Human MK (Midkine) ELISA Kit (HUES03174)

SKU:
HUES03174
€539

Description

ELISA Kit Technical ManualMSDS

Human MK (Midkine) 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 MK . 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 MK 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 MK, 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 MK. The concentration of Human MK 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 MK in samples. No significant cross-reactivity or interference between Human MK 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
102.233
2.289
2.2612.178
51.43
1.456
1.4431.36
2.50.807
0.793
0.80.717
1.250.438
0.464
0.4510.368
0.630.228
0.226
0.2270.144
0.320.181
0.167
0.1740.091
0.160.122
0.14
0.1310.048
00.079
0.087
0.083--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.521.493.370.491.343.53
Standard deviation0.040.080.130.030.070.18
C V (%)7.695.373.866.125.225.10

Recovery

The recovery of Human MK 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)84-9791
EDTA plasma (n=5)89-9994
Cell culture media (n=5)83-9890

Linearity

Samples were spiked with high concentrations of Human MK 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 (%)85-9884-9590-104
Average (%)929095
1:4Range (%)90-10483-9585-97
Average (%)958891
1:8Range (%)87-10285-9886-100
Average (%)939292
1:16Range (%)91-10381-9583-95
Average (%)988689

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 MK (Midkine) ELISA Kit (HUES03174) 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 MK (Midkine) ELISA Kit (HUES03174) Protein Information

UniProt Protein Function:MDK: Developmentally regulated, secreted growth factor homologous to pleiotrophin (PTN), which has heparin binding activity. Binds anaplastic lymphoma kinase (ALK) which induces ALK activation and subsequent phosphorylation of the insulin receptor substrate (IRS1), followed by the activation of mitogen-activated protein kinase (MAPK) and PI3-kinase, and the induction of cell proliferation. Involved in neointima formation after arterial injury, possibly by mediating leukocyte recruitment. Also involved in early fetal adrenal gland development. Belongs to the pleiotrophin family.
UniProt Protein Details:

Protein type:Secreted; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 11p11.2

Cellular Component: cell projection; cytoplasm; extracellular region

Molecular Function:heparin binding; growth factor activity

Biological Process: response to drug; nervous system development; cell migration; Notch signaling pathway; dentate gyrus development; behavioral fear response; cerebellar granular layer development; short-term memory; adrenal gland development; positive regulation of transcription, DNA-dependent; response to glucocorticoid stimulus; signal transduction; positive regulation of cell division; response to wounding; cerebral cortex development; negative regulation of neuron apoptosis; cell differentiation; defecation; regulation of behavior

NCBI Summary:This gene encodes a member of a small family of secreted growth factors that binds heparin and responds to retinoic acid. The encoded protein promotes cell growth, migration, and angiogenesis, in particular during tumorigenesis. This gene has been targeted as a therapeutic for a variety of different disorders. Alternatively spliced transcript variants encoding multiple isoforms have been observed. [provided by RefSeq, Jul 2012]
UniProt Code:P21741
NCBI GenInfo Identifier:127116
NCBI Gene ID:4192
NCBI Accession:P21741.1
UniProt Secondary Accession:P21741,Q2LEK4, Q9UCC7,
UniProt Related Accession:P21741
Molecular Weight:9,512 Da
NCBI Full Name:Midkine
NCBI Synonym Full Names:midkine (neurite growth-promoting factor 2)
NCBI Official Symbol:MDK
NCBI Official Synonym Symbols:MK; ARAP; NEGF2
NCBI Protein Information:midkine; retinoic acid inducible factor; amphiregulin-associated protein; midgestation and kidney protein; neurite outgrowth-promoting factor 2
UniProt Protein Name:Midkine
UniProt Synonym Protein Names:Amphiregulin-associated protein; ARAP; Midgestation and kidney protein; Neurite outgrowth-promoting factor 2; Neurite outgrowth-promoting protein
Protein Family:Midkine
UniProt Gene Name:MDK
UniProt Entry Name:MK_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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