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Limulus Amebocyte Lysate (LAL) Test Methods

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. Endotoxin. Factor C. Activated Factor C. b - Glucan.

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Limulus Amebocyte Lysate (LAL) Test Methods

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  1. Limulus Amebocyte Lysate (LAL) Test Methods

  2. LAL Test Methods • The gel-clot method • The kinetic turbidimetric method • The chromogenic methods (kinetic and endpoint)

  3. Gel-Clot Turbidimetric Chromogenic

  4. Biochemical Reaction 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

  5. 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

  6. positive cloudy negative Reading the Gel-Clot Test

  7. 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

  8. Turbidimetric Methods light DETECTOR

  9. 0.5 EU/ml 0.25 EU/ml 0.125 EU/ml optical density 0.0625 EU/ml 0.03125 EU/ml threshold OD time Kinetic Data - OD vs time

  10. 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

  11. 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)

  12. Standard Curve (Kinetic Test)

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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)

  18. 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)

  19. 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)

  20. 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

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

  22. 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

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