81-96 85

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Neprilysin (CD10) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Neprilysin (CD10) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Neprilysin (CD10) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 78-104% 90-99% 82-94% 98-105%
EDTA plasma(n=5) 86-94% 78-102% 84-101% 80-103%
heparin plasma(n=5) 78-92% 91-98% 91-105% 79-102%

Stability

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Reagents and materials provided

Reagents Quantity Reagents Quantity
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard 2 Standard Diluent 1×20mL
Detection Reagent A 1×120µL Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
TMB Substrate 1×9mL Stop Solution 1×6mL
Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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Magazine Citations
13 Elevated Plasma levels of Neutral Endpeptidase in Hypertension Patients Treated with Angiotensin Converting Enzyme Inhibitors Ahajournals:Source
Biol Pharm Bull Effect of High Glucose Levels on Amyloid β Production in Retinas of Spontaneous Diabetes Mellitus Otsuka Long-Evans Tokushima Fatty Rats PubMed: 25832640
Eur J Heart Fail.  Soluble neprilysin does not correlate with outcome in heart failure with preserved ejection fraction Pubmed:26725876
8 A Test in Context: Neprilysin: Function, Inhibition, and Biomarker. pubmed:27491909
European Journal of Heart Failure Effects of sacubitril/valsartan on neprilysin targets and the metabolism of natriuretic peptides in chronic heart failure: a mechanistic clinical study Pubmed: 30520545
European Journal of Heart Failure Increased granulocyte membrane neprilysin (CD10) expression is associated with better prognosis in heart failure Pubmed: 30828920
Clinical Research in Cardiology Soluble neprilysin, NT-proBNP, and growth differentiation factor-15 as biomarkers for heart failure in dialysis patients (SONGBIRD) Pubmed: 32002632
Neurotoxicology Involvement of hippocampal agmatine in β1-42 amyloid induced memory impairment, neuroinflammation and BDNF signaling disruption in mice Pubmed: 32522471
European Heart Journal P1620 Association of granulocyte neprilysin (CD10) expression with prognosis in heart failure with reduced ejection fraction
Clin Biochem Finding a reliable assay for soluble neprilysin Pubmed:35331754
Scientific Reports The potential role of serum tau protein (MAPT), neuronal cell adhesion molecule (NrCAM) and neprilysin (NEP) in neurodegenerative disorders development in …
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