Human DNase-I (Deoxyribonuclease I) ELISA Kit (HUES02950)

SKU:
HUES02950
€299

Description

ELISA Kit Technical ManualMSDS

Human DNase-I (Deoxyribonuclease I) 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 DNase-I . 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 DNase-I 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 DNase-I, 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 DNase-I. The concentration of Human DNase-I 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 Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human DNase-I in samples. No significant cross-reactivity or interference between Human DNase-I 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
(ng/mL)
O.D Average Corrected
102.424
2.482
2.4532.362
51.637
1.641
1.6391.548
2.50.942
0.914
0.9280.837
1.250.518
0.55
0.5340.443
0.630.284
0.284
0.2840.193
0.320.208
0.184
0.1960.105
0.160.135
0.155
0.1450.054
00.085
0.097
0.091--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.531.213.890.521.243.97
Standard deviation0.040.070.150.030.060.16
C V (%)7.555.793.865.774.844.03

Recovery

The recovery of Human DNase-I 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)89-10395
EDTA plasma (n=5)93-108100
Cell culture media (n=5)85-9992

Linearity

Samples were spiked with high concentrations of Human DNase-I 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 (%)100-11591-10386-99
Average (%)1059693
1:4Range (%)90-10584-9988-100
Average (%)989193
1:8Range (%)91-10287-10186-99
Average (%)969393
1:16Range (%)89-10385-9783-94
Average (%)949089

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 DNase-I (Deoxyribonuclease I) ELISA Kit (HUES02950) 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 DNase-I (Deoxyribonuclease I) ELISA Kit (HUES02950) Protein Information

UniProt Protein Function:DNASE1: Among other functions, seems to be involved in cell death by apoptosis. Binds specifically to G-actin and blocks actin polymerization. Defects in DNASE1 are a cause of susceptibility to systemic lupus erythematosus (SLE). A chronic, inflammatory and often febrile multisystemic disorder of connective tissue. It affects principally the skin, joints, kidneys and serosal membranes. It is thought to represent a failure of the regulatory mechanisms of the autoimmune system. Belongs to the DNase I family.
UniProt Protein Details:

Protein type:Deoxyribonuclease; Motility/polarity/chemotaxis; Secreted; Secreted, signal peptide; EC 3.1.21.1

Chromosomal Location of Human Ortholog: 16p13.3

Cellular Component: extracellular region; nuclear envelope; nucleus

Molecular Function:protein binding; deoxyribonuclease I activity; actin binding; endodeoxyribonuclease activity

Biological Process: apoptosis; DNA catabolic process, endonucleolytic

Disease: Systemic Lupus Erythematosus

NCBI Summary:This gene encodes a member of the DNase family. This protein is stored in the zymogen granules of the nuclear envelope and functions by cleaving DNA in an endonucleolytic manner. At least six autosomal codominant alleles have been characterized, DNASE1*1 through DNASE1*6, and the sequence of DNASE1*2 represented in this record. Mutations in this gene have been associated with systemic lupus erythematosus (SLE), an autoimmune disease. A recombinant form of this protein is used to treat the one of the symptoms of cystic fibrosis by hydrolyzing the extracellular DNA in sputum and reducing its viscosity. Alternate transcriptional splice variants of this gene have been observed but have not been thoroughly characterized. [provided by RefSeq, Jul 2008]
UniProt Code:P24855
NCBI GenInfo Identifier:118919
NCBI Gene ID:1773
NCBI Accession:P24855.1
UniProt Secondary Accession:P24855,Q14UU9, Q14UV0, B4DV35,
UniProt Related Accession:P24855
Molecular Weight:Calculated: 19kDa/31kDaObserved: 37kDa
NCBI Full Name:Deoxyribonuclease-1
NCBI Synonym Full Names:deoxyribonuclease I
NCBI Official Symbol:DNASE1
NCBI Official Synonym Symbols:DNL1; DRNI
NCBI Protein Information:deoxyribonuclease-1; Dornase alfa; DNase I, lysosomal; human urine deoxyribonuclease I
UniProt Protein Name:Deoxyribonuclease-1
UniProt Synonym Protein Names:Deoxyribonuclease I; DNase I; INN: Dornase alfa
Protein Family:Deoxyribonuclease
UniProt Gene Name:DNASE1
UniProt Entry Name:DNAS1_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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