Limulus amebocyte lysate lal test methods
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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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Limulus amebocyte lysate lal test methods

Limulus Amebocyte Lysate (LAL) Test Methods


Lal test methods

LAL Test Methods

  • The gel-clot method

  • The kinetic turbidimetric method

  • The chromogenic methods

    (kinetic and endpoint)


Limulus amebocyte lysate lal test methods

Gel-Clot

Turbidimetric

Chromogenic


Biochemical reaction

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


The gel clot method

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

Reading the Gel-Clot Test


Turbidimetric methods

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 methods1

Turbidimetric Methods

light

DETECTOR


Kinetic data od vs time

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


Kinetic turbidimetric method

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 method1

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

Standard Curve (Kinetic Test)


Kinetic turbidimetric method2

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

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

  • 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

  • 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

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

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

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

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 dilution1

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


Select a sensitivity

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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