LAL testing

Endotoxin Testing:

Endotoxin Testing

Endotoxin:

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

Endotoxin:

Endotoxin What is it ? a lipopolysaccaride

Endotoxin:

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

Endotoxin:

Endotoxin Why is it important? a pyrogen survives sterilization

Endotoxin Testing:

Endotoxin Testing Which products are tested? How is testing done?

Endotoxin Testing:

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

Endotoxin Testing:

Endotoxin Testing How is testing done? Rabbit pyrogen test LAL test

Rabbit Pyrogen Test:

Rabbit Pyrogen Test Standard method for most of the 20th century Substance being tested injected in rabbit’s ear

LAL Test:

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

LAL:

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

Horseshoe Crabs Being Bled:

Horseshoe Crabs Being Bled

Horseshoe Crab Blood:

Horseshoe Crab Blood

Types of LAL Test:

Types of LAL Test Gel Clot Turbidimetric Colorimetric

Gel Clot Method:

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.

Turbidimetric Method:

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.

Colorimetric Method:

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

Colorimetric Reaction:

Colorimetric Reaction Endotoxin 1. Proenzyme → Enzyme Enzyme 2. Substrate → Peptide + p-NA

Comparison of Methods:

Comparison of Methods Gel Clot Chromogenic Endpoint Chromogenic Kinetic Turbidimetric Semi- quantitative Quantitative Quantitative Quantitative Simple Least expensive, Requires 37 degree bath Requires spectrophotometer or plate reader Requires incubating plate or tube reader Requires incubating plate or tube reader Manually read and recorded Can be automated, allows electronic data storage Can be automated, allows electronic data storage Can be automated, allows electronic data storage Sensitive down to 0.03 EU/ml Sensitive down to 0.1 EU/ml Sensitive down to .005 EU/ml Sensitive down to .001 EU/ml * * (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)

Notes:

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