Endotoxin is widely found in the cell walls of Gram-negative bacteria, such as E. coli. In the process of expressing recombinant protein, E. coli produces macroendotoxin. Endotoxin, as a heat source, leads to inflammatory and febrile reactions in the organism, so when the recombinant protein is used in some animal experiments, endotoxin needs to be removed in order to avoid causing adverse reactions in the animals and thus affecting the experimental results.
Table 1. Effects of endotoxin in different experiments.
|Application of recombinant protein||High endotoxin||Low endotoxin|
|In vitro enzyme activity test||no effect||no effect|
|Protein antigen, preparation of antibodies||no effect||no effect|
|In vitro cell differentiation culture||influenced||Requires low endotoxins <1EU/ug|
|Other animal in vivo experiments||big influence||Requires low endotoxin <0.1EU/ug|
|Evaluation of mouse efficacy||influenced||Requires low endotoxin|
Creative BioMart' recombinant protein endotoxin testing service mainly uses chromogenic LAL analysis and Gel Clot LAL Assay.
Chromogenic LAL analysis is an improvement on the LAL/TAL method, which uses a chromogenic substrate to detect the presence of endotoxin in solution. The dynamic color development method does not monitor the change in turbidity of the solution, but monitors the color change of the solution. The more endotoxin contained in the test solution, the faster the solution turns yellow. The endpoint colorimetric method uses a fixed test time, and the yellow intensity is used to distinguish the level of endotoxin. The Chromogenic LAL Analysis Service is designed to detect endotoxins through a discoloration reaction caused by the interaction between LAL and endotoxin. The mechanism of this reaction is a complex enzyme-linked reaction, as shown in the figure below. In the presence of LPS, factor C in LAL/TAL is activated, thereby activating factor B. Factor B activates procoagulase to thrombin. Finally, thrombin cuts the chromogenic substrate into a yellow product. We measure endotoxin content by color depth.
Fig1. Mechanism of Chromogenic LAL Assay.
The Gel Clot LAL Test Service is designed to detect endotoxins through the formation of gels through the interaction of LAL and endotoxins. The mechanism of this reaction is a complex enzyme-linked reaction, as shown in the figure below. In the presence of LPS, factor C in LAL / TAL is activated, thereby activating factor B. Factor B activates procoagulase to thrombin. Finally, lectin cleaves lectins into lectins, which bind together to form a clot. And we determine the presence or absence of endotoxin by forming a gel.
Fig2. Mechanism of Gel Clot LAL Assay.
Creative BioMart offers a corresponding endotoxin testing service. You can purchase the corresponding endotoxin removal kit and related accessory products according to the needs of your own samples. We guarantee that all instruments, water, reagents, and consumables used in the experiment are free of endotoxins, and the experiment is conducted in a clean room to ensure that low levels of endotoxins are returned to the sample. In addition, we can also provide you with related services including:
|Project name||Endotoxin testing in recombinant protein|
|Testing purpose||Endotoxins are widespread in the cell walls of Gram-negative bacteria (such as E. coli). During the expression of recombinant proteins, E. coli produces large endotoxins. As a heat source, endotoxin can cause inflammation and fever reactions in the body. Therefore, when using recombinant protein in some animal experiments, it is necessary to remove endotoxin to avoid causing adverse reactions in the animal body, thereby affecting the experimental results.|
|Testing cycle||3-5 days.|
|Service including||We provide you with raw data and test reports.|