Mix a sample with LAL reagent in a test tube and incubate it using a block heater at 37 ± 1 °C, 60 ± 2 minutes, without subjecting to vibration. Upon completion of heating, immediately but slowly tilt the tube through 180°. If a gel has formed and maintains its integrity without deformation or collapse, the result can be determined positive, while it is negative if no gel has formed. During the test, a series of samples is diluted multiple times (usually 2-fold) to check if the result is positive in each sample. The maximum valid dilution or the minimum
concentration determined positive is referred to as the endpoint.
Invert the tube through about 180˚.
(Turn the tube upside down.)
The reagent is available in a single-type kit with reaction vials containing pre-dispensed reagent for a single measurement, and a multi-type kit for dispensing the required amount of the dissolved reagent into reaction vials. The single test kit is ideal for an assay with a few samples, and the multi test kit for a larger number of samples.
Single test is dispensed beforehand in a reaction tube. Multi test is used by dissolving Lysate reagent in endotoxin test water and dispensing necessary quantity of it in a reaction tube or plate.
A multi-type kit is used by dispensing 0.1 mL of dissolved LAL reagent into reaction tubes, which is then mixed after having 0.1 mL of the sample added. A single-type kit can be used by adding 0.2 mL of the sample to the reaction vial with pre-dispensed, lyophilized LAL reagent.
This technique uses synthetic chromogenic substrate cleavage to detect the activation of LAL reagent induced by endotoxin. Since the yellow color of p-nitroaniline is measured by absorbance at approx. 405 nm, the technique is not applicable if the sample has considerable absorbance at approx 405 nm.
This technique uses the change in gel turbidity to detect the activation of LAL reagent induced by endotoxin.
It cannot be applied to samples with considerable turbidity.