Human Serpin E1/PAI-1 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human Serpin E1 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human Serpin E1 in cell culture supernates, cell lysates, and plasma. It contains Sf 21-expressed recombinant human Serpin E1 and has been shown to accurately quantitate the recombinant factor in its active and latent forms as well as in the vitronectin complexes. However, the kit does not detect recombinant human Serpin E1 in complexes with recombinant human uPA and recombinant human tPA. Results obtained using natural human Serpin E1 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human Serpin E1.

Preparation and Storage

Background: Serpin E1/PAI-1

The human serpin superfamily consists of at least 35 members that target not only serine proteases, but also selected cysteine proteases and non-protease proteins. Serpins bind the protease active site resulting in a major conformational rearrangement that traps the enzyme in a covalent acyl-enzyme intermediate. As protease inhibitors, serpins have an array of functions including regulating blood clotting, the complement pathway, extracellular matrix remodeling, and cell motility. They are also involved in activities that extend beyond their ability to inhibit proteases. For instance, they may also regulate blood pressure, angiogenesis, or act as storage/transport proteins.

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.