Human TPA ELISA Kit, Fluorescent (ab229408)
Key features and details
- One-wash 90 minute protocol
- Sensitivity: 7.27 pg/ml
- Range: 9.77 pg/ml - 20000 pg/ml
- Sample type: Cell culture supernatant, Cit plasma, EDTA Plasma, Hep Plasma, Serum
- Detection method: Fluorescent
- Assay type: Sandwich (quantitative)
- Reacts with: Human
Overview
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Product name
Human TPA ELISA Kit, Fluorescent
See all Tissue Plasminogen Activator kits -
Detection method
Fluorescent -
Precision
Intra-assay Sample n Mean SD CV% Human serum 5 3.7% Inter-assay Sample n Mean SD CV% Human serum 3 10.5% -
Sample type
Cell culture supernatant, Serum, Hep Plasma, EDTA Plasma, Cit plasma -
Assay type
Sandwich (quantitative) -
Sensitivity
7.27 pg/ml -
Range
9.77 pg/ml - 20000 pg/ml -
Recovery
Sample specific recovery Sample type Average % Range Cell culture supernatant 119 116% - 123% Serum 104 100% - 107% Hep Plasma 97 94% - 99% EDTA Plasma 84 82% - 86% Cit plasma 99 97% - 104% -
Assay time
1h 30m -
Assay duration
One step assay -
Species reactivity
Reacts with: Human
Does not react with: Goat, Cow, Pig -
Product overview
Tissue Plasminogen Activator (TPA) in vitro CatchPoint SimpleStep ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of Tissue Plasminogen Activator (TPA) protein in human serum, plasma and cell culture supernatant.
This CatchPoint SimpleStep ELISA kit has been optimized for Molecular Devices Microplate Readers. Click here for a list of recommended Microplate Readers.
If using a Molecular Devices’ plate reader supported by SoftMax® Pro software, a preconfigured protocol for these CatchPoint SimpleStep ELISA Kits is available with all the protocol and analysis settings at www.softmaxpro.org.The CatchPoint SimpleStep ELISA employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. CatchPoint HRP Development Solution containing the Stoplight Red Substrate is added. During incubation, the substrate is catalyzed by HRP generating a fluorescent product. Signal is generated proportionally to the amount of bound analyte and the intensity is measured in a fluorescence plater reader at 530/570/590 nm Excitation/Cutoff/Emission.
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Notes
Tissue Plasminogen Activator (Tissue-type plasminogen activator or TPA) is a circulating serine protease involved in the breakdown of clots. TPA converts inactive plasminogen to active plasmin; in turn plasmin degrades the fibrin matrix in clots. In addition, plasmin can cleave TPA at Arg-310 with results in a two chain disulphide linked TPA that has even greater proteolytic activity. TPA is synthesized in many tissues and is secreted into most extracellular body fluids. Recombinant TPA is used medically to resolve or prevent blood clots in ischemic stroke or myocardial infarction.
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Platform
Pre-coated microplate (12 x 8 well strips)
Properties
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Storage instructions
Store at +4°C. Please refer to protocols. -
Components 1 x 96 tests 100X Stoplight Red Substrate 1 x 120µl 10X Human TPA Capture Antibody 1 x 600µl 10X Human TPA Detector Antibody 1 x 600µl 10X Wash Buffer PT (ab206977) 1 x 20ml 500X Hydrogen Peroxide (H2O2, 3%) 1 x 50µl Antibody Diluent 4BI 1 x 6ml Human TPA Lyophilized Recombinant Protein 2 vials Plate Seals 1 unit Sample Diluent NS (ab193972) 1 x 50ml SimpleStep Pre-Coated Black 96-Well Microplate 1 unit Stoplight Red Substrate Buffer 1 x 12ml -
Research areas
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Function
Converts the abundant, but inactive, zymogen plasminogen to plasmin by hydrolyzing a single Arg-Val bond in plasminogen. By controlling plasmin-mediated proteolysis, it plays an important role in tissue remodeling and degradation, in cell migration and many other physiopathological events. Play a direct role in facilitating neuronal migration. -
Tissue specificity
Synthesized in numerous tissues (including tumors) and secreted into most extracellular body fluids, such as plasma, uterine fluid, saliva, gingival crevicular fluid, tears, seminal fluid, and milk. -
Involvement in disease
Note=Increased activity of TPA results in increased fibrinolysis of fibrin blood clots that is associated with excessive bleeding. Defective release of TPA results in hypofibrinolysis that can lead to thrombosis or embolism. -
Sequence similarities
Belongs to the peptidase S1 family.
Contains 1 EGF-like domain.
Contains 1 fibronectin type-I domain.
Contains 2 kringle domains.
Contains 1 peptidase S1 domain. -
Domain
Both FN1 and one of the kringle domains are required for binding to fibrin.
Both FN1 and EGF-like domains are important for binding to LRP1.
The FN1 domain mediates binding to annexin A2.
The second kringle domain is implicated in binding to cytokeratin-8 and to the endothelial cell surface binding site. -
Post-translational
modificationsThe single chain, almost fully active enzyme, can be further processed into a two-chain fully active form by a cleavage after Arg-310 catalyzed by plasmin, tissue kallikrein or factor Xa.
Differential cell-specific N-linked glycosylation gives rise to two glycoforms, type I (glycosylated at Asn-219) and type II (not glycosylated at Asn-219). The single chain type I glycoform is less readily converted into the two-chain form by plasmin, and the two-chain type I glycoform has a lower activity than the two-chain type II glycoform in the presence of fibrin.
N-glycosylation of Asn-152; the bound oligomannosidic glycan is involved in the interaction with the mannose receptor.
Characterization of O-linked glycan was studied in Bowes melanoma cell line. -
Cellular localization
Secreted > extracellular space. - Information by UniProt
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Alternative names
- Alteplase
- DKFZp686I03148
- Plasminogen activator tissue
see all -
Database links
- Entrez Gene: 5327 Human
- Omim: 173370 Human
- SwissProt: P00750 Human
- Unigene: 491582 Human
Images
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Other - Human TPA ELISA Kit, Fluorescent (ab229408)
SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.
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Example of human Tissue Plasminogen Activator (TPA) standard curve in Sample Diluent NSThe Tissue Plasminogen Activator (TPA) standard curve was prepared as described in Section 10. Raw data generated on SpectraMax M4 Multi-Mode Microplate Reader is shown in the table. Background-subtracted data values (mean +/- SD) are graphed.
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Titration of human serum and plasma (heparin) within the working range of the assayBackground-subtracted data values (mean +/- SD, n =2) are graphed.
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Interpolated concentrations of TPA in human serum and plasmaThe concentrations of TPA were measured in duplicate and interpolated from the TPA standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean TPA concentration was determined to be 4,710 pg/mL in serum and 4,270 pg/mL in plasma (heparin).
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Interpolated concentrations of TPA in human serum from 10 male donorsSerum from 10 apparently healthy male donors was measured in duplicate. The mean TPA concentration was determined to be 2,741 pg/mL with a range of 1,836- 4,012 pg/mL in male donors.
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Comparison of secreted TPA in unstimulated and PHA-stimulated human PBMCPBMC were grown in the absence or presence of phytohemagglutinin (PHA) for 2 days. TPA was measured in 4-fold diluted cell culture supernatants of unstimulated and PHA stimulated PBMC. Measured values were interpolated from the TPA Standard Curve diluted in Sample Diluent NS and DF corrected. Mean +/-SD, n=2, are graphed.
