Human MK (Midkine) CLIA Kit (HUES01193)

SKU:
HUES01193
€599

Description

ELISA Kit Technical ManualMSDS

Human MK (Midkine) 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 MK . 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 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 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 MK. The concentration of Human MK 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 range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

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 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
(pg/mL)
RLU Average Corrected
200050725
60717
5572155693
100021971
25977
2397423946
50011389
10675
1103211004
2504991
5597
52945266
1252666
2550
26082580
62.51371
1251
13111283
31.25649
699
674646
027
29
28--

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 (pg/mL)108.90304.98911.70113.64283.10904.35
Standard deviation10.9032.4873.9412.7029.9273.79
C V (%)10.0110.658.1111.1810.578.16

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)95-111102
EDTA plasma (n=5)90-10095
Cell culture media (n=5)93-10698

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 (%)85-9886-9891-107
Average (%)919298
1:4Range (%)96-110103-11696-108
Average (%)103109101
1:8Range (%)102-11988-10189-104
Average (%)1099595
1:16Range (%)90-10595-11288-99
Average (%)9710394

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 MK (Midkine) CLIA Kit (HUES01193) 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 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL 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 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.
    7. 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.
    8. Add 100 µL 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 100 µL 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 MK (Midkine) CLIA Kit (HUES01193) 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:
CLIA
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