The Human Plasminogen Activator Inhibitor-1 (PAI-1) ELISA research-use-only kit is to be used for the quantitative determination of human PAI-1 in samples (e.g., serum, EDTA and heparin plasma, cell culture supernatants, etc.) using 96-well plates and a microplate reader. The assay will recognize both natural and recombinant human PAI-1.
Performance characteristics • Sensitivity: <30 pg/mL • Standard curve range: 62.5–4,000 pg/mL • Sample types: serum, plasma (EDTA, heparin), cell culture supernatants • Species cross-reactivity: human • Sample volume: 100 μL (1:2) • Total assay incubation time: 5 hrs
Principle of the method The Human PAI-1 kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). A monoclonal antibody specific for Human PAI-1 has been coated onto the wells of the microtiter strips provided. During the first incubation, standards of known human PAI-1 content, controls, and unknown samples are pipetted into the coated wells. After washing, polyclonal biotinylated second antibody, is added. After washing, streptavidin-peroxidase (enzyme) is added. This binds to the biotinylated antibody to complete the four-member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of Human PAI-1 present in the original specimen.
Background information Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA), which convert plasminogen to plasmin. PAI-1 is a member of serine protease inhibitor superfamily, also known as Serpin E1. PAI-1 inhibits the serine proteases tPA and uPA/urokinase. It has been also demonstrated that PAI-1 activates the JAK/Stat signaling pathway and stimulates cell migration by binding to the low-density lipoprotein receptor-related protein.
PAI-1 is synthesized and secreted by many tissue and cell types. Free PAI-1 is relatively unstable in its active form and readily converts into a latent, inactive form by spontaneous insertion of its reactive center loop into the β-sheet core of the protein. It was demonstrated that binding of vitronectin to PAI-1 could stabilize the active form by preventing the conformation change. The active form of PAI-1 binds tightly with uPA and tPA in a 1:1 ratio. After the formation of an initial docking complex, the proteases cleave the reactive center loop of PAI-1 to form a stable, covalent complex, resulting in the inactivation of the targeted serine protease.
Each ELISA kit is validated for sensitivity, specificity, precision, and lot-to-lot consistency. See product insert for more information on validation.
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