QuantiChromTM Urea Assay Kit
Urea is mainly produced in the liver and secreted by the kidneys. It is the main end product of protein catabolism in animals. At the same time, it is the basic mechanism for removing toxic ammonia from the body. The determination of urea is very useful for clinicians in the assessment of renal function in patients. In general, elevated levels of urea are associated with inflammation of the kidneys, their ischemia, obstruction of the urinary tract and digestive tract and some non-renal diseases, e.g. congestive heart failure, liver disease and diabetes. A reduced level indicates acute hepatic failure or may be the result of too intense parenteral administration of fluids. A simple, direct and automated procedure for measuring the concentration of urea or blood urea nitrogen in biological samples is becoming popular in the field of research and drug discovery. The BioAssay Systems' set is intended for measuring the concentration of urea directly in biological samples, without pre-treatment. An improved way of so-called the Jung method uses a chromogenic reagent that forms a colored complex with urea. The intensity of color, measured at 520nm, is directly proportional to the concentration of urea in the sample. The optimized formula significantly reduces the disturbances caused by substances contained in raw samples.
- Sensitive and accurate - use 5 μL of sample. Limit of detection range: 0.08 mg / dL (13μM) to 100mg / dL urea in the test using a 96-well plate.
- Simple, high performance - the procedure consists of adding a working reagent and incubating for 20 min. The test can be automated as a high-performance method for testing thousands of samples per day.
- Increased reagent stability and versatility - the optimized test formula significantly increases the stability of the reagent and signal. Tests can be done in 96-well plates or cuvettes.
- Low interference in biological samples - no need for pre-preparation. The tests can be performed directly on raw biological samples, e.g. in the presence of lipids and proteins.
- Direct tests: urine in serum, plasma, milk, tissue / cell cultures, bronchoalveolar fluid
- Pharmacology: the effect of drugs on urea metabolism
- Environment: determination of urea in sewage, soil, etc.
Procedure using a 96-well plate
- Preparation of reagents: Bring the reagents to room temperature. Prepare a sufficient amount of the working reagent by combining equal volumes of Reagent A and Reagent B, shortly before starting the test. Use the Working Reagent within 20 minutes after mixing Reagents A and B.
- Serum and plasma samples can be determined directly (n = 1). Urine samples should be diluted 50 times in distilled water before starting the test (n = 50). Transfer 5 μL of water (blank), 5 μL of standard (50 mg / dL) and 5 μL of samples in duplicate to wells of a 96-well plate with a transparent bottom. In samples with low urea content (<5 mg / dL), eg tissue / cell extracts, media, bronchoalveolar fluids etc., transfer 50 μL of water (blank), 50 μL 5 mg urea / dL (50 mg / dL standard) diluted in distilled water) and 50 μL of the sample in duplicate, into separate wells.
- Add 200 μL of working reagent and shake slightly to mix.
- Incubate 20 min (50 min for samples with a low urea content) at room temperature Read the optical density at 520nm. For samples with a low urea content, read the OD at a wavelength of 430 nm.
Procedure using a cuvette
Prepare samples as described for the procedure using a 96-well plate. Transfer 20 μL of water, standard (50mg / dL) and samples to appropriately labeled tubes. For samples with a low urea content, use 5 mg / dL and 200 μL standard, instead of 20 μL. Add 1000 μL of working reagent and shake slightly to mix. Incubate 20 min (50 min), then read the OD at 520 nm (OD340 nm).