Human NADH Dehydrogenase, Quinone 1 (NQO1) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to NQO1. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to NQO1. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain NQO1, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of NQO1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Flavin-containing quinone reductase that catalyzes two-electron reduction of quinones to hydroquinones using either NADH or NADPH as electron donors. In a ping-pong kinetic mechanism, the electrons are sequentially transferred from NAD(P)H to flavin cofactor and then from reduced flavin to the quinone, bypassing the formation of semiquinone and reactive oxygen species (PubMed:8999809, PubMed:9271353) (By similarity). Regulates cellular redox state primarily through quinone detoxification. Reduces components of plasma membrane redox system such as coenzyme Q and vitamin quinones, producing antioxidant hydroquinone forms. In the process may function as superoxide scavenger to prevent hydroquinone oxidation and facilitate excretion (PubMed:8999809, PubMed:9271353, PubMed:15102952). Alternatively, can activate quinones and their derivatives by generating redox reactive hydroquinones with DNA cross-linking antitumor potential (PubMed:8999809). Acts as a gatekeeper of the core 20S proteasome known to degrade proteins with unstructured regions. Upon oxidative stress, interacts with tumor suppressors TP53 and TP73 in a NADH-dependent way and inhibits their ubiquitin-independent degradation by the 20S proteasome (PubMed:15687255, PubMed:28291250).

GENE ID	1728
SWISS PROT	P15559
SYNONYMS	DHQU; DIA4; DTD; NMOR1; NMORI; QR1; Azoreductase; DT-diaphorase; Menadione reductase; Phylloquinone reductase; Quinone reductase 1; NAD(P)H:quinone oxidoreductase 1

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level NQO1 were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level NQO1 were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 0.124ng/mL.
ASSAY RANGE	0.312-20ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of NQO1. No significant cross-reactivity or interference between NQO1 and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between NQO1 and all analogs, therefore, cross reactivity may still exist.