Human ENPP-2/Autotaxin Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human ENPP-2 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human ENPP-2 in cell culture supernates, serum, plasma, urine, and human milk. It contains NS0-expressed recombinant human ENPP-2 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human ENPP-2 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 ENPP-2.

Preparation and Storage

Background: ENPP-2/Autotaxin

ENPP-2, also known as Autotaxin, belongs to the ectonucleotide pyrophosphatase/phosphodiesterase (NPP) family. Some NPPs hydrolyze phosphates from nucleotides and their derivatives. ENPP-2 shares 40 - 50% identity to ENPP1 & 3, all of which contain a N-terminal intracellular domain, a single transmembrane domain and a large extracellular domain that includes a catalytic domain, two somatomedin-B-like domains, and a C-terminal nuclease-like domain. Unlike ENPP-1 and ENPP-3, ENPP-2 has weak activity against nucleotides, but exhibits a lysophospholipase D activity which allows the formation of lysophosphatidic acid (LPA) and choline from lysophosphatidylcholine. The hydrolysis of nucleotides and lysophospholipids by ENPP-2 is mediated by a single catalytic site. Evidence shows LPA and sphingosine 1-phosphate to be specific inhibitors of ENPP-2. ENPP-2 was originally found to stimulate tumor cell motility and has since been found to enhance tumor invasion and metastasis and to be up-regulated in several types of carcinomas including breast and lung.

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.