Human VE-Cadherin (Vascular Endothelial Cadherin) CLIA Kit (HUES01138)

SKU:
HUES01138
€599

Description

ELISA Kit Technical ManualMSDS

Human VE-Cadherin (Vascular Endothelial Cadherin) 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 VE-Cadherin . 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 VE-Cadherin 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 VE-Cadherin, 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 VE-Cadherin. The concentration of Human VE-Cadherin 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 VE-Cadherin in samples. No significant cross-reactivity or interference between Human VE-Cadherin 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
200053319
58123
5572155693
100022442
25506
2397423946
50011238
10826
1103211004
2505251
5337
52945266
1252676
2540
26082580
62.51311
1311
13111283
31.25644
704
674646
028
28
28--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)98.14196.64869.5989.94205.80861.05
Standard deviation11.5618.4384.3511.5717.4377.06
C V (%)11.789.379.7012.868.478.95

Recovery

The recovery of Human VE-Cadherin 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)91-10899
EDTA plasma (n=5)94-107100
Cell culture media (n=5)93-10698

Linearity

Samples were spiked with high concentrations of Human VE-Cadherin 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 (%)94-10786-10088-101
Average (%)1009393
1:4Range (%)102-11493-108102-116
Average (%)10899109
1:8Range (%)96-11095-11094-111
Average (%)101103101
1:16Range (%)95-10793-10989-99
Average (%)10110094

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 VE-Cadherin (Vascular Endothelial Cadherin) CLIA Kit (HUES01138) 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 VE-Cadherin (Vascular Endothelial Cadherin) CLIA Kit (HUES01138) Protein Information

UniProt Protein Function:CDH5: Cadherins are calcium dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types. This cadherin may play a important role in endothelial cell biology through control of the cohesion and organization of the intercellular junctions. It associates with alpha-catenin forming a link to the cytoskeleton. Acts in concert with KRIT1 to establish and maintain correct endothelial cell polarity and vascular lumen. These effects are mediated by recruitment and activation of the Par polarity complex and RAP1B. Required for activation of PRKCZ and for the localization of phosphorylated PRKCZ, PARD3, TIAM1 and RAP1B to the cell junction. Interacts via cadherin 5 domain with PTPRB. Interacts with TRPC4. Interacts with KRIT1. Endothelial tissues and brain.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Cell adhesion; Membrane protein, integral

Chromosomal Location of Human Ortholog: 16q22.1

Cellular Component: adherens junction; tight junction; membrane; integral to membrane; plasma membrane; intercellular junction; cell junction

Molecular Function:protein binding; beta-catenin binding; calcium ion binding; protein phosphatase binding; receptor binding

Biological Process: intercellular junction assembly and maintenance; negative regulation of cell proliferation; vascular endothelial growth factor receptor signaling pathway; homophilic cell adhesion; blood vessel maturation

NCBI Summary:This gene is a classical cadherin from the cadherin superfamily and is located in a six-cadherin cluster in a region on the long arm of chromosome 16 that is involved in loss of heterozygosity events in breast and prostate cancer. The encoded protein is a calcium-dependent cell-cell adhesion glycoprotein comprised of five extracellular cadherin repeats, a transmembrane region and a highly conserved cytoplasmic tail. Functioning as a classic cadherin by imparting to cells the ability to adhere in a homophilic manner, the protein may play an important role in endothelial cell biology through control of the cohesion and organization of the intercellular junctions. An alternative splice variant has been described but its full length sequence has not been determined. [provided by RefSeq, Jul 2008]
UniProt Code:P33151
NCBI GenInfo Identifier:322510142
NCBI Gene ID:1003
NCBI Accession:P33151.5
UniProt Secondary Accession:P33151,Q4VAI5, Q4VAI6,
UniProt Related Accession:P33151
Molecular Weight:
NCBI Full Name:Cadherin-5
NCBI Synonym Full Names:cadherin 5, type 2 (vascular endothelium)
NCBI Official Symbol:CDH5
NCBI Official Synonym Symbols:7B4; CD144
NCBI Protein Information:cadherin-5; 7B4 antigen; VE-cadherin; cd144 antigen; endothelial-specific cadherin; vascular endothelial cadherin; cadherin 5, type 2, VE-cadherin (vascular epithelium)
UniProt Protein Name:Cadherin-5
UniProt Synonym Protein Names:7B4 antigen; Vascular endothelial cadherin; VE-cadherin; CD_antigen: CD144
Protein Family:Cadherin
UniProt Gene Name:CDH5
UniProt Entry Name:CADH5_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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