The Bacterial Endotoxins Test (BET) using Limulus Amebocyte Lysate (LAL) is a method that utilizes the coagulation reaction between amebocytes from horseshoe crabs and trace amounts of bacterial endotoxin to determine whether the endotoxin level in a sample complies with the specified limit. Compared with the rabbit pyrogen test, the LAL test offers the advantages of being simpler, faster, more sensitive, and more reproducible. However, the test can be affected by various factors. Therefore, the following aspects should be carefully considered when performing an LAL test.
1. LAL Reagent Sensitivity
Pharmacopoeial requirements specify that the sensitivity of each batch of LAL reagent should be verified before use. Due to differences among manufacturers, LAL reagents with the same labeled sensitivity may produce different results when testing the same sample. Therefore, verifying the sensitivity of the LAL reagent in advance can help prevent erroneous conclusions caused by variations in reagent performance.
2. Operating Conditions
2.1 Operator Proficiency
Before operation, hands should be disinfected with alcohol. Before opening an ampoule, the neck should first be scored with an ampoule file, regardless of whether it is a color-dot or color-ring breakable ampoule. The neck should then be carefully snapped open by hand. Do not use forceps to strike the ampoule, as this may cause glass fragments to fall into the ampoule.
Important: During ampoule opening, avoid using forceps to strike the ampoule, as glass fragments may contaminate the reagent.
During the reconstitution of the LAL reagent, sample dilution, and sample addition, all volumes should be measured accurately. Solutions should be added slowly to avoid introducing air bubbles. Avoid moving the hands back and forth over the opening of ampoules or test tubes. After adding the sample, immediately seal the tube with sealing film and place it promptly in a constant-temperature water bath.
The entire operation should be performed quickly and carefully; otherwise, false-positive results may occur.
2.2 Temperature
Temperature has a significant effect on LAL reagent sensitivity. As the temperature increases and the incubation time is prolonged, the sensitivity of the LAL reagent may also increase, potentially resulting in false-positive results. Excessively high temperatures may damage the LAL reagent.
The temperature should be controlled at (37 ± 1) °C, with an incubation time of (60 ± 2) min.
2.3 Test Tube Rack and Water Bath
A dedicated test tube rack is recommended. The diameter of the rack openings should closely match the diameter of the ampoules or test tubes to minimize lateral movement.
The water bath should be placed on a stable, level surface and should not be tilted. During incubation, the tubes should not be removed from the water bath for frequent observation.
Avoid Vibration: Vibration should be avoided, because the gel formed by the reaction between the LAL reagent and bacterial endotoxin can become deformed when disturbed, potentially resulting in a false-negative interpretation.
3. Other Considerations
3.1 Sample Dilution
When using an LAL reagent with higher sensitivity, the sample may need to be diluted to a greater dilution factor to ensure that the test results are within the appropriate detection range.
3.2 Thorough Cleaning and Sterilization of All Apparatus
All laboratory glassware and other apparatus used for the test should be thoroughly cleaned and appropriately sterilized. Particular attention should be paid to equipment that has been exposed to acids, alkalis, detergents, washing powders, or cleaning solutions.
Such equipment should be thoroughly rinsed to remove any residual cleaning agents, as trace residues may interfere with or destroy extremely small amounts of endotoxin and consequently affect the accuracy of the test results.