CHEMISTRY REAGENT KITS
                             ----->GLUCOSE(GOD-POP )END POINT

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PRINCIPLE
Glucose     Glucose    Gluconic
+ 02          oxidase     acid
                                 + H2O2
2H2O2 -------------> Coloured
+ phenol Peroxidase      Quinoneimine
+amino-4                    + 4H20
antipyrine

REAGENTS SUPPLIED
Cat. No. A166

5x100 mL

1. Enzyme Powder (for 100 mL) 133 mmol/L
2. Standard 100 mGs/dL (3 mL) 138 mmol/L
3. Buffer Ready to use (100 mL) 5 Bottles
Cat. No. A 167

10x500 mL

1. Enzyme Powder (for500mL) 10 Vials
2. Standard 100 mGs/dL (5 mL) 1 Vial
3. Buffer (500 mL) 10 Bottles
Composition

Concentrations of ready to use solutions.

1. Enzyme reagent

0.125 mMol/L

Amino-4-antipyrine

30.000 U/L

Glucose oxidase 10.000 U/L
Peroxidase 100 mMol/L
Phosphate buffers  
2.   Standard

100 mgs/dL

Glucose (5.55 mMol/L) 16 mMol/L
3.   Buffer  
Sodium Phenolate 16 mMol/L

Preparation of Working Reagent

Cat. No. A166 5x100 mL
Transfer 1 vial Enzyme powder to one bottle of (100 mL Reagent No.3 (Working Buffer). Mix gently to dissolve which is ready to use.
Cat. No. A167 10x500 mL

Transfer 1 vial Enzyme powder to one bottle of 500 mL Reagent No.3 (Working Buffer). Mix gently to dissolve which is ready to use.

STABILITY
At 2-8°C up to expiry date mentioned on the pack. Working Reagent is stable for 60 days at 2-8°C away from light.

SAMPLE
It is recommended to collect venous blood in a flouride oxalate mixture. Common anticoagulants have no interference with this assay. Longer storage of sample may cause glycolysis resulting in low glucose values. (All samples should be handled as potential infective agents as no laboratory methods make conclusive findings for its safety. Therefore, adequate protective laboratory measures should be taken while handling such materials).

SYSTEM PARAMETERS
Reaction

End-point

Temperature 37°C
Wavelength 500 nM ± 20
Standard Concentration 100 mGs/dL
Absorbance Range

0-1 °A

Cuvette Path Length

1 cM

Reagent Volume mL 1000 uL 500 uL
Sample Volume mL 10 uL 5uL
Reaction Time 15Mins
Linearity 500mGs/dL
Max. limit of Blank Reagent 0.100
Final Colour Stability

30Mins

MANUAL METHOD

 

 

3mL Cuvette
1. Pipette into 3 Test tubes BLACK
mL
STD
mL
TEST
mL
Working Reagent

3.00

3.00 3.00
Standard - 0.02 -
Sample - - 0.02

2.Mix well. Incubate for 15 minutes at 37C or 20 minutes at 30C.
3.Read the O.D. of the Test and Standard against Blank at 500 nM (480-520 nM) or    GREEN filter. The final colour is stable for at least 30 minutes when protected from    bright light.
Note : Volumes can be increased by adding 2 mL distilled water to each tube after incubation.

MICRO METHOD FOR DISCRETE ANALYSERS

For Cuvette Volume.................   1.0mL 0.5mL
Working Reagent........................ 1000 µL 500 µL
Sample or Standard......................       10µL      5µL

Mix well. Incubate at 37C for 15 minutes. Read at 500 nM (480-520 nM). The final colour is stable for at least 30 minutes when protected from bright light.
NOTE: Programme the analyser using system parameters. A specific programme data sheet may be provided for each analyser upon request.

RESULTS
Computer
                                            O.D.Test
Glucose in mGs/dL =     -------------------------- x 100
                                           O.D.STD.

                                           O.D.Test
Glucose in mMoI/L =     ------------------- x 5.55
                                           O.D.STD.

EXPECTED VALUES
Serum or Plasma:
Fasting                                         70-110 mGs/dL
2 Hrs after food/Glucose             <140 mGs/dL
C.S.F.: 
Non diabetic                     >50 mGs/dL
                                        (50 to 70 mGs/dL)
As with all diagnostic methods, the final diagnosis should not be made on the result of a single test as well as laboratory diagnosis must be confirmed with clinical manifestations.

URINE TEST
Perform the test on pure or diluted urine in distilled water. Proceed to the calculation indicated for the serum. Multiply the result obtained by the dilution factor applied.

LIMITATIONS
This assay is linear to 500 mGs/dL (27.75 mMol/ L). For values higher than 500 mGs/dL perform a new test on sample diluted in a 0.9% sodium chloride solution. Multiply the result by the dilution factor applied e.g. 5 for a dilution of 1 to 5. Glucose oxidase is specific to p D-Glucose only. Invitro contamination of glucose, or any oxidiser like hydrogen peroxide make false results. Use of contaminated pipette or water leads to elevated blank absorbance. No specific activity of enzyme is possible at higher temperature or contamination of chemicals.

QUALITY CONTROL
To ensure adequate quality control, each kit should be tested against a standard control sera. It should be realised that the use of quality control material checks both instrument and reagent function together. Factors which might affect the performance of this test include proper instrument function, temperature control, cleanliness of glasswares and accuracy of pipetting. It is appropriate to establish each laboratory's accuracy constant and interpret values accordingly. Similarly, laboratory findings should be established by clinical manifestations.

WARNING
This reagent system is for invitro use only. This reagent system is containing preservatives and components that have not established for safety if contacted on broken skin or eye or taken orally. In case of such incidents wash off with plenty of water, or consult a physician.

BIBLIOGRAPHY

  1. TRINDER.P. Ann. Clin. Biochem 1969 6.24
  2. EMERSON.1. Org. Chem 1943.8.417.