Rat Hydroxyacyl Coenzyme A Dehydrogenase Beta (HADHb) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to HADHb. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to HADHb. 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 HADHb, 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 HADHb 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

Mitochondrial trifunctional enzyme catalyzes the last three of the four reactions of the mitochondrial beta-oxidation pathway. The mitochondrial beta-oxidation pathway is the major energy-producing process in tissues and is performed through four consecutive reactions breaking down fatty acids into acetyl-CoA. Among the enzymes involved in this pathway, the trifunctional enzyme exhibits specificity for long-chain fatty acids. Mitochondrial trifunctional enzyme is a heterotetrameric complex composed of two proteins, the trifunctional enzyme subunit alpha/HADHA carries the 2,3-enoyl-CoA hydratase and the 3-hydroxyacyl-CoA dehydrogenase activities, while the trifunctional enzyme subunit beta/HADHB described here bears the 3-ketoacyl-CoA thiolase activity.

GENE ID	171155
SWISS PROT	Q60587
SYNONYMS	MTPB; Mitochondrial Trifunctional Protein,Beta Subunit; 3-Ketoacyl-Coenzyme A Thiolase/Enoyl-Coenzyme A Hydratase(Trifunctional Protein)Beta Subunit; Beta-ketothiolase

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 HADHb were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level HADHb 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 0.135ng/mL.
ASSAY RANGE	0.312-20ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of HADHb. No significant cross-reactivity or interference between HADHb and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between HADHb and all analogs, therefore, cross reactivity may still exist.