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An Overview of the Gel-clot Endotoxin Testing Method

The Gel-clot Endotoxin Testing Method

The gel-clot method is based on the clotting properties of the Limulus amoebocyte lysate (LAL), which reacts with endotoxins present in the sample. This method is primarily used for qualitative or semi-quantitative analysis and NJ Labs strictly follows USP <85> for endotoxin testing.  Here is an overview of this method:

  1. Sample Preparation: The test sample is put into solution, and then diluted if necessary. If the sample is already a liquid it can just be diluted.  A Maximum Valid Dilution can then be calculated.  It will also be necessary to reconstitute and dilute the CSE or Control Standard Endotoxin, which is an accepted Reference Standard of known Endotoxin value, usually reconstituted to 1000EU/ml and then diluted down to a working solution for sample testing.
  2. LAL Reagent Preparation: The Limulus amoebocyte lysate (LAL) reagent is prepared by reconstituting a lyophilized form of the lysate with a specific volume of water or a reconstitution buffer provided by the manufacturer. The LAL reagent contains enzymes and clotting factors necessary for the reaction.
  3. Test Setup: There are different steps involved depending upon whether you are performing a routine test, setting up a standard curve or a method validation.  For purposes of this discussion, for routine testing all that is necessary to do is to perform the test in duplicate using 2λ Negative Control, the unspiked diluted sample and a positive product control. Multiple test tubes are prepared, each containing a different concentration of the test sample. Control tubes are also included. The control tubes consist of positive and negative controls. The positive control contains a known amount of endotoxin, while the negative control is LAL Reagent Water which does not contain any endotoxin.  All tubes, reagents and consumables are also Pyrogen or Endotoxin Free.
  4. Reaction: At NJ Labs we use a 100ul volume of LAL reagent, added to each tube, including the control tubes. The tubes are then incubated at 37C +/- 1C in a non-circulating water bath for a temperature at 37°C for a period of 60 minutes +/- 2 minutes.
  5. Gel Clot Formation: If endotoxins are present in the sample, they will interact with the coagulogen in the LAL reagent. This interaction activates a clotting cascade, resulting in the formation of a gelatinous clot. The gel formation is visually observed and assessed by inverting the tube 180 degrees in one fluid motion to avoid disturbing the clot.
  6. Interpretation: After the incubation period, the tubes are examined for gel formation. If a gel clot is visible, it indicates the presence of endotoxins. But all the positive controls need to be positive and negative controls negative for the test to be accepted. Therefore, the gel-clot method is primarily used to confirm the presence or absence of endotoxins rather than quantifying their concentration.

It’s important to note that the gel-clot method is susceptible to interference from certain substances, such as high protein concentrations, complex formulations, or some types of preservatives. For example, (1→3)-ß-D-glucan interference. In such cases, other methods like the chromogenic or turbidimetric method may be more suitable. The choice of method depends on the specific requirements and characteristics of the sample being tested.

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