Requirements
Ø Take the Depyrogenate glassware (250deg C temperature at least 1 hour).
Ø Be ensure all material are Pyrogen free.
Ø 10x75 mm depyrogenated incubation glass tubes
Ø 16x100 mm depyrogenated dilution glass tubes
Ø 0.125 EU/ml Bacterial Endotoxin test kit from Charles River
Ø 20-200µl depyrogenated micropipette tips
Ø 100-1000µl depyrogenated micropipette tips
Ø Dry heat block calibrated at 37±10C temperature
Method :1
Ø Water Endotoxin Release Limit (ERL) = 0.25 EU/ml
Ø Water potency (C)=1 mg/1ml.
Ø (LAL ) Lysate Sensitivity (λ)= 0.125 EU/ml
Ø Select the MVD (Maximum Valid Dilution) method.
Ø Calculate as below
Test Sample:
MVD = ERL X C/ λ
= 0.25 x 1/0.125
=2 (dilution factor)
Ø Perform 2 dilution of water.(it means 1 dilution water sample and 1 dilution LRW).
Ø Next take the 50µl of sample, 50µl of LAL reagent water , and 100µl of Lysate (LAL).
Ø Incubate at 37±10C at 1 hour .
Ø Observe the reading.
ERL Verification :
= Lambda X Dilution factor = ERL
=0.125 X 2 = 0.25
Negative Control:
Ø Next take the 100 µl of LRW and 100µl of Lysate (LAL).
Ø Incubate at 37±10C 1 hour .
Ø Observe the reading.
Positive Control :
Ø Take the 100 µl (ERL X 2) CSE and 100µl of Lysate (LAL).
Ø Incubate at 37±10C at 1 hour .
Ø Observe the reading
Conclusion:
After incubation read the test tubes by gently inverting them at 1800. The presence of a gel clot that remains in the bottom of the test tube after inversion indicates a positive test. The test is negative if the clot breaks apart or if the contents of the tube remain liquid
Method :2
Ø Water Endotoxin Release Limit (ERL) = 0.125 EU/ml
Ø Water potency (C)=1 mg/1ml.
Ø (LAL ) Lysate Sensitivity (λ)= 0.125 EU/ml
Ø Select the MVD (Maximum Valid Dilution) method.
Ø Calculate as below
Test Sample:
MVD = ERL X C/ λ
= 0.125 x 1/0.125
= 1(dilution factor)
Ø Perform 1 dilution of water. (not required dilution)
Ø Ø Next take the 50µl of sample, 50µl of LAL reagent water , and 100µl of Lysate (LAL).
Ø Incubate at 37±10C at 1 hour .
Ø Observe the reading.
ERL Verification :
= Lambda X Dilution factor = ERL
=0.125 X 1 = 0.125
Negative Control:
Ø Next take the 100 µl of LRW and 100µl of Lysate (LAL).
Ø Incubate at 37±10C at 1 hour .
Ø Observe the reading.
Positive Control :
Ø take the 100 µl (ERL X 2) CSE and 100µl of Lysate (LAL).
Ø Incubate at 37±10C at 1 hour .
Ø Observe the reading
Conclusion:
After incubation read the test tubes by gently inverting them at 1800. The presence of a gel clot that remains in the bottom of the test tube after inversion indicates a positive test. The test is negative if the clot breaks apart or if the contents of the tube remain liquid
Method :3
Ø Water Endotoxin Release Limit (ERL) = 0.125 EU/ml
Ø Water potency (C)=1 mg/1ml.
Ø (LAL ) Lysate Sensitivity (λ)= 0.03125 EU/ml
Ø Select the MVD (Maximum Valid Dilution) method.
Ø Calculate as below
Test Sample:
MVD = ERL X C/ λ
=0.125 x 1/0.03125
= 4 (dilution factor)
Ø Perform 4 dilution of water.(it means 1 dilution water sample and 3 dilution LRW).
Ø Ø Next take the 50µl of sample, 50µl of LAL reagent water , and 100µl of Lysate (LAL).
Ø Incubate at 37±10C at 1 hour .
Ø Observe the reading.
ERL Verification :
= Lambda X Dilution factor = ERL
=0.03125 X 4 = 0.125
Negative Control:
Ø Next take the 100 µl of LRW and 100µl of Lysate (LAL).
Ø Incubate at 37±10C at 1 hour .
Ø Observe the reading.
Positive Control :
Ø take the 100 µl (ERL X 2) CSE and 100µl of Lysate (LAL).
Ø Incubate at 37±10C at 1 hour .
Ø Observe the reading
Conclusion:
After incubation read the test tubes by gently inverting them at 1800. The presence of a gel clot that remains in the bottom of the test tube after inversion indicates a positive test. The test is negative if the clot breaks apart or if the contents of the tube remain liquid
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