AMMONIA KIT (Kinetic Method)
INTENDED USE
The reagent kit is intended for the in vitro quantitative determination of Ammonia.
SUMMARY
Ammonia (NH₃) is a reagent used for the quantitative determination of ammonium ions in plasma/serum using the enzymatic method of using Glutamate Dehydrogenase (GLDH) and NADPH.
PRINCIPLE
Ammonia combines with α-ketoglutarate to form glutamate in presence of GLDH. This results in a decrease in absorbance at 340 nm, which is directly proportional to the concentration of ammonia.
Reaction:
NH₃ + α-ketoglutarate + NADH → Glutamate + NAD⁺
MATERIALS REQUIRED
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Clean & Dry Glassware
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Laboratory Glass Pipettes or Micropipettes & Tips
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Photometer
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Stopwatch
STORAGE & STABILITY
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Store the unopened kit at 2–8°C and use until expiry date.
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PREPARATION OF REAGENT & STABILITY
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Reagent R1 and R2 to be mixed at 4:1 ratio.
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The final reagent is stable at 2–8°C till expiry date mentioned on kit.
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Once mixed, stable for 8 hours at 2–8°C.
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Unmixed standard reagent should be stored at 2–8°C.
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PROCEDURE
Pipette into a clean dry test tube labelled as Standard (S) and Test (T):
| Additions | Standard | Test |
|---|---|---|
| Working Reagent | 1.0 ml | 1.0 ml |
| Standard | 10 µl | – |
| Sample | – | 10 µl |
Incubate at 37°C for 5 minutes.
Add 10 µL of Standard to Standard tube and 10 µL of sample to Test tube.
Mix well and read absorbance at 340 nm.
After 30 seconds, take first absorbance A₁.
Take second absorbance A₂ after 60 seconds.
Calculate ΔA/min.
CALCULATION
Ammonia (µg/dL) =Δ
NORMAL VALUE
Plasma: 17–80 µg/dL
Expected range varies by population and age.
NOTES
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EDTA plasma or Heparinized plasma
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Blood sample should be taken in sterile vials and stored on ice.
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Separate plasma from cells within 20 minutes after collection.
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Avoid contamination with ammonia from detergents or glassware.
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Avoid rubber stoppers.
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Ammonia in plasma samples may be frozen for 2 hours at −20°C.
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GENERAL SYSTEM PARAMETERS
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Type of Reaction: Kinetic (Decreasing)
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Wavelength: 340 nm
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Temperature: 37°C
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Cuvette Path length: 1 cm
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Zero setting: Distilled Water
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Read Time: 30 seconds
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Sample Volume: 10 µL
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Reagent Volume: 1000 µL
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Total Volume: 1010 µL
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Light path: 1 cm
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LINEARITY
The assay is linear up to 1500 µg/dL.
If values exceed, dilute sample with distilled water. -
LIMITATIONS / PRECAUTIONS
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Ammonia is highly volatile; avoid contamination.
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Do not use glassware washed with ammonia-containing detergents.
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Avoid hemolysis.
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QUALITY CONTROL
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Use known normal and abnormal QC serum.
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