Human PDPN (Podoplanin) ELISA Kit (HUES02772)

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HUES02772
€539

Description

ELISA Kit Technical ManualMSDS

Human PDPN (Podoplanin) 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 PDPN . 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 PDPN 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 PDPN, 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 PDPN. The concentration of Human PDPN 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 PDPN in samples. No significant cross-reactivity or interference between Human PDPN 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
102.194
2.196
2.1952.138
51.405
1.407
1.4061.349
2.50.822
0.808
0.8150.758
1.250.383
0.417
0.40.343
0.630.233
0.215
0.2240.167
0.320.155
0.139
0.1470.09
0.160.101
0.107
0.1040.047
00.055
0.059
0.057--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.541.144.860.551.214.69
Standard deviation0.030.060.230.030.060.25
C V (%)5.565.264.735.454.965.33

Recovery

The recovery of Human PDPN 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)92-10999
EDTA plasma (n=5)92-10698
Cell culture media (n=5)96-108101

Linearity

Samples were spiked with high concentrations of Human PDPN 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 (%)93-10988-9986-97
Average (%)1009392
1:4Range (%)88-10483-9583-95
Average (%)958788
1:8Range (%)92-10381-9383-96
Average (%)978589
1:16Range (%)86-9985-9687-102
Average (%)929193

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 PDPN (Podoplanin) ELISA Kit (HUES02772) 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 PDPN (Podoplanin) ELISA Kit (HUES02772) Protein Information

UniProt Protein Function:PDPN: May be involved in cell migration and/or actin cytoskeleton organization. When expressed in keratinocytes, induces changes in cell morphology with transfected cells showing an elongated shape, numerous membrane protrusions, major reorganization of the actin cytoskeleton, increased motility and decreased cell adhesion. Required for normal lung cell proliferation and alveolus formation at birth. Induces platelet aggregation. Does not have any effect on folic acid or amino acid transport. Does not function as a water channel or as a regulator of aquaporin-type water channels. Belongs to the podoplanin family. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral

Chromosomal Location of Human Ortholog: 1p36.21

Cellular Component: external side of plasma membrane; filopodium; filopodium membrane; integral to plasma membrane; lamellipodium; microvillus membrane; plasma membrane; ruffle

Molecular Function:amino acid transmembrane transporter activity; folic acid transporter activity; water channel activity; water transporter activity

Biological Process: alveolus development; amino acid transport; cell morphogenesis; cell motility involved in cell locomotion; cell proliferation; cell-cell adhesion; folic acid transport; lung development; lymphangiogenesis; platelet activation; positive regulation of cell motility; prostaglandin metabolic process; regulation of cell shape; signal transduction; water transport

NCBI Summary:This gene encodes a type-I integral membrane glycoprotein with diverse distribution in human tissues. The physiological function of this protein may be related to its mucin-type character. The homologous protein in other species has been described as a differentiation antigen and influenza-virus receptor. The specific function of this protein has not been determined but it has been proposed as a marker of lung injury. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Jul 2008]
UniProt Code:Q86YL7
NCBI GenInfo Identifier:215274223
NCBI Gene ID:10630
NCBI Accession:Q86YL7.3
UniProt Secondary Accession:Q86YL7,O60836, O95128, Q7L375, Q8NBQ8, Q8NBR3, A9Z1Y2 B2R6J8, E9PB68, F6QWX5,
UniProt Related Accession:Q86YL7
Molecular Weight:12,421 Da
NCBI Full Name:Podoplanin
NCBI Synonym Full Names:podoplanin
NCBI Official Symbol:PDPN  
NCBI Official Synonym Symbols:T1A; GP36; GP40; Gp38; OTS8; T1A2; TI1A; T1A-2; AGGRUS; HT1A-1; PA2.26  
NCBI Protein Information:podoplanin
UniProt Protein Name:Podoplanin
UniProt Synonym Protein Names:Aggrus; Glycoprotein 36; Gp36; PA2.26 antigen; T1-alpha; T1A
Protein Family:Podoplanin
UniProt Gene Name:PDPN  
UniProt Entry Name:PDPN_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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