Human D betaH (Dopamine- beta-Hydroxylase) CLIA Kit (HUES00699)

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HUES00699
€599

Description

ELISA Kit Technical ManualMSDS

Human D betaH (Dopamine- beta-Hydroxylase) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human D betaH . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human D betaH 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 D betaH, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human D betaH. The concentration of Human D betaH in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

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

Typical Data

As the RLU 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)
RLU Average Corrected
2032549
35415
3398233950
1011483
13457
1247012438
55543
4957
52505218
2.52381
2667
25242492
1.251489
1277
13831351
0.63921
813
867835
0.31580
666
623591
030
34
32--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.922.226.940.862.437.09
Standard deviation0.120.180.800.110.230.48
C V (%)13.048.1111.5312.799.476.77

Recovery

The recovery of Human D betaH 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)93-107101
EDTA plasma (n=5)99-111105
Cell culture media (n=5)90-10697

Linearity

Samples were spiked with high concentrations of Human D betaH 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 (%)87-9995-111102-117
Average (%)92101109
1:4Range (%)98-11799-11394-106
Average (%)106107100
1:8Range (%)88-10187-10088-100
Average (%)939394
1:16Range (%)102-11685-9990-102
Average (%)1089097

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 CLIA 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 Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human D betaH (Dopamine- beta-Hydroxylase) CLIA Kit (HUES00699) 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Human D betaH (Dopamine- beta-Hydroxylase) CLIA Kit (HUES00699) Protein Information

UniProt Protein Function:DBH: Conversion of dopamine to noradrenaline. Homotetramer composed of two non-covalently bound disulfide-linked dimers. Activity is enhanced by nerve growth factor (in superior cervical ganglia and adrenal medulla). Trans-synaptic stimulation with reserpine, acetylcholine and glucocorticoids. Belongs to the copper type II ascorbate-dependent monooxygenase family.
UniProt Protein Details:

Protein type:Membrane protein, integral; Motility/polarity/chemotaxis; EC 1.14.17.1; Oxidoreductase; Apoptosis; Amino Acid Metabolism - tyrosine; Cell cycle regulation

Chromosomal Location of Human Ortholog: 9q34

Cellular Component: membrane; cytoplasm; integral to membrane; extracellular region

Molecular Function:copper ion binding; L-ascorbic acid binding; dopamine beta-monooxygenase activity; catalytic activity

Biological Process: fear response; dopamine catabolic process; maternal behavior; response to amphetamine; cytokine production; locomotory behavior; response to pain; behavioral response to ethanol; leukocyte mediated immunity; glucose homeostasis; memory; regulation of cell proliferation; norepinephrine biosynthetic process; synaptic transmission; catecholamine biosynthetic process; homoiothermy; positive regulation of vasoconstriction; visual learning; blood vessel remodeling; leukocyte migration

Disease: Dopamine Beta-hydroxylase Deficiency, Congenital

NCBI Summary:The protein encoded by this gene is an oxidoreductase belonging to the copper type II, ascorbate-dependent monooxygenase family. The encoded protein, expressed in neuroscretory vesicles and chromaffin granules of the adrenal medulla, catalyzes the conversion of dopamine to norepinephrine, which functions as both a hormone and as the main neurotransmitter of the sympathetic nervous system. The enzyme encoded by this gene exists exists in both soluble and membrane-bound forms, depending on the absence or presence, respectively, of a signal peptide. Mutations in this gene cause dopamine beta-hydroxylate deficiency in human patients, characterized by deficits in autonomic and cardiovascular function, including hypotension and ptosis. Polymorphisms in this gene may play a role in a variety of psychiatric disorders. [provided by RefSeq, Aug 2017]
UniProt Code:P09172
NCBI GenInfo Identifier:158517849
NCBI Gene ID:1621
NCBI Accession:P09172.3
UniProt Related Accession:P09172
Molecular Weight:
NCBI Full Name:Dopamine beta-hydroxylase
NCBI Synonym Full Names:dopamine beta-hydroxylase
NCBI Official Symbol:DBH  
NCBI Official Synonym Symbols:DBM; ORTHYP1  
NCBI Protein Information:dopamine beta-hydroxylase
UniProt Protein Name:Dopamine beta-hydroxylase
UniProt Synonym Protein Names:Dopamine beta-monooxygenaseSoluble dopamine beta-hydroxylase
Protein Family:Dopamine beta-hydroxylase
UniProt Gene Name:DBH  
UniProt Entry Name:DOPO_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
CLIA
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