ADMA ELISA KIT
INTENDED USE
The ADMA ELISA Kit is intended for the quantitative determination of asymmetric dimethylarginine (ADMA) in rodent EDTA-plasma or serum and in cell culture media. It is for research use only.
INTRODUCTION
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NOsynthase. It is formed during proteolysis of methylated proteins and removed by renal excretion or metabolic degradation by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Several cell types, including human endothelial and tubular cells are capable of synthesizing and metabolizing ADMA. Elevated ADMA concentrations in the blood are found in numerous diseases associated with endothelial dysfunction. For example, elevated ADMA levels in blood of dialysis patients correlate significantly with the degree of arteriosclerosis and cardiovascular risk. Furthermore, elevated ADMA levels are found in patients with hypercholesterolemia, hypertension, arteriosclerosis, chronic renal failure and chronic heart failure, and are associated with restrictions in endothelial vasodilatation.
During the last years, the important clinical relevance of the regulation of vascular tone and structure by nitric oxide (NO) has been shown. Moreover, there were reports that human endothelial cells produce ADMA as well as nitric oxide, which points to an endogenous endothelial NO-regulation by ADMA. Therefore it was assumed that hypertension, arteriosclerosis and immunological dysfunction in patients with chronic renal failure are connected to a dysfunction of the L-arginin/NO-metabolism and to ADMA accumulation. The reasons for the deregulation of the L-arginin/NO-metabolism could only partially be elucidated. Certainly, there are multiple factors involved in the Larginin/ NO-metabolism regulation as for example elevation of free superoxide radicals (O2-), ADMA accumulation and reduced NO-synthase activity.
Prospective clinical studies of the last years demonstrate the increased importance of ADMA as a novel cardiovascular risk factor.
PRINCIPLE OF THE TEST
ADMA ELISA Kit
The ADMA ELISA Kit is intended for the quantitative determination of asymmetric dimethylarginine (ADMA) in rodent EDTA-plasma or serum and in cell culture media. This ADMA ELISA Kit assay is based on the method of competitive enzyme linked immunoassays. The sample preparation includes the addition of a derivatization-reagent for ADMA coupling. Afterwards, the treated samples and the polyclonal ADMA-antiserum are incubated in wells of microplate coated with ADMA-derivative (tracer). During the incubation period, the target ADMA in the sample competes with the tracer immobilized on the wall of the microtiter wells for the binding of the polyclonal antibodies. The ADMA in the sample displaces the antibodies out of the binding to the tracer. Therefore the concentration of the tracer-bound antibody is inverse proportional to the ADMA concentration in the sample. During the second incubation step, a peroxidase-conjugated antibody is added to each microtiter well to detect the anti-ADMA antibodies. After washing away the unbound components, tetramethylbenzidine (TMB) is added as a substrate for peroxidase. Finally, the enzymatic reaction is terminated by an acidic stop solution. The color changes from blue to yellow and the absorbance is measured in the photometer at 450nm. The intensity of the yellow color is inverse proportional to the ADMA concentration in the sample; this means high ADMA concentration in the sample reduces the concentration of tracer-bound antibodies and lowers the photometric signal.
A dose response curve of absorbance unit (optical density, OD at 450 nm) vs. concentration is generated using the values obtained from the standard. ADMA present in the patient samples is determined directly from this curve.
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