ENDOTOXIN TESTING USP 85

Amrutha Raiker• Introduction to bacterial endotoxins• Types of Endotoxin tests• Procedure

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.

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

• Limit test• Semi QuantitativeGel Clot

• End Point Turbidometric

• Kinetic Turbidometric

Photometric I:

Turbidometric method

• End point Chromogenic

• Kinetic Chromogenic

Photometric II:

Chromogenic Method

DIFFERENT KINDS OF BET

Set UP• BET Kit• Heating

Block

Reconstitute CSE vial• Standard

Preparations

• Dilution Calculations

Sample Preparations • MVD• Lysate

Calculations

PROCEDURE OF BET

Control Standard Endotoxin(CSE) Dilution

CONFIRMATION OF LYSATE SENSITIVITY

CSE Dilutions +Lysate

Incubation Results

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

LYSATE SENSITIVITY

• Geometric Mean End Point Concentration= Antilog (Se/f)Where,

Se is Sum of log endpoint concentrationf 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

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.

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.

PRODUCT INTERFERENCE FACTOR

Sample BET water

MVD/4 CSE Lysate

Negative Product Control

50µl 50µl 4 λ50µl2 λ 50µl1 λ 50µl0.5λ 50µl

50µl

Positive Product Control

NA 50µl 4 λ50µl2 λ 50µl1 λ 50µl0.5λ 50µl

100µl

Positive Water Control

50µl NA 4 λ50µl2 λ 50µl1 λ 50µl0.5λ 50µl

100µl

Negative Water Control

100µl NA NA 100µl

BET TEST RESULTS Sample

Control Standard Endotoxin

LAL Reagent Water

Product Lysate Results

Negative Water

ControlNot

Applicable 100µl Not Applicable

100µl Negative

Positive Control 50µl of 4ʎ 50µl Not

Applicable100µl Positive

Product Positive Control

50µl of 4ʎ Not Applicable

50µl 100µl Positive

Product Not Applicable

50µl 50µl 100µl Negative

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

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

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.

REFERENCES

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