Limulus Amebocyte Lysate (LAL) Test Methods

LAL Test Methods

• The gel-clot method

• The kinetic turbidimetric method

• The chromogenic methods (kinetic and endpoint)

Gel-Clot

Turbidimetric

Chromogenic

Biochemical Reaction

Modified from Iwanaga et al., 1985

Gelation

Coagulogen Coagulin

Endotoxin

Factor B Factor G

Clotting Enzyme

Activated Factor(s) B/G

Activated Clotting Enzyme

Factor C Activated Factor C -Glucan

Turbidimetric

The Gel-Clot Method

• Simplest and most widely used

• The USP referee method

• The labeled gel-clot reagent sensitivity () is the least concentration of endotoxin to cause a solid clot under standard conditions

Reading the Gel-Clot Test

positive

negative

cloudy

Turbidimetric Methods

• As coagulin molecules coalesce forming particles, the reaction mixture becomes turbid

• The rate of increase in turbidity is a function of endotoxin concentration

Turbidimetric Methods

light

DETECTOR

Kinetic Data - OD vs time

opticaldensity

timethreshold OD

0.25 EU/ml0.125 EU/ml0.0625 EU/ml

0.03125 EU/ml

0.5 EU/ml

Kinetic Turbidimetric Method

• The threshold OD (onset OD) is used as a point of reference for data collection

• The greater the endotoxin concentration, the shorter the time taken to reach the onset OD

Kinetic Turbidimetric Method

• The onset time is the time that it takes (in seconds) for the reaction to reach the onset OD

• Standard curves are constructed by plotting log10(onset time) on log10(endotoxin concentration)

Standard Curve (Kinetic Test)

Kinetic Turbidimetric Method

• Calculate sample endotoxin content by comparing with standards

• Take sample onset time and reference against standard curve to determine its endotoxin content

Interference

• Most samples, at some concentration, interfere with the LAL reaction

• Interference is caused by– sample interaction with the LAL reagent – sample interaction with endotoxin

Inhibition

• Inhibition is a reduction in sensitivity of the assay which causes an underestimation of the concentration of endotoxin

• Inhibition controls (PPC’s) prevent misinterpretation of negative results

Enhancement

• Enhancement is an increase in the sensitivity of the assay which causes an overestimation of the concentration of endotoxin

• Positive product controls (PPC’s) prevent misinterpretation of positive results in the photometric methods

False Positives

• Enhancement is not a false positive!• A false positive test is a positive in the

absence of endotoxin• False positives are rare

– trypsin (all methods)– activated serine proteases

(chromogenic)– beta-glucans (suspected, all methods)

Positive Product Control

• All LAL tests must have a control to demonstrate that the sample itself does not cause a false negative result

• A known quantity of endotoxin is added to a portion of the sample under test to provide an inhibition or positive product control (PPC)

Remove Interference

• Dilute with LRW first• Use a more sensitive LAL reagent or

method to increase the MVD• Reconstitute LAL with Pyrosol

(strongly buffered products outside the pH range, highly concentrated electrolytes, or for sample/endotoxin interactions)

Maximum Valid Dilution

• The maximum valid dilution (MVD) is the greatest possible dilution at which the limit can be detected

• This is the dilution used for the pass/fail test

• The MVD increases with increasing test sensitivity

• If is 0.125 EU/mL and the unknown has an endotoxin limit of 2 EU/mL, calculate the MVD:

Maximum Valid Dilution

Limit in EU/mL in EU/mL

= 2 EU/mL0.125 EU/mL

= 16

Limit in EU/mL in EU/mL

= 2 EU/mL0.03125 EU/mL

= 64

Select a Sensitivity

• Sensitivity is lowest point on curve• Consider the endotoxin limit and MVD• Perform preliminary tests• If interference cannot be overcome

without exceeding the MVD of a product, go to a more sensitive reagent or method