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 Turbidimetric Endotoxin Factor C Activated Factor C b-Glucan Factor B Activated Factor(s) B/G Factor G Clotting Enzyme Activated Clotting Enzyme Coagulogen Coagulin Gelation Turbidimetric Modified from Iwanaga et al., 1985

The Gel-Clot Method Simplest and most widely used The USP referee method The labeled gel-clot reagent sensitivity (l) is the least concentration of endotoxin to cause a solid clot under standard conditions

Reading the Gel-Clot Test positive cloudy negative

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 optical density time threshold OD 0.25 EU/ml 0.125 EU/ml 0.0625 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

Maximum Valid Dilution If l is 0.125 EU/mL and the unknown has an endotoxin limit of 2 EU/mL, calculate the MVD: Limit in EU/mL 2 EU/mL = 16 = l in EU/mL 0.125 EU/mL Limit in EU/mL 2 EU/mL = 64 = l in EU/mL 0.03125 EU/mL

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