introduction Bacterial Endotoxin according to USP85. different procedure involved.

1. ENDOTOXIN TESTING USP 85
Amrutha Raiker
• Introduction to bacterial endotoxins
• Types of Endotoxin tests
• Procedure

2. INTRODUCTION TO ENDOTOXIN
• Bacterial Endotoxins are
Lipopolysaccrides(LPS), cause Fever and
Diseases
• Endotoxins bind with limulous
amoebocyte lysate to form clot, principle
for bacterial endotoxin test.

3. Bacterial Endotoxin Test
Gel Clot Method
•Based on Gel Formation
Turbidometric method
development of turbidity after cleavage of endogenous substrate
Chromogenic method
Development of colour by cleavage of synthetic peptide
chromogen complex

4. • Limit test
• Semi Quantitative
Gel Clot
• End Point Turbidometric
• Kinetic Turbidometric
Photometric I:
Turbidometric
method
• End point Chromogenic
• Kinetic Chromogenic
Photometric II:
Chromogenic Method
DIFFERENT KINDS OF BET

5. Set UP
• BET Kit
• Heating Block
Reconstitute
CSE vial
• Standard
Preparations
• Dilution
Calculations
Sample
Preparations
• MVD
• Lysate
Calculations
PROCEDURE OF BET

6. Control Standard Endotoxin(CSE)
Dilution

7. CONFIRMATION OF LYSATE
SENSITIVITY
CSE Dilutions
+Lysate
Incubation Results

8. LYSATE SENSITIVITY (D)
50µl of 2,1,0.5,0.25 λ of CSE and negative (0)
50µl of Limulus Amebocyte Lysate(LAL) reagent water
100µl of lysate
Incubate 1 hour 37
Firm gel Positive and no intact gel negative result. Lowest concentration should
be negative

9. LYSATE SENSITIVITY
• Geometric Mean End Point Concentration= Antilog (Se/f)
Where,
Se is Sum of log endpoint concentration
f is number of replicate test tube (usually 4)
• λ measured is EU/ml
• Value should not be less then 0.5λ and more then 2 λ
• Thus confirm labelled sensitivity

10. MAXIMUM VALID DILUTION (MVD)
Maximum Valid Dilution = Endotoxin Limit × Product Concentration
Lysate Sensitivity
Entotoxin Limit = threshold human pyrogenic dose of
endotoxin/ kg of body weight
maximum recommended human dose
of product / kg of body wt.

11. EXAMPLE FOR CALCULATION OF MVD
• The limit for a vaccine could be 5EU/kg of body
• For a 70kg person the EU limit would be 5*70=350units of EU
• Therefore according to the formula MVD will be 11,666.666
• Thus it can be diluted so many times.

12. PRODUCT INTERFERENCE FACTOR
Sample BET water MVD/4 CSE Lysate
Negative Product
Control
50µl 50µl 4 λ50µl
2 λ 50µl
1 λ 50µl
0.5λ 50µl
50µl
Positive Product
Control
NA 50µl 4 λ50µl
2 λ 50µl
1 λ 50µl
0.5λ 50µl
100µl
Positive Water Control 50µl NA 4 λ50µl
2 λ 50µl
1 λ 50µl
0.5λ 50µl
100µl
Negative Water
Control
100µl NA NA 100µl

13. BET TEST RESULTS
Sample
Control
Standard
Endotoxin
LAL
Reagent
Water
Product Lysate Results
Negative
Water
Control
Not
Applicable
100µl
Not
Applicable
100µl
Negative
Positive
Control 50µl of 4ʎ 50µl
Not
Applicable
100µl
Positive
Product
Positive
Control
50µl of 4ʎ Not
Applicable
50µl 100µl
Positive
Product
Not
Applicable
50µl 50µl 100µl
Negative

14. CHROMOGENIC ENDOTOXIN
QUANTIFICATION
A small volume of the sample (10μL) is combined
with the Limulus Amebocyte Lysate, and
endotoxins in the sample activate the proteolytic
activity of Factor C.
When the chromogenic substrate is added, the
activated protease catalyzes the cleavage of p-
nitroalinine (pNA), resulting in yellow color that
can be quantitated by measuring the absorbance
at 405nm (A405) and extrapolating against a
standard curve.
A standard curve is created using the E. coli
endotoxin standard included with each kit to
calculate endotoxin levels as low as 0.1 EU/mL,
where one endotoxin unit/mL (EU/mL) equals
approximately 0.1ng endotoxin/mL of solution
www.thermofisher.com

15. TURBIDOMETRIC QUATIFICATION OF
ENDOTOXIN
• This assay is performed in a 96-well plate
allowing many samples to be processed at
one time
• Kinetic Turbidimetric Assay is measured at
the 340 nm wavelength and has a
sensitivity range from 0.01 to 100.0 EU/ml.
http://www.lonza.com

16. TURBIDOMETRIC QUATIFICATION OF
ENDOTOXIN
• A sample is mixed with the reconstituted LAL reagent, placed in the incubating
absorbance plate reader, and automatically monitored over time for the appearance of
turbidity
• In the presence of endotoxin, the lysate will begin to gel, causing the solution to
become cloudy or turbid
• The time required for the change is inversely proportional to the amount of endotoxin
present.
• The concentration in unknown samples can be calculated from a standard curve.

17. REFERENCES
• www.thermofisher.com
• http://www.lonza.com
• Ryan, J. Endotoxins and Cell Culture. Corning Technical Bulletin. (2008).