This immunoassay kit allows for the in vitro quantitative determination of bovine carnitine concentrations in cell culture supernates, serum, plasma and other biological fluids.

Bovine Carnitine ELISA Kit


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Background

Carnitine is a quaternary ammonium compound biosynthesized from the amino acids lysine and methionine.[1] In living cells, it is required for the transport of fatty acids from the cytosol into the mitochondria during the breakdown of lipids (or fats) for the generation of metabolic energy. It is often sold as a nutritional supplement. Carnitine was originally found as a growth factor for mealworms and labeled vitamin Bt. Carnitine exists in two stereoisomers: its biologically active form is L-carnitine, while its enantiomer, D-carnitine, is biologically inactive.
Carnitine transports long-chain acyl groups from fatty acids into the mitochondrial matrix, so that they can be broken down through β-oxidation to acetate to obtain usable energy via the citric acid cycle. In some organisms such as fungi, the acetate is used in the glyoxylate cycle for gluconeogenesis and formation of carbohydrates. Fatty acids must be activated before binding to the carnitine molecule to form acyl-carnitine. The free fatty acid in the cytosol is attached with a thioester bond to coenzyme A (CoA). This reaction is catalyzed by the enzyme fatty acyl-CoA synthetase and driven to completion by inorganic pyrophosphatase.

Features

<PRINCIPLE OF THE ASSAY>
The microtiter plate provided in this kit has been pre-coated with an antibody specific to carnitine. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for carnitine and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain carnitine, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of carnitine in the samples is then determined by comparing the O.D. of the samples to the standard curve.

< SPECIFICITY>
This assay recognizes recombinant and natural bovine carnitine. No significant cross-reactivity or interference was observed.

<DETECTION RANGE>
1.56 ng/ml-100 ng/ml.
The standard curve concentrations used for the ELISA’s were 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.12 ng/ml, 1.56 ng/ml.

<SENSITIVITY>
The minimum detectable dose of bovine carnitine is typically less than 0.39 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Composition

  Reagent Quantity
1. Assay plate 1
2. Standard 2
3. Sample Diluent 1 x 20 ml
4. Biotin-antibody Diluent 1 x 10 ml
5. HRP-avidin Diluent 1 x 10 ml
6. Biotin-antibodyt 1 x 120μl
7. HRP-avidin 1 x 120μl
8. Wash Buffer 1 x 20 ml (25×concentrate)
9. TMB Substrate 1 x 10 ml
10. Stop Solution 1 x 10 ml
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To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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