Purpose:
2. Scope:
3. Responsibilities:
1. Microbiologist
2. Lab Assistant
4. Abbreviations:
MVD Maximum Valid Dilution
MVC Maximum Valid Concentration
ELC Endotoxin Limit Concentration
EU Endotoxin Units
LAL Lymulus Amebocyte Lysate
Std Standard
ET Endotoxin
DF Dilution Factor
5. MATERIALS
A) General
1. Hot air oven.
2. Glass pipettes (1,2,5,10 ml)
3. Micropipetter (0.1 ml or 100 ul)
4. Sterile and pyrogen-free micro-tips
5. Borosilicate glass tubes
6. 0.1N Hcl
7. 0.1N Nacl
8. Photocells (full and half size)
9. Spectrophotometer
10. Vortex mixer
11. Incubator
12. Ice water bath or suitable ice water container for test tube rack
13. Tube stands S/S
14. Beakers
15. LAL water
16. Acetic acid 0.8 M
17. 70% alcohol
18. Aluminium foil depyrogenated
19. Deep freezer (-20oC)
20. Refrigerator (2-8oC)
21. Water tray in an incubator for tube stand
22. Forceps
B) LAL Test Reagents
1) Chromogenic Lysate (Toxicolor systems etc).
2) Respective Endotoxin Standard.
3) Diazo Coupling Reagent (Set of four bottles).
Note: All reagents must be stored in refrigerator at 2-8oC.
WASHING & DEPYROGENATION OF MATERIALS
1) After proper cleaning, rinse the test tubes thoroughly with distilled water.
2) Wrap the test tubes properly. Aluminum foil.
3) Depyrogenate the test tubes in an oven at 250oC for not less than one hour.
PREPARATION OF ACETIC ACID 0.8M
Dissolve 45.6 ml of Acetic acid in one liter of distilled water. The final concentration of Acetic acid is 0.8M. This solution can be stored for three months.
RECONSTITUTION OF REAGENTS
Opening Reagent Vials
Remove the plastic cover. Wipe off with 70% Alcohol around the rubber cap and top portion of every vial. Remove the aluminium cap with sterile and pyrogen free forceps and then cover with depyrogenated aluminium foil to avoid any Endotoxin contamination.
A) Endotoxin (ET-2)
(2.8 ml LAL water vial is provided with Endotoxin vial, concentration is mentioned on label)
1) Pour whole quantity of LAL water into the ET vial and cover with it foil.
2) Vortex mix vigorously for at least 10 seconds.
Storage: Store reconstituted Endotoxin solution at 4oC in a refrigerator for 14 days.
The solution can also be stored at –20oC for a month.
Avoid freezing during storage.
Note: Stir every time vigorously before use.
B) Toxicolor Lysate LS-6
(Buffer vial and LAL water is provided with Lysate. Sensitivity is mentioned on certificate)
1) After taking from refrigerator, pour whole quantity of buffer and LAL water into the lysate vial as soon as possible.
2) Then quickly stir to dissolve. Avoid air bubbling during stirring.
3) Place the vial in ice water bath for 2-3 minutes before use.
Note: Be sure that reagent is completely dissolved. This reagent must be reconstituted just before use. The reagent is extremely sensitive and must be consumed at one time. Storage should be avoided but can be stored at - 20oC in 0.05 ml dispensed quantities in small test tubes. Use stored lysate if the color is not changed. Reconstituted lysate may only be deep frozen once.
C) Diazo Coupling Reagent
Four bottles are provided with one set, marked as 7s, 8s, 9s and 10s respectively.
1) Transfer whole quantity of bottle no. 7s into bottle No. 8s.
2) Then add 12 ml distilled water into each of bottle No. 9s and 10s. Ultimately we will have three bottles 8s, 9s, 10s, which are used stepwise to block the reaction.
PRE-TEST PREPARATIONS
A) pH of the sample
The pH of the sample is adjusted by pyrogen free 0.1N NaoH or 0.1N Hcl. The pH of the sample should be between 6.0-8.0.
B) Test Tubes Settings
Arrange test tubes in two stands as under;
1) Stand 1 - Test tubes for sample and standard dilutions.
2) Stand 2 -Test tubes for reaction.
PROCEDURE
A) Sample Preparations
Take 0.05ml well-mixed sample into small test tubes.
If required, make 1/10 dilution of the sample with Pyrogen free water as
under;
1) Take 4.5 ml of pyrogen free water in the test tube.
2) Then add 0.5 ml of well-mixed sample.
3) Vortex to mix for few seconds.
4) Take 0.1 ml into small test tube for further process.
B) Standard Preparation
Make dilution of the Endotoxin (concentration 0.470 EU/ml) according to the product limit.
For making 0.235 EU/ ml ( If the Product limit is 0.25) proceed as follows;
1) Take 0.05 ml of the reconstituted Endotoxin in the test tube after stirring.
2) Add 0.05 ml of pyrogen free water and vortex to mix. Now the final dilution is 0.235EU/ml.
3) Take 0.05ml of step 2 into small test tube for further process.
C) Blank Preparation
Pour 0.05 ml of pyrogen free water (Being used in the test) in small test tube as a blank for further process.
D) Lysate Addition
1) Place the tube stand for small test tubes (containing the tubes of blank, standard and diluted samples in ice water bath or suitable ice water container
2) Add 0.05ml of lysate to all of the tubes as soon as possible.
3) Stir the contents of every tube soon after the addition of lysate for few seconds. Avoid foaming.
E) INCUBATION
Soon after the addition of lysate, place the test tube rack in the incubator set at 32.5+2.5oC for 30 minutes. The tube rack can be placed in the water container placed in the incubator.
F) BLOCKING THE REACTION
After completion of the incubation period, place tube rack in ice water bath then block the reaction immediately by one of the two methods mentioned below:
1) By Acetic Acid
Add 0.4 ml of 0.8M Acetic acid into each tube and stir to mix.
2) By Diazo Coupling Reagent
Three bottles of the reagent are used as under;
a- Add 0.25 ml from bottle No 8s to each tube and stir to mix.
b- Add 0.25 ml from bottle No 9s to each tube and stir to mix.
c- Add 0.25 ml from bottle No 10s to each tube and stir to mix.
G) ABSORBANCE
1) Measurement at 405nm
If 0.8M Acetic acid is used to block the reaction then absorbance reading is taken at 405nm.
Note: The readings. Glass photocell is used for reading at 405nm. Because the volume of the tube content is not sufficient the distilled water is added to each tube and is stirred to mix.
2) Measurement at 545nm
When Diazo coupling reagent is used for blockage of the reaction then the reading is taken at 545nm. Note all the readings.
Note: Distilled water is used for reference in both cases.
I) MANUAL MEASUREMENT OF ENDOTOXIN
Following Formula is used to calculate the results
Conc. of Std. In Solution X ( SampleABS – BlankABS)
Endotoxin/ml = X DF
In product Std. ABS – BlankABS
J) CALCULATIONS OF MVC, MVD AND ELC
1) MVC = M / K
Where is the lowest sensitivity of lysate, M is the maximum dose / kg body weight and K is constant having value equal to 5.
2) MVD = Concentration of Product in 1 ml / MVC
3) ELC = X MVD
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