Human PAI1 (Plasminogen Activator Inhibitor 1) ELISA Kit (HUES03013)

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HUES03013
€539

Description

ELISA Kit Technical ManualMSDS

Human PAI1 (Plasminogen Activator Inhibitor 1) 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 PAI1 . 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 PAI1 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 PAI1, 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 PAI1. The concentration of Human PAI1 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 PAI1 in samples. No significant cross-reactivity or interference between Human PAI1 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
102.506
2.516
2.5112.443
51.753
1.769
1.7611.693
2.50.978
0.952
0.9650.897
1.250.458
0.494
0.4760.408
0.630.282
0.278
0.280.212
0.320.189
0.165
0.1770.109
0.160.12
0.128
0.1240.056
00.066
0.07
0.068--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human PAI1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PAI1 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.551.444.920.561.504.68
Standard deviation0.030.080.270.030.090.20
C V (%)5.455.565.495.366.004.27

Recovery

The recovery of Human PAI1 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)89-10495
EDTA plasma (n=5)86-10093
Cell culture media (n=5)86-10092

Linearity

Samples were spiked with high concentrations of Human PAI1 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 (%)86-9797-11194-106
Average (%)92103100
1:4Range (%)89-10480-9482-97
Average (%)958688
1:8Range (%)91-10684-9782-94
Average (%)978988
1:16Range (%)90-10280-9488-102
Average (%)958695

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 PAI1 (Plasminogen Activator Inhibitor 1) ELISA Kit (HUES03013) 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 PAI1 (Plasminogen Activator Inhibitor 1) ELISA Kit (HUES03013) Protein Information

UniProt Protein Function:SERPINE1: a secreted protein that acts as 'bait' for tissue plasminogen activator, urokinase, and protein C. Its rapid interaction with TPA may function as a major control point in the regulation of fibrinolysis. Belongs to the serpin family. Interacts with VTN. Binds LRP1B; binding is followed by internalization and degradation. Plasma levels of PAI-1 and VCAM-1 together may be useful in predicting post-operative recurrence in patients with colorectal cancer.
UniProt Protein Details:

Protein type:Secreted, signal peptide; Motility/polarity/chemotaxis; Secreted

Chromosomal Location of Human Ortholog: 7q22.1

Cellular Component: extracellular matrix; extracellular space; extracellular region; plasma membrane

Molecular Function:serine-type endopeptidase inhibitor activity; protein binding; protease binding; receptor binding

Biological Process: circadian rhythm; transcription initiation from RNA polymerase II promoter; platelet activation; extracellular matrix organization and biogenesis; positive regulation of blood coagulation; transcription, DNA-dependent; negative regulation of blood coagulation; negative regulation of smooth muscle cell migration; defense response to Gram-negative bacterium; positive regulation of receptor-mediated endocytosis; regulation of cell proliferation; positive regulation of interleukin-8 production; fibrinolysis; positive regulation of angiogenesis; platelet degranulation; negative regulation of fibrinolysis; transforming growth factor beta receptor signaling pathway; gene expression; positive regulation of transcription from RNA polymerase II promoter; regulation of receptor activity; angiogenesis; chronological cell aging; blood coagulation; negative regulation of cell adhesion mediated by integrin; negative regulation of cell migration; positive regulation of inflammatory response

Disease: Plasminogen Activator Inhibitor-1 Deficiency

NCBI Summary:This gene encodes a member of the serine proteinase inhibitor (serpin) superfamily. This member is the principal inhibitor of tissue plasminogen activator (tPA) and urokinase (uPA), and hence is an inhibitor of fibrinolysis. Defects in this gene are the cause of plasminogen activator inhibitor-1 deficiency (PAI-1 deficiency), and high concentrations of the gene product are associated with thrombophilia. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2009]
UniProt Code:P05121
NCBI GenInfo Identifier:129576
NCBI Gene ID:5054
NCBI Accession:P05121.1
UniProt Secondary Accession:P05121,B7Z4S0, F8WD53,
UniProt Related Accession:P05121
Molecular Weight:43,404 Da
NCBI Full Name:Plasminogen activator inhibitor 1
NCBI Synonym Full Names:serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1
NCBI Official Symbol:SERPINE1  
NCBI Official Synonym Symbols:PAI; PAI1; PAI-1; PLANH1  
NCBI Protein Information:plasminogen activator inhibitor 1; serpin E1; endothelial plasminogen activator inhibitor; serine (or cysteine) proteinase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1
UniProt Protein Name:Plasminogen activator inhibitor 1
UniProt Synonym Protein Names:Endothelial plasminogen activator inhibitor; Serpin E1
UniProt Gene Name:SERPINE1  
UniProt Entry Name:PAI1_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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