Overview

  • Product name
    Human TPA ELISA Kit, Fluorescent
    See all TPA 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, Heparin Plasma, EDTA Plasma, Citrate 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%
    Heparin Plasma 97 94% - 99%
    EDTA Plasma 84 82% - 86%
    Citrate Plasma 99 97% - 104%

  • Assay time
    1h 30m
  • Assay duration
    One step assay
  • Species reactivity
    Reacts with: Human
    Does not react with: Mouse, Rat, Rabbit, Goat, Guinea pig, Hamster, Cow, Dog, 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. 

  • 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.

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform
    Pre-coated microplate (12 x 8 well strips)

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Human TPA Capture Antibody 1 x 600µl
    10X Human TPA Detector Antibody 1 x 600µl
    Human TPA Lyophilized Recombinant Protein 2 vials
    Antibody Diluent 4BI 1 x 6ml
    10X Wash Buffer PT (ab206977) 1 x 20ml
    Stoplight Red Substrate Buffer 1 x 12ml
    100X Stoplight Red Substrate 1 x 120µl
    500X Hydrogen Peroxide (H2O2, 3%) 1 x 50µl
    Sample Diluent NS (ab193972) 1 x 50ml
    SimpleStep Pre-Coated Black 96-Well Microplate 1 unit
    Plate Seals 1 unit
  • Research areas
  • 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
    modifications
    The 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
  • Alternative names
    • Alteplase
    • DKFZp686I03148
    • Plasminogen activator tissue
    • Plasminogen activator tissue type
    • PLAT
    • Reteplase
    • t PA
    • T Plasminogen Activator
    • t-PA
    • T-plasminogen activator
    • Tissue plasminogen activator (t PA)
    • Tissue type plasminogen activator
    • Tissue-type plasminogen activator chain B
    • tPA
    • TPA_HUMAN
    • TPA1
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab229408 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • ELISA Protocol Summary
  • The 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.

  • Background-subtracted data values (mean +/- SD, n =2) are graphed.

  • The 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).

     

  • Serum 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.

  • PBMC 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.

Protocols

References

ab229408 has not yet been referenced specifically in any publications.

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