Comparing the LAL Method with the Pyrogen Test on Rabbits

When the goal is to check the content of endotoxins that exist in a sample, The LAL (Limulus Amebocyte Lysate ) method has largely replaced the pyrogen test that used to be conducted via the use of rabbits not only in the experiments that are conducted in research, but also in the pharmaceutical and food industry as well as other industries. Bacterial endotoxins are named this way as they are substances that form part of the cellular wall of the Gram-negative bacteria, which cause toxicity in humans and many animals. As we will see in this article, there are various reasons for which the LAL method is used for the detection of bacterial endotoxins as a preferable method to the one with rabbits.

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The pyrogen test on rabbits is based on the measurement of the increase in the rabbit's temperature upon being injected with a product that could contain a contaminant of the pyrogen type. The pyrogens, as their name suggests, refer to all the substances that cause an increase in fever, also known as pyrexia. Upon entering into contact with pyrogens, rabbits have an increase in their temperature, just like humans. For this reason and since they are animals used in laboratories for different purposes, they were chosen to conduct this test.

The disadvantages of the pyrogen test making use of rabbits for the determination of bacterial endotoxins are numerous:

• It is a long test and therefore, the temperature of the animal needs to be measured during the 3 hours following the injection, at approximately 30-minute intervals.

• 3 animals need to be used for each dissolution to be tested. It is necessary that these animals did not present an increase in temperature in the two previous weeks and the same animals should not be used until after two days have passed following the test. Moreover, various animals that have a stable temperature should be chosen for the control group, which is why it is necessary to have a great quantity of rabbits to conduct the test, especially in industries with a great volume of production. Therefore, it can be regarded as a laborious test.

• If it is thought that many other substances which are both endogenous and exogenous could be causing the increase in the rabbits' temperature, this could prove to be a highly limiting factor to use this test for the determination of endotoxins in a sample. Although it is difficult that this test gives a false positive as a result due to the endogenous substances because of the great number of subjects used for the test, it is very common to see that it provides wrong results when the samples are found to be contaminated with a virus, fungus or any other type of substance which could cause fever in all the animals used in the test.

• It is an inadequate method to determine pyrogens in medicines, such as steroids, those that are used in chemotherapy and others that belong to a great group of substances, which, when they are administered, could cause the organisms to respond with mechanisms that lead to an increase in temperature, this being another one of the highly limiting factors of this test when research with medicines are conducted or when it comes to the control of endotoxins in the pharmaceutical industry.

• A very important disadvantage is the fact that the content of endotoxins that are present in a sample cannot be quantified via this test, which only offers a qualitative result.

• There are products, such as those that contain blood plasma, where endotoxins are found in an inactive form which does not lead to fever when they are inoculated, but which, with the passage of time, due to the metabolic processes that the plasma goes through in the organism, becomes toxic. On these occasions, the pyrogen method on rabbits gives a false negative result.

The LAL method

The LAL method takes its name from the fact that it is conducted with the hemolymph of the horseshoe crab, whose scientific name is Limulus Polyphemus. The hemolymph of this crab goes through a process of clotting in the presence of bacterial endotoxins, which is the pyrogen that has the highest probability of being found as a contaminant to the laboratory material and reagents. The clotting of the hemolymph in the Limulus crab was discovered in the 60s and, after several years, the same research team developed a test for the qualitative and quantitative determination of endotoxins using an amebocyte lysate of the horseshoe crab. This test rapidly started to be used as the method for the determination of bacterial endotoxins, offering the advantages of having a low limit of detection, a better specificity, a lower cost, a lower variability of the results and the possibility of quantifying the endotoxin content. Although this test also uses animals, due to its ethical consequences, the hemolymph extraction does not affect the life of the crabs.

For all the reasons that have been previously discussed, the LAL test is the currently recommended method by the main pharmaceutical companies and international bodies which regulate the quality control of medicine and food. There are cases, such as the Hepatitis B vaccine, where the pyrogen test on rabbits still continues to be used for the detection of endotoxins. However, it has been studied that, by conducting the LAL test with the gel-clot method and by preparing the vaccine samples in an adequate manner, it is more convenient to use the LAL method also in the samples where there is interference that causes false results. Although the LAL test is specific to endotoxins and does not detect other pyrogen substances, endotoxins are the most common and harmful pyrogens in many medicines, which is why a negative result of the LAL test is sufficient to determine that the sample is suitable to use.

Since the demand for reagents and kits to conduct the LAL test started, different companies all around the world have been working in this field. The brand Pyrostar of the company Wako is exclusively dedicated to providing researchers with all the reagents, equipment and laboratory material necessary for the determination of bacterial endotoxins via the Limulus Amebocyte Lysate test. To this end, it offers kits for conducting the test through the different methods of detection, which are the methods of gelation, turbidimetry and colorimetry, both in qualitative and quantitative manners.

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