Determination of Bacterial Endotoxin

Endotoxins are associated with bacteria. Also known as Lipopolysaccharide (LPS), these large molecules are located in the cell membrane component of gram-negative bacteria. During viral infections, the cell membrane is broken down (cell lysis) and millions of LPS are released into the lysate. A single E. coli cell contains approximately 2 million LPS. These endotoxins can cause severe reactions in both people and animals. A strong immune response can result in systemic inflammatory response syndrome (SIRS), fever, sepsis and sepsis shock, and even death. Endotoxins have a high toxicity even at low concentrations and play an important role in the development of serious diseases.

Bacterial Endotoxin Tests (BET) are performed to detect levels of endotoxins using white blood cells from the horseshoe crab. The 3 methods used most often in BET are the gel-clot technique, the turbidimetric technique, and the chromogenic technique. While they all test for endotoxins, the best method to use depends on whether you need quantitative detailed results, or you simply want to detect whether endotoxins are present.

GEL-CLOT TECHNIQUE: This is the simplest test to perform for presence. Simply mix the sample of lysate TS and look for clots to form when endotoxins are present. The minimum concentration required to clot under standard conditions is listed on the sensitivity of the lysate TS. However, to ensure the integrity of the test you must first run tests to confirm that the labeled sensitivity is valid. You should do this when starting any new lot or there is any change in test conditions. Second, you must test for any interfering factors whenever any condition changes that could influence the results of the test.

TURBIDIMETRIC TECHNIQUE: This technique is a photometric assay measuring increases in reactant turbidity (absorbance or transmission). This is a quantitative test rather than simply a test for whether endotoxins are present. There are 2 types of tests. Endpoint-turbidimetric tests measure turbidity at the end of an incubation period. Kinetic-turbidimetric tests measure either the time it takes to get to a predetermined level, or the rate of development. Because this test is quantitative, it offers a greater sensitivity over a wider range.

CHROMOGENIC TECHNIQUE: This color test measures the chromophore released from chromogenic peptide from the reaction of endotoxins with lysate. This technique is similar to the turbidimetric technique in that there are again 2 types of tests. Endpoint-chromogenic gives a measurement at the end, or kinetic-chromogenic which measures over time. This test also requires pretests to ensure that there is valid criteria used and no interference with the actual test. Again, this test is quantitative for greater sensitivity. Endotoxins are difficult to remove because there is a high variability of molecular weights and they are relatively stable.

In addition, endotoxins are insensitive to changes in temperature and pH. The principle of bacterial endotoxin test is an important step required by the FDA for drugs in their final stage of formation. The medical world needs the innovative detection techniques available in BET as many therapeutics are developed in bacteria. BET tests are a critical step in vaccine development, immunotherapy, and drug and medical development.