Catechol-O-Methyltransferase (COMT), BioAssay™ ELISA Kit is a sandwich ELISA for quantitative detection of human COMT in cell culture supernates, cell lysates, serum and plasma (heparin, EDTA).
Catechol-O-methyltransferase (COMT) is one of several enzymes that degrade catecholamines such as dopamine, epinephrine, and norepinephrine. In humans, catechol-O-methyltransferase protein is encoded by the COMT gene. As the regulation of catecholamines is impaired in a number of medical conditions, several pharmaceutical drugs target COMT to alter its activity and therefore the availability of catecholamines. In the brain, COMT-dependent dopamine degradation is of particular importance in brain regions with low expression of the presynaptic dopamine transporter (DAT), such as the prefrontal cortex. This process is supposed to take place in postsynaptic neurons, as, in general, COMT is located intracellularly in the central nervous system (CNS).
Assay Range:31.2-2000pg/ml
Sensitivity:<10pg/ml
Specificity:Natural and recombinant human COMT
Crossreactivity:There is no detectable cross-reactivity with other relevant proteins
Test Principle:This human COMT BioAssay™ ELISA Kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. A monoclonal antibody from mouse specific for COMT has been precoated onto 96-well plates. The detection antibody is a biotinylated detection polyclonal antibody from goat specific for COMT. The kit contains recombinant human COMT with immunogen: E. coli, G52-271. To measure human COMT, add standards and samples to the wells, then add the biotinylated detection antibody. Wash the wells with PBS or TBS Buffer and then add Avidin-Biotin-Peroxidase Complex. Wash away the unbound Avidin-Biotin-Peroxidase Complex with PBS or TBS Buffer and then add TMB. TMB is substrate to HRP and will be catalyzed to produce a blue color product, which changes into yellow after adding the acidic stop solution. The density of yellow is linearly proportional to the human COMT amount of sample captured in plate. Read the density of the yellow product in each well using a plate reader and benchmark the sample wells’ readings against the standard curve to determine the concentration of human COMT in the samples.
Storage and Stability:Store all components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Assay Summary:1. Add 100ul of samples and standards and incubate the plate at 37°C for 90 minutes or at RT for 2 hours. Do not wash
2. Add 100ul biotinylated antibody and incubate the plate at 37°C for 60 minutes or at RT for 90 minutes. Wash plate 3 times with 0.01M PBS or TBS
3. Add 100ul of ABC working solution and incubate the plate at 37°C for 30 minutes or at RT for 40 minutes. Wash plate 5 times with 0.01M PBS or TBS
4. Add 90ul of TMB color developing agent and incubate the plate at 37°C in dark for 25-30 minutes or at RT for 30 minutes
