UREA (NED METHOD):-

The reagent set is intended for in vitro Quantitative determination of Urea in Serum and plasma.

CLINICAL SIGNIFICANCE:
Urea is the end product of the protein metabolism. It is synthesized in the liver from ammonia produced by the deamination of amino acids. It is transported by blood to the kidneys where it is excreted. Increased values are found in renal failure, urinary tract obstruction, shock, congestive heart failure and burns. Decreased levels are found in liver failure and pregnancy.

PRINCIPLE:
Urea forms with ortho–phthalaldehyde and Naphthylethylenediamine in the acidic medium a coloured complex. The value of colour formed is directly proportional to the urea concentration in the test sample and is measured by a fixed time method at 550 nm.

REACTION:
Urea + ODA → NH₃ + H₂O
NH₃ + NED → Orange Color Complex.

CONTENTS:
Reagent 1 : ODA Reagent
Reagent 2 : NED Reagent
Reagent 3 : Urea Standard, 50 mg/dl

MATERIALS REQUIRED BUT NOT PROVIDED:

• Clean Dry Glassware

• Laboratory Glass Pipettes or Micropipettes & Tips

• Bio–Chemistry Analyse

• SAMPLES:

• Serum, Heparinized/EDTA Plasma. Urea is reported to be stable in the serum for 5 days when stored at 2–8°C.

  PREPARATION OF REAGENT & STABILITY:
  All the reagents are ready for use and stable till the expiry date mentioned on the label when stored at 2–8°C.

  GENERAL SYSTEM PARAMETERS:
  Reaction type: End point
  Wavelength: 550 nm (520–550 nm) (Increasing)
  Temperature: 37°C
  Reagent Volume: 1.0 ml
  Sample Volume: 10 µl
  Zero setting: Against Reagent Blank
  Light path: 1 cm.

• PROCEDURE:
  Pipette into clean dry test tubes labeled as Standard (S) and Test (T)

• Addition sequence	(S)	(T)

  ODA Reagent

  1.0 ml

  1.0 ml

  Sample

  —

  10 µl

  Standard

  10 µl

  —

  Mix well and incubate at 37°C for 5 minutes

  NED Reagent

  0.05 ml

  0.05 ml

  Mix well and read the absorbance A₁ of the standard and test after exactly 5 minutes. Read A₂ after exactly 10 minutes. The absorbance reading to be recorded at 550 nm.
  Finally, take the difference A₂–A₁ for both the standard and test.

  For Standard: ΔA = A₂S – A₁S
  For Test: ΔA = A₂T – A₁T

  CALCULATION:
  Urea Concentration (mg/dl) =
  (ΔAT / ΔAS) × 50

  NORMAL VALUE:
  Serum/plasma: 15–40 mg/dl
  Each laboratory should establish its own normal range depending on the population.

  LINEARITY:
  The method is linear upto 200 mg/dl. The value exceeding 200 mg/dl should be diluted appropriately with distilled water and the values obtained multiplied by dilution factor.

  QUALITY CONTROL:
  For accuracy it is necessary to run known controls with every assay.

  LIMITATION & PRECAUTIONS:

  1. Storage condition of the reagent and kit should be strictly followed.

  2. Avoid contamination of reagents.

  3. All glassware must be dry and free from detergent or debris.

  BIBLIOGRAPHY:

  1. Goodwin, J., Hart, T., Am. J. Chem., 26 (1977) 707

  2. CODE NO.	PACK SIZE	Reagent 1	Reagent 2	Reagent 3
     Z18	100 ml	1 x 100 ml	1 x 50 ml	1 x 3.0 ml

      

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