INTENDED USE
NS Biotec AST reagent is intended for the in vitro quantitative determination of aspartate aminotransferase (EC 2.6.1.2) activity in serum on both automated and manual systems.
ASSAY PRINCIPLE
In 1955, Karmen et al described the first kinetic determination of AST activity in serum, using a coupled reaction of malate dehydrogenase (MDH) and NADH. This assay system was critically evaluated and optimized in 1960 by Henry et al. A modification of this method incorporates lactate dehydrogenase into AST assay mixtures in order to accelerate the lag phase by exhaustion of endogenous ketoacids.
AST reagent is based on the recommendation of the IFCC. The series of reactions involved in the assay system are as follows:
1. The amino group is enzymatically transferred by AST present in the specimen from aspartate to the carbon atom of 2-oxoglutarate yielding oxaloacetate and L-glutamate.
2. Oxaloacetate is reduced to malate by MDH present in the reagent with the simultaneous oxidation of NADH to NAD.
The rate of oxidation of the coenzyme NADH is proportional to the AST activity in the specimen. It is determined by measuring the decrease in absorbance at 334 / 340 / 365 nm correspondingly. Lactate dehydrogenase is included in the reagent to convert endogenous pyruvate in the specimen to lactate during the lag phase prior to measurement.
LINEARITY
When run as recommended, the assay is linear up to 400 U/l or 6.63 kat/l.
If result exceeds 400 U/l or 6.63 kat/l, specimen should be diluted 1+5 with 0.9% NaCl solution and reassayed. Multiply the result by 6.
SENSITIVITY
The sensitivity is defined as the lower detection limit represents the lowest measurable AST/GOT activity that can be distinguished from zero.
When run as recommended the sensitivity of this assay is 5 U/l or 0.75
Stability
All reagents are ready for use and stable up to the expiry date given on label when stored at 2–8°C.
Warning & Precautions
NS Biotec AST reagent is for in vitro diagnostic use only. Normal precautions exercised in handling laboratory reagents should be followed.
Warm up working solution to the corresponding temperature before use.
The reagent and sample volumes may be altered proportionally to accommodate different spectrophotometer requirements.
Valid results depend on an accurately calibrated instrument, timing, and temperature control.
Don’t use the reagent if it is turbid or if the absorbance is less than 1.0 at 340 nm.
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