Endotoxin is a general term for toxic substances present in Gram-negative bacteria and is a component in the cell walls of many Gram-negative bacteria, also known as lipopolysaccharide (LPS). Endotoxin is released when bacteria die, lyse, or destroy bacterial cells artificially, and also occurs during normal cell growth and division. The toxic effects of endotoxins of various bacteria are weak and roughly the same, which can cause fever, microcirculation disturbance, endotoxin shock, and disseminated intravascular coagulation. Endotoxin is detected by either a gel method or a photometric method, but both rely on Limulus (Horseshoe Crab) blood as a raw material for the Limulus reagent. Due to the influence of national policies, the raw material horseshoe crab used in endotoxin detection reagents is listed as a national second-class protected animal, which means that the raw materials of endotoxin detection reagents will be strictly controlled. With the gradual reduction of horseshoe crab resources and the development of recombinant technology, a new generation of recombinant factor C (rFC) endotoxin detection method was officially introduced into Pharmacopoeia.
Figure 1. The coagulation cascade in the horseshoe crab amoebocyte lysate. (Ding J L, et al., 2010)
Factor C is a protein sensitive to bacterial endotoxin in a traditional Limulus reagent, which can selectively recognize endotoxin. The rFC method is to express the rFC of Limulus orientalis in the form of genetic recombination. The rFC bound and activated by endotoxin can cleave the substrate to obtain a free fluorescent group. The release is directly proportional to the concentration of endotoxin, allowing quantitative detection of endotoxin. The rFC endotoxin detection method has attracted more and more attention due to its characteristics of rapidity, simplicity, and non-animal origin. It is expected to replace the Limulus reagent and become the main method for endotoxin detection. The rFc method only needs one step to enzymatically digest the fluorescent substrate to generate fluorescence, and feedback on the endotoxin content according to the amount of substrate digestion, which fully complies with the three principles of experimental animals: reduction, replacement, optimization, and at the same time can meet the endotoxin detection reproducibility. Therefore, the advantages of adopting rFC instead of LAL are obvious.
Figure 2. Horseshoe crab in a shallow water of Atlantic ocean.
In addition, Limulus blood is a complex component. For different individuals, Limulus blood collected at different times and places will be different, which leads to certain batch-to-batch fluctuations in the Limulus reagent. The rFC method can guarantee its batch-to-batch stability due to its controllable production process. Enable enterprises to achieve standardized testing. Nowadays, the demand for endotoxin detection is increasing sharply, but the resources of Limulus are shrinking sharply. This relationship between supply and demand also brings great risks to the use of Limulus reagent. The rFC method is based on its infinitely repeatable production process, which can ensure a continuous and stable supply.
rFC endotoxin detection reagent does not depend on animal-derived components, has high sensitivity, and high specificity, and can be stably and continuously provided as an endotoxin determination reagent that replaces the Limulus reagent. Using this reagent for endotoxin detection has considerable advantages. rFC endotoxin detection reagents are chemically synthesized, and will only become cheaper and cheaper in the future with the increase in production. With the advantages of technology and cost, it is historically inevitable to completely replace LAL reagents. In the future, the rFC endotoxin detection method will be the development direction of the traditional Limulus reagent endotoxin detection method, which has been accepted by many international regulatory agencies.
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