How to perform the water BET?

Ø  Take the Depyrogenate glassware (250⁰ temperature at least 1 hour).

Ø  Be ensure all material are Pyrogen free.

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  You will do 2 dilution of water.(it means 1 dilution water sample and 1 dilution LRW).

Ø  Next take the 100 µl of sample and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ 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 37⁰ 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 37⁰ at 1 hour .

Ø  Observe the reading

  

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  You will do 1 dilution of water. (not required dilution)

Ø  Next take the 100 µl of sample and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ 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 37⁰ 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 37⁰ at 1 hour .

Ø  Observe the reading

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  You will do 4 dilution of water.(it means 1 dilution water sample and 3 dilution LRW).

Ø  Next take the 100 µl of sample and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ 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 37⁰ 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 37⁰ at 1 hour .

Ø  Observe the reading