Human ADAM10 (A Disintegrin And Metalloprotease 10) ELISA Kit (HUES01500)

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HUES01500
€539

Description

ELISA Kit Technical ManualMSDS

Human ADAM10 (A Disintegrin And Metalloprotease 10) ELISA Kit

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 ADAM10 . 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 ADAM10 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 ADAM10, 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 ADAM10. The concentration of Human ADAM10 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range62.50-4000 pg/mL
Sensitivity37.50 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human ADAM10 in samples. No significant cross-reactivity or interference between Human ADAM10 and analogues was observed.

Typical Data

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.

Concentration
(pg/mL)
O.D Average Corrected
40002.403
2.439
2.4212.346
20001.578
1.63
1.6041.529
10000.923
0.917
0.920.845
5000.463
0.489
0.4760.401
2500.282
0.27
0.2760.201
1250.174
0.17
0.1720.097
62.500.118
0.132
0.1250.05
00.066
0.084
0.075--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human ADAM10 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ADAM10 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)183.39507.831750.20190.85537.661716.83
Standard deviation10.7328.3452.8611.0922.9693.91
C V (%)5.855.583.025.814.275.47

Recovery

The recovery of Human ADAM10 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=5)85-9590
EDTA plasma (n=5)86-9891
Cell culture media (n=5)92-10498

Linearity

Samples were spiked with high concentrations of Human ADAM10 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)
1:2Range (%)90-10691-10490-105
Average (%)989698
1:4Range (%)92-10687-10082-91
Average (%)999287
1:8Range (%)88-10181-9385-97
Average (%)948891
1:16Range (%)87-10183-9687-100
Average (%)948994

Kit Components & Storage

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.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human ADAM10 (A Disintegrin And Metalloprotease 10) ELISA Kit (HUES01500) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL 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.
    7. Aspirate or decant the solution from the plate and add 350µL 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL 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.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human ADAM10 (A Disintegrin And Metalloprotease 10) ELISA Kit (HUES01500) Protein Information

UniProt Protein Function:ADAM10: Cleaves the membrane-bound precursor of TNF-alpha at '76-Ala-|-Val-77' to its mature soluble form. Responsible for the proteolytical release of soluble JAM3 from endothelial cells surface. Responsible for the proteolytic release of several other cell-surface proteins, including heparin-binding epidermal growth- like factor, ephrin-A2 and for constitutive and regulated alpha- secretase cleavage of amyloid precursor protein (APP). Contributes to the normal cleavage of the cellular prion protein. Involved in the cleavage of the adhesion molecule L1 at the cell surface and in released membrane vesicles, suggesting a vesicle-based protease activity. Controls also the proteolytic processing of Notch and mediates lateral inhibition during neurogenesis. Responsible for the FasL ectodomain shedding and for the generation of the remnant ADAM10-processed FasL (FasL APL) transmembrane form. Also cleaves the ectodomain of the integral membrane proteins CORIN and ITM2B. May regulate the EFNA5-EPHA3 signaling.
UniProt Protein Details:

Protein type:Cell adhesion; Cell surface; EC 3.4.24.81; Membrane protein, integral; Motility/polarity/chemotaxis; Protease; Vesicle

Chromosomal Location of Human Ortholog: 15q21.3

Cellular Component: cell surface; cytoplasm; focal adhesion; Golgi apparatus; Golgi-associated vesicle; membrane; nucleus; plasma membrane

Molecular Function:endopeptidase activity; metalloendopeptidase activity; metallopeptidase activity; protein binding; protein homodimerization activity; protein kinase binding

Biological Process: constitutive protein ectodomain proteolysis; ephrin receptor signaling pathway; extracellular matrix disassembly; in utero embryonic development; membrane protein ectodomain proteolysis; monocyte activation; negative regulation of cell adhesion; neutrophil degranulation; Notch receptor processing; Notch signaling pathway; PMA-inducible membrane protein ectodomain proteolysis; positive regulation of cell growth; positive regulation of cell migration; positive regulation of cell proliferation; protein amino acid phosphorylation

Disease: Alzheimer Disease 18; Reticulate Acropigmentation Of Kitamura

NCBI Summary:Members of the ADAM family are cell surface proteins with a unique structure possessing both potential adhesion and protease domains. This gene encodes and ADAM family member that cleaves many proteins including TNF-alpha and E-cadherin. Alternate splicing results in multiple transcript variants encoding different proteins that may undergo similar processing. [provided by RefSeq, Feb 2016]
UniProt Code:O14672
NCBI GenInfo Identifier:29337031
NCBI Gene ID:102
NCBI Accession:O14672.1
UniProt Secondary Accession:O14672,Q10742, Q92650, B4DU28,
UniProt Related Accession:O14672
Molecular Weight:84kDa
NCBI Full Name:Disintegrin and metalloproteinase domain-containing protein 10
NCBI Synonym Full Names:ADAM metallopeptidase domain 10
NCBI Official Symbol:ADAM10  
NCBI Official Synonym Symbols:RAK; kuz; AD10; AD18; MADM; CD156c; CDw156; HsT18717  
NCBI Protein Information:disintegrin and metalloproteinase domain-containing protein 10
UniProt Protein Name:Disintegrin and metalloproteinase domain-containing protein 10
UniProt Synonym Protein Names:CDw156; Kuzbanian protein homolog; Mammalian disintegrin-metalloprotease; CD_antigen: CD156c
Protein Family:Disintegrin and metalloproteinase domain-containing protein
UniProt Gene Name:ADAM10  
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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