Description
TIMP1, also known as TIMP-1 (Tissue Inhibitor of Metallo Proteinases 1), is an endogenous extracellular inhibitor of MMPs (matrix metalloproteinases) belonging to the TIMP family, of which four isoforms are known (TIMP1 to TIMP4). TIMP1 plays a crucial role in Extracellular Matrix (ECM) composition, and is a key modulator of extracellular matrix degradation. It regulates matrix metalloproteinases (MMPs) by forming 1:1 complexes with MMPs (such as MMP-2 and MMP-9), and irreversibly inactivates them. In culture supernatants and biological fluids, TIMP1 exists either as the free form TIMP1 (uncomplexed), or as tight TIMP1-MMP complexes. TIMPs maintain the balance between matrix destruction and formation. A MMPs/TIMPs imbalance leads to excessive ECM deposition and to fibrogenic processes.
Assay principle
Human TIMP1 is measured using a sandwich immunoassay involving two specific anti-human TIMP1 antibodies, respectively labeled with HTRF donor and acceptor dyes. The intensity of the signal is directly proportional to the concentration of TIMP1 present in the sample.
Assay Protocol
The assay protocol, using a 384-well small volume white plate or low volume 96 well plate (20 µL final), is described on the right. 16µl of cell supernatant, sample, or standard are dispensed directly into the detection plate for detection by HTRF® reagents. The antibodies labeled with the HTRF donor and acceptor can be pre-mixed and added in a single dispensing step to further streamline the assay procedure. The assay can be run in 96- to 384-well plates by simply resizing each addition volume proportionally. High levels of TIMP-1 are found in saliva, so the necessary PPE precautions to prevent any contamination must be taken (e.g. mask and gloves).
TIMP1 expression in two human cell lines
IMR90 (human fetal lung myofibroblast, 1.6 x106 cells) and LX2* (human hepatic stellate cells, 2.7 x106 cells) were respectively plated in T75 culture flasks (duplicates) in DMEM containing 10% FCS. After 3 days, the medium was changed to DMEM containing 0.2% BSA, and then the cells were either left unstimulated or stimulated for 3 days with TGFβ1 (10 ng/mL).The supernatants from each flask were collected and hTIMP1 was quantified after a 1/16 dilution in diluent from the kit following the hTIMP1 kit protocol.*Provided by EMD Millipore (product number #SCC064)
Correlation between HTRF Human TIMP1 assay kit and a commercial ELISA
A side-by-side comparison between the HTRF Human TIMP1 assay kit and a commercial ELISA was performed on cell supernatants of MRC5, IMR90, and LX-2* cells, either unstimulated or treated with TGF-β1 for 48h (in FBS-free medium + 0.2% BSA).The two methods are very well correlated, as demonstrated by the value of the correlation coefficient (r) which was close to 1.*Provided by EMD Millipore (product number #SCC064)
