TEST PROCEDURE FOR SPECTROPHOTOMETER
The following is a general procedure for use on a manual
spectrophotometer.
Procedure Conditions
Wavelength 540
nm
Temperature 37°
C
Mode Endpoint
Sample to Reagent Ratio
TEST PROCEDURE
(1)
A. Label
reagent tubes, as "BLANK", "CONTROL" and
"PATIENT".
B.
Incubate reagent
tubes at 37° C for 5 minutes.
C.
Add 0.8 ml of SGOT
reagent to each tube.
D.
Add 100 ml of normal control serum and to the "CONTROL" tube. Cap the tube and mix well by inversion.
E.
Add 100 ml of patient serum sample to the "PATIENT" tube. Cap the tube and mix well by inversion.
F.
Incubate reagent
tubes at 37° C for 60 minutes.
G.
Add 0.5 ml of color
developer A to each reagent tube. Cap
the tubes and mix well by inversion.
H.
Let reagent tubes
stand at room temperature for 20 minutes.
I.
Add 2 ml of color
developer B to each reagent tube. Cap
the tubes and mix well by inversion. Let
tube stand at room temperature of 5 minutes.
J.
Wipe the reagent
tubes clean with a lint‑free tissue.
K.
Place the
"BLANK" tube in the test well and adjust the photometer to zero
absorbance.
L.
Place the
"CONTROL" and "PATIENT" tubes in the test well and record
the absorbance of the "CONTROL" and "PATIENT" samples.
Calculation Patient SGOT concentration =
absorbance
of "PATIENT" sample
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑---------------------- X
mean assay value of control
absorbance
of "CONTROL" sample
EXPECTED VALUES Normal range: 8 ‑ 40 U/L at 37°
C
NOTE: 1. a. Careful
control of temperature and timing is essential for the accuracy and precision
of test results.
b. The final reaction color is stable for 30
minutes.
INTENDED USE
These reagents are for the quantitative determination of
Serum Glutamate‑Oxalacetate Transaminase (SGOT) enzyme activity in serum.
SUMMARY AND EXPLANATION
Glutamate Oxalacetate Transaminase (GOT) is one of the
amino transferase enzymes which catalyze the reversible reaction of amino acids
and alpha‑ketoglutaric acid by the transfer of the amino group. GOT is present in large amounts in heart,
liver muscle and kidney tissue.
Consequently the determination of serum GOT could serve as a valuable aid
in different diagnoses.
In 1957 Reitman and Frankel (1) presented a colorimetric
approach to measure GOT activity. An end
product of the transamination reaction, oxalacetate, is reacted with dinitro
phenylhydrazine (DNPH) to form the hydrazone complex. This hydrazone is reacted with an alkaline
diluent to form a colored complex which can be measured quantitatively in a
spectrophotometer or a colorimeter. The Biotron Diagnostics method is the
modification of Reitman and Frankel.
TEST PRINCIPLE
The enzyme glutamate oxalacetate transaminase (GOT)
catalyzes an exchange of an amino group of asparate for an alpha‑keto
group of alpha ketoglutarate. The end
products formed in this reaction are oxalacetate and glutamate.
The oxalacetate formed partially decomposes to pyruvate
in a constant ratio under the condition of the test. Dinitrophenylhydrazine is
added to form the hydrazones of the keto acids present. These hydrazones are sequentially reacted
with sodium hydroxide to form a color which can be read by a spectrophotometer
or colorimeter.
MATERIALS PROVIDED
SGOT (AST) Reagent 100 ml
Color developer A 60 ml
Color developer B 120 ml
Standard/Control/Calibrator
REAGENTS
For In Vitro Diagnostic use.
1. SGOT (AST)
Reagent contains 0.5 ml phosphate buffer containing 0.02 M alphaketoglutaric
acid, 0.2 M dl‑asparate and sodium azide as a preservative.
2. Color
Developer A contains 0.003 M 2,4‑dinitrophenylhydrazine, 1 M hydrochloric
acid, preservative.
3. Color
Developer B contains 1.6% sodium hydroxide.
CAUTION! Do not
take these reagents internally or allow them to come in contact with the body.
STORAGE
Store reagents in refrigerator at 2‑8° C. All reagents are stable till
the expiration date stated on the label when stored in refrigerator.
ADDITIONAL MATERIALS REQUIRED
1. Blood
analyzer, spectrophotometer, or colorimeter
2. Pipetting
devices
3. Commercially
available assayed control serum
SAMPLE AND PREPARATION
Freshly drawn serum sample.
EXPECTED VALUES
The normal range of SGOT is 8 to 40 U/L at 37° C.
The above range is intended as a guide. Each laboratory should establish its own
normal range.
PERFORMANCE
1. Precision ‑
The precision study was done by
(a) repetitive assay (N=23) of normal
serum. The assay yielded a mean of 17
U/L and a standard deviation of 3 U/L and a coefficient of variation of 17.6%.
(b) 8 day reproducibility. A pool serum specimen yielded a mean of 14
U/L a standard deviation of 2 U/L and a coefficient of variation of 14.3%.
2. Accuracy ‑
The accuracy study was done by running 28 specimens on Dade (trademark of Dade
Diagnostics Inc.) SGOT kit and Biotron Diagnostics Kit on Starsar III
(registered trademark of Gilford Instruments).
The study yielded a regression equation of Biotron = 1.027 * reference
method - 2.06 and a correlation of 0.91.
LIMITATIONS OF THE PROCEDURE
This procedure is linear from 0 to 120 U/L at 37° C.
QUALITY CONTROL
Standard practice for quality control should be applied
to this system. Commercially available
lyophilized controls can be used. Daily quality control must fall within 2
standard deviations of the established value.
If correlation is not obtained and repetition of the assay excludes
error in technique, the following steps should be taken:
1. Calibrate
the instrument according to manufacturer's instructions.
2. Check the
cleanliness of the reagent tube.
3. Check the
expiration date of the reagent package.
4. Contact
BiotronDiagnostics Technical Services Department in Indianapolis, IN.
REFERENCES
1. Reitman, S.
and Frankel, S., American Journal of Clinical Pathology, 28:56‑63, 1957.
2. Henry, R.
J., Cannon, D. C. and Winkleman, J. W., "Clinical Chemistry, Principles
and Technics," 2nd Ed., 1974, Harper and Row, New York, 884‑889.
3. Kin
Diagnostics Laboratory Data, Indianapolis, IN, 1981.