Endotoxin Testing

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Endotoxin Testing
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Endotoxin

• What is it ?
• Where does it come from ?
• Why is it important?

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Endotoxin

• What is it ?
• a lipopolysaccaride

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Endotoxin

• Where does it come from?
• the cell membrane of Gram negative bacteria

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Endotoxin

• Why is it important?
• a pyrogen
• survives sterilization

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Endotoxin Testing

• Which products are tested?
• How is testing done?

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Endotoxin Testing

• Which products are tested?
• injectable drugs and medical devices
• which will contact blood or spinal fluid
• includes raw materials, water and
• in process monitoring

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Endotoxin Testing

• How is testing done?
• Rabbit pyrogen test
• LAL test

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Rabbit Pyrogen Test

• Standard method for most of the 20th century
• Substance being tested injected in rabbits ear

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LAL Test

• Developed in 1960s by Drs. Bang and Levin
• Based on clotting reaction of horseshoe crab
  blood to endotoxin
• Faster, more economical, more sensitive than
  rabbit pyrogen test

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LAL

• Limulus - genera of crab
• Amebocyte - crab blood cell from which
  active component is derived
• Lysate - component is obtained by separating
  amebocytes from the plasma and then lysing them

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Horseshoe Crabs Being Bled
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Horseshoe Crab Blood
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Types of LAL Test

• Gel Clot
• Turbidimetric
• Colorimetric

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Gel Clot Method

• Original method
• The official referee test
• The specimen is incubated with LAL of a known
  senstivity. Formation of a gel clot is positive
  for endotoxin.

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Turbidimetric Method

• A kinetic method
• The specimen is incubated with LAL and either the
  rate of increase in turbidity or the time taken
  to reach a particular turbidity is measured
  spectrophotometrically
• and compared to a standard curve.

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Colorimetric Method

• Endotoxin catalyzes the activation of a proenzyme
  in LAL which will cleave a colorless substrate to
  produce a colored endproduct which can be
  measured spectrophotmetrically and compared to a
  standard curve.
• Can be kinetic or endpoint

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Colorimetric Reaction

• Endotoxin
• 1. Proenzyme ? Enzyme
• Enzyme
  
• 2. Substrate ? Peptide p-NA

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Comparison of Methods
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Steps in Chromogenic Endpoint Procedure

• Read SOP (reagents are prepared)
• Label tubes and make dilutions of stock standard
• Pipette blanks, standards unknowns into
  prewarmed plate following template
• Start timer as LAL is added to first well.
• Add quickly with repeat pipettor to all
  wells being used mix.

• At T 10 mins add prewarmed chromogenic
  substrate in same manner as above
• At T 16 mins. Add stop reagent in same manner
  as above
• Read plate

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Template
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Notes

• Glassware, tips,and water must be
• endotoxin- free.
• Temperatures and timing are critical.