Human NEP(Neprilysin)ELISA Kit (HUES01937)
- Product Type:
- ELISA Kit
- 96 Assays
- ELISA Type:
- Tested Sample Types:
- Serum, plasma and other biological fluids
|Detection Range:||0.16-10 ng/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Human NEP in samples. No significant cross-reactivity or interference between Human NEP and analogues was observed.|
This ELISA kit uses Sandwich-ELISA as the method. 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 appropriate 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 to each micro plate well successively 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 adding 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. The concentration of Human NEP in samples can be calculated by comparing the OD of the samples to the standard curve.
|UniProt Protein Function:||CD10: Thermolysin-like specificity, but is almost confined on acting on polypeptides of up to 30 amino acids. Biologically important in the destruction of opioid peptides such as Met- and Leu-enkephalins by cleavage of a Gly-Phe bond. Able to cleave angiotensin-1, angiotensin-2 and angiotensin 1-9. Involved in the degradation of atrial natriuretic factor (ANF). Displays UV- inducible elastase activity toward skin preelastic and elastic fibers. Inhibited in a dose dependent manner by opiorphin. Belongs to the peptidase M13 family.|
|UniProt Protein Details:|
Protein type:EC 3. 4. 24. 11; Membrane protein, integral; Protease
Chromosomal Location of Human Ortholog: 3q25. 2
Cellular Component: axon; brush border; cytoplasm; dendrite; focal adhesion; plasma membrane; synapse; synaptic vesicle
Molecular Function:endopeptidase activity; exopeptidase activity; metalloendopeptidase activity; metallopeptidase activity; peptide binding; protein binding; zinc ion binding
Biological Process: angiotensin maturation; beta-amyloid metabolic process; creatinine metabolic process; kidney development; peptide metabolic process; proteolysis; sensory perception of pain
Disease: Membranous Nephropathy, Susceptibility To
|NCBI Summary:||This gene encodes a common acute lymphocytic leukemia antigen that is an important cell surface marker in the diagnosis of human acute lymphocytic leukemia (ALL). This protein is present on leukemic cells of pre-B phenotype, which represent 85% of cases of ALL. This protein is not restricted to leukemic cells, however, and is found on a variety of normal tissues. It is a glycoprotein that is particularly abundant in kidney, where it is present on the brush border of proximal tubules and on glomerular epithelium. The protein is a neutral endopeptidase that cleaves peptides at the amino side of hydrophobic residues and inactivates several peptide hormones including glucagon, enkephalins, substance P, neurotensin, oxytocin, and bradykinin. This gene, which encodes a 100-kD type II transmembrane glycoprotein, exists in a single copy of greater than 45 kb. The 5' untranslated region of this gene is alternatively spliced, resulting in four separate mRNA transcripts. The coding region is not affected by alternative splicing. [provided by RefSeq, Jul 2008]|
|NCBI GenInfo Identifier:||128062|
|NCBI Gene ID:||4311|
|NCBI Accession:||P08473. 2|
|UniProt Secondary Accession:||P08473,Q3MIX4, A8K6U6, D3DNJ9,|
|UniProt Related Accession:||P08473|
|Molecular Weight:||85,514 Da|
|NCBI Full Name:||Neprilysin|
|NCBI Synonym Full Names:||membrane metallo-endopeptidase|
|NCBI Official Symbol:||MME|
|NCBI Official Synonym Symbols:||NEP; SFE; CD10; CALLA; CMT2T|
|NCBI Protein Information:||neprilysin|
|UniProt Protein Name:||Neprilysin|
|UniProt Synonym Protein Names:||Atriopeptidase; Common acute lymphocytic leukemia antigen; CALLA; Enkephalinase; Neutral endopeptidase 24. 11; NEP; Neutral endopeptidase; Skin fibroblast elastase; SFE; CD_antigen: CD10|
|UniProt Gene Name:||MME|
|UniProt Entry Name:||NEP_HUMAN|
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
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|
|C V (%)||6.12||5.17||4.95||5.66||4.67||3.24|
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 (%)|
|EDTA plasma (n=5)||96-112||102|
|Cell culture media (n=5)||94-110||101|
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.
|Serum (n=5)||EDTA plasma (n=5)||Cell culture media (n=5)|
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
|Micro ELISA Plate(Dismountable)||8 wells ×12 strips||-20°C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100×)||1 vial, 120 µL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 µL||-20°C(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4°C, 6 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||4°C(shading light)|
|Stop Solution||1 vial, 10 mL||4°C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
- Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37 °C.
- Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 90 µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37 °C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.