The Detection of Endotoxins via the LAL Test, the Gel Clot Method

The Limulus Amebocyte Lysate test is recommended in international pharmacopoeias as the method for detecting bacterial toxins both in the raw materials used for the production of medicines and for the final products.

This test is also useful for the cosmetics industry and in food production as it is the method recommended by the FDA (Food and Drug Administration) for the detection of pyrogens.

It has already been a few decades since an effort was made by the scientific community and the society in general to minimize the use of animals in laboratory tests. One of the main advantages of the LAL test that the crabs, from which the hemolymph is extracted to prepare the LAL reagent, remain alive.

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Bacterial endotoxins

The Gram-negative bacteria have an external membrane formed by lipopolysaccharides. This structure is toxic to other superior organisms, such as animals and humans. These lipopolysaccharides are known as endotoxins in order to differentiate them from the other toxins that could be secreted by the bacteria, but do not form a part of their structure, called exotoxins. When the bacteria multiply or are destroyed, part of these endotoxins passes into the environment, thus performing their pathogenic function.

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The toxicity mechanism that the endotoxins trigger is caused by the lipid fraction of the lipopolysaccharides. For example, when the lysis of the bacteria within an organism takes place, the response in the presence of the lipids that go into the bloodstream can be through the activation of the complement system. This lipid fraction leads to the release of different cytokines, such as interleukins 1 and 8. The production of the tumour necrosis factor is probably also activated. The infection produced is associated with inflammatory processes and can pose a great danger for the infested organism. Interleukins 1 are a series of cytokines that the organism releases as an immune response and against the inflammation. This signal leads to the migration of neutrophils towards the place where the infection has occurred, producing chemotaxis. This facilitates the occurrence of phagocytosis; however, in some cases, depending on the state of the immune system of the individual and the level of infection, the bacteria could lead to a generalized sepsis, along with the risks that are brought about by the sepsis. It is known that there are many cases where the Gram-negative bacteria have caused death by systemic infection in higher mammals.

Among the Gram-negative bacteria, we come across some that are widely known, such as Salmonella and Escherichia coli and others that are not that known, such as Shigella and Neisseira.

The Limulus Amebocyte Lysate Test

The Limulus Polyphemus crab is one of the animals that have survived on land since prehistoric time with origins that date back more than 200 million years. This resistant animal experiences coagulation in its haemolymph due to the presence of bacterial endotoxins. Upon the discovery of this fact in the 60s, research was continued until managing to take advantage of this reaction to conduct in vitro detection tests for endotoxins in different environments. The Limulus Polyphemus belongs to a group of Horseshoe Crabs, which live in the Atlantic coast in the northern part of the American continent, including the Gulf of Mexico.

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What is responsible for the coagulation of the hemolymph in Limulus Polyphemus crabs are the amebocytes, the main components of the hemolymph of this animal. In invertebrates, the amebocytes fulfil the function of white blood cells in vertebrates. They defend the organism against pathogens; therefore, they respond to the influx of endotoxins from the bacteria by releasing a series of enzymes. Scientists studied this phenomenon until ascertaining that, if a lysate of the amebocytes extracted from the Horseshoe Crab in a watery environment is diluted, this could serve to detect very small quantities of endotoxins.

The amebocytes contain procoagulant enzymes that trigger a chain of reactions. The final product of these chain reactions is a gel comprised by coagulated proteins. The enzymatic response is produced when the amebocytes enter into contact with the endotoxins. This mechanism is often compared to the trypsin that also triggers a chain of reactions to finally form the thrombin, the agent responsible for the coagulation of blood in humans.

The LAL test is only valid for detecting endotoxins and not any other type of pyrogen (a name that is given to any compound that could cause fever). In many occasions, this test is used with the goal of detecting other pyrogens, which is wrong. While bacterial endotoxins, as they are highly resistant to heat and to different chemical reagents used for sterilization, are one of the most common pyrogens left present after the common measures of sanitation in industries, a negative LAL test only indicates the absence of endotoxins and not the absence of other pyrogen micro-organisms. Conducting this test can only lead to the confirmation of an environment free of pyrogens when this test is accompanied by other analyses and specific sanitary measures to eradicate the rest of the contaminating micro-organisms.

To use the LAL test, a confirmation needs to be conducted for the specific industrial process where the test will be applied. FDA regulates the confirmation of this test, encountering many records in the literature of this procedure, above all for injectable medications.

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Gel-Clot Method to quantify endotoxins via the LAL test

The LAL test can be conducted by using different methods to measure the process of gelation that occurs as a response from the amebocytes against the endotoxins. These methods are the so-called Gel-Clot method, turbidimetric and chromogenic methods. Here, we will focus on the Gel-clot method or gelation.

The Gel-Clot method is based on the presence or absence of a gel clot in your sample tube. The gelation occurs when proteins are coagulated due to the presence of endotoxins. The detection limit of the tests depends on the manufacturer of the kit that contains the LAL reagent. Using the Gel-Clot method, the detection limit is normally between 0.01 and 0.03 endotoxin units per one millilitre of the solution used in the test. This means that a solid gel does not come to be formed below this concentration of endotoxins when moving the test tube. A criterion used in the method of gelation is to turn the test tube 180º and ascertain that the gel remains intact. The Gel-Clot method can be used in a qualitative manner, yielding positive results or negative ones if the gel is not formed. The method can also be used in a semi-quantitative fashion.

Wako Chemicals USA, Inc. markets an LAL test, known as PYROSTAR™ ES-F that works via the Gel-Clot method. With the use of this test, the interference of β-1, 3-glucan, another polysaccharide that is found in the cellular wall of bacteria and fungi, is removed. In small amounts, this polysaccharide can interfere with the measurement of endotoxins. This test already has the β-1, 3-glucan in a bigger concentration than there usually is in the environment where the analysis of endotoxins takes place. In higher concentrations, this compound does not trigger the reactions that lead to the formation of the coagulant. The use of this test is restricted to research-related purposes. The procedure is simple, but it should be conducted with a lot of precaution in order to avoid the contamination of the samples. After incubating the tubes where the test is conducted at 37°C for one hour, the tubes are turned face down to check if the gel is formed or not. This is a qualitative test.

In addition to PYROSTAR™ ES-F, Wako provides investigators with a complete line of accessories and reagents for the purpose of LAL testing. Among the offered products, you can find control standard endotoxins, a solution for the extraction of endotoxins on medical devices and the Toxinometer® measurement system. You can also find other tests designed for endotoxin determination via chromogenic methods.