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

Bovine Oxaloacetic Acid ELISA Kit


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Background

Oxaloacetic acid is an organic compound with the chemical formula HO2CC(O)CH2CO2H. It also has other names and its conjugate base is called "oxaloacetate." This four-carbon dicarboxylic acid is a protonated variant of oxaloacetate, which is an intermediate of the citric acid cycle and gluconeogenesis. Oxaloacetate forms upon oxidation of L-malate, catalysed by malate dehydrogenase, and reacts with Acetyl-CoA to form citrate, catalysed by citrate synthase. It also forms in the mesophyll of plants by the condensation of CO2 with phosphoenolpyruvate, catalysed by PEP Carboxykinase. It can arise from pyruvate via an anaplerotic reaction.
The enol forms of oxaloacetic acid are particularly stable, so much so that the two isomers have different melting points (152 °C cis, 184 °C trans). The enol proton has a pKa value of 13.02. The enzyme fumarase A from E. coli catalyses the conversion between the keto and enol forms. Oxaloacetate is unstable in solution, decomposing to pyruvate by decarboxylation over a period of hours (room temperature) or days (0 °C). Refrigerated storage of the solid is therefore recommended.

Features

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

<DETECTION RANGE>
3.12 ng/ml-200 ng/ml.
The standard curve concentrations used for the ELISA’s were 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.12 ng/ml.

<SENSITIVITY>
The minimum detectable dose of bovine oxaloacetic acid is typically less than 0.78 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
Product List

Product Name Cat# Quantity Price

Species : 3.12 ng/ml - 200 ng/ml.
Assay range : 100 µL(Cell Culture Supernates, Plasma,Serum)
Assay sample volume : 6-8 hr.{3 hr


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