Detection and Removal of Endotoxins

Endotoxins are found attached with bacteria. It is a toxin molecule in which bacterial structure can easily recognize the immune system. Endotoxins are not secreted by bacteria to produced adverse effects to the host. During the plasmid DNA culture, it should be free from all contaminants. When outcome of microbiological culture are contaminated it may result in death of an organism. For instance, if ovalbumin is contaminated with endotoxins, allergic response arises. Special care should be taken to avoid the presence of contaminants in culture.  Depyrogenation oven is used for maintaining contaminant free microbial culture. Growth rate of endotoxin is reduced on the basis of time and temperature.

Endotoxin acceptable limit

They are measured in terms of EU, Endotoxin Unit. 100 pg of bacterial lipopolysaccharide which is present in 105 bacteria is represented as one EU. When EU is more than 5 EU/kg body weights of humans, they are infected. Their presence is indicated by fever, low BP, heart rate increases and low urine excretion. Permissible limit of endotoxin set by FDA is 0.2 EU/kg for intrathecal injection drug, it is 5 EU/kg for non for intrathecal injection drug and it should be 0.2 to 0.5 EU/kg for a sterile water.

Detection of endotoxins

Endotoxins are detected by LAL assay test. LAL is Limulus amebocyte lysate and are extracted from horseshoe crab, Limulus polyphemus blood cells. LAL interacts with gram negative bacteria’s cell membrane component like lipopolysaccharide or with endotoxin produced by bacteria. This helps in endotoxin detection and also in quantification of bacterial endotoxins by LAL assay test.

Limulus amebocyte lysate assay

Animal blood cells called amebocyte is mobile. When bacterial endotoxin interacts with cell membrane, it results in coagulation. Coagulation has bacterial infections. Horseshoe crab pericardium blood is removed. To separate the blood from serum it has to be centrifuged. Cell lysis occurs by swelling and then they burst. As a result of cell lysis chemicals are released, which are then subjected to freeze drying and purified. To confirm the presence of endotoxins lysate is mixed with water. Coagulation occurs as a result of contamination. There are three methods of LAL assay test.

• Gel clot
• Turbidimetric
• Chromogenic

Source of LAL can be either blood or cerebro spinal fluid. Series of LAL actions are triggered by 1, 3 beta D glucan. Eventually Limulus amebocyte lysate and 1, 3 beta D glucan are pathogen associated patterns of molecular organism.