Human t-Plasminogen Activator/tPA Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human t-Plasminogen Activator/tPA Immunoassay is a 4.5 hour solid phase ELISA designed to measure tPA levels in cell culture supernates, serum, plasma, saliva, urine, and human milk. It contains CHO cell-expressed recombinant human tPA and antibodies raised against the recombinant protein. Results obtained for naturally occurring human tPA showed linear curves that were parallel to the standard curves obtained using the Quantikine Human tPA Immunoassay standards. These results indicate that this kit can be used to determine relative mass values for natural human tPA.

Preparation and Storage

Background: t-Plasminogen Activator/tPA

Tissue Plasminogen Activator (tPA), also known as PLAT, is a 64-69 kDa extracellular glycoprotein that belongs to the peptidase S1 family of serine proteases. The biological effects of tPA include blood clot degradation, vascular remodeling, synaptic plasticity, and neurodegeneration in the brain following trauma. Human tPA is secreted as a 530 amino acid (aa) single chain polypeptide (36-562 aa) that contains an amino-terminal fibrin-"finger"-like domain, an epidermal growth factor-like domain, two kringle domains and a C-terminal serine protease catalytic domain (1, 2). The partially active single chain tPA is cleaved between Arg310-Ile311 by Plasmin, Kallikrein/KLKB1, and Coagulation Factor X/Xa to generate the mature two-chain disulfide-linked polypeptide. This mature form of tPA is 10-fold more catalytically active than the single chain (3, 4). From aa 36-562, human tPA shares 81% and 72% identity with mouse and rat tPA, respectively. Human tPA is synthesized and secreted by fibroblasts, vascular endothelial cells, melanoma cells, and neural cells (5). The biological activity of tPA is tightly controlled; freely circulating tPA is sequestered by the serine protease inhibitors Serpin I1 and Serpin E1/PAI-1 (6). tPA is also rapidly cleared from the extracellular and vascular space through Low-Density Lipoprotein Receptor-related Protein-1 mediated endocytosis (7, 8). In the vasculature, circulating tPA binds to blood clot formations. Here tPA converts its primary substrate Plasminogen into Plasmin, an enzyme that subsequently degrades the fibrin matrix of the clot (9, 10). Increased expression or activity of tPA is associated with excessive bleeding, while reduced tPA activity has been implicated in thrombosis and embolism formation. In the brain, tPA is expressed in neurons, astrocytes, microglia, and vascular parenchymal endothelial cells (5, 11). Changes in tPA expression in the brain have been shown following stroke, hypoxia, excitotoxic trauma, and stress-induced cognitive decline (5, 12, 13). The proteolytic activity of tPA is targeted against proteins in brain extracellular matrix (ECM). tPA-mediated breakdown of the ECM is involved in promoting synaptic plasticity, including neurite outgrowth and synapse remodeling (14). In addition to its proteolytic function, tPA has also been shown to have a number of critical non-proteolytic functions, including activation of microglia and modulation of neurotransmission (5, 15, 16). tPA is also involved in angiogenesis and breakdown of the blood-brain barrier (17-20). The Quantikine Human t-Plasminogen Activator/tPA Immunoassay is a 4.5 hour solid phase ELISA designed to measure tPA levels in cell culture supernates, serum, plasma, saliva, urine, and human milk. It contains CHO cell-expressed recombinant human tPA and antibodies raised against the recombinant protein. Results obtained for naturally occurring human tPA showed linear curves that were parallel to the standard curves obtained using the Quantikine Human tPA Immunoassay standards. These results indicate that this kit can be used to determine relative mass values for natural human tPA.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1、Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2、Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3、Add 50 μL of Assay Diluent to each well.

4、Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5、Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6、Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7、Aspirate and wash 5 times.

8、Add 100 μL Substrate Solution to each well.

9、Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.