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This immunoassay kit allows for the in vitro quantitative determination of rat CACT concentrations in serum, plasma and other biological fluids.

Rat carnitine-acylcarnitine translocase [CACT] ELISA Kit


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Background

Carnitine-acylcarnitine translocases are responsible for transporting both carnitine andcarnitine-fatty acid complexes into and out of the mitochondria, across the inner mitochondrialmembrane. This enzyme is required as fatty acids cannot cross the mitchondrial membraneswithout assistance. The fatty acid is firstly bound to CoA to cross the external mitochondrialmembrane. It then switches the CoA for carnitine by the use of the enzyme carnitine palmitoyltransferase I. The complex then uses facilitated diffusion by Carnitine-acylcarnitine translocaseto enter the mitochondrial matrix. Here, the acylcartinine complex is disrupted by cartininepalmitoyl transferase II and the fatty acid rebinds to CoA. Cartinine then diffuses back acrossthe membrane by a second carnitine-acylcarnitine translocase into the inter-mitochondiralmembrane space. This is the cartinine shuttle system.
A disorder is associated with carnitine-acylcarnitine translocase deficiency. This disorderprevents the shuttle-like action of cartinine from assisting fatty acids across the mitochondiralmembrane and therefore there is decreased fatty acid metabolism. The result of this is anincreased number of fat droplets within muscles and liver, decreased tolerance to long termexcerise, inability to fast for more than a few hours, muscle weakness and wasting, strong acidic smell on breath (due to protein breakdown)

Feature and Advantages

<PRINCIPLE OF THE ASSAY>
The microtiter plate provided in this kit has been pre-coated with an antibody specific to CACT. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for CACT 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 CACT, 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 CACT 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 rat CACT. No significant cross-reactivity or interference was observed.

<DETECTION RANGE>
15.6 pg/ml-1000 pg/ml.
The standard curve concentrations used for the ELISA’s were 1000pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.2 pg/ml, 15.6 pg/ml.

<SENSITIVITY>
The minimum detectable dose of rat CACT is typically less than 3.9 pg/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.

Kit component

  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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