Bacterial endotoxin is a component of the cell wall of Gram-negative bacteria and has a variety of biological activities. Its chemical structure is lipopolysaccharide (LPS). Endotoxin is the main pyrogen substance, which can cause hair ripening, microcirculatory disturbance, endotoxin shock, and disseminated intravascular coagulation. Bacterial endotoxin testing (BET) is an essential safety requirement in the pharmaceutical and biomedical industries. If endotoxin enters the patient's blood in sufficient concentration, it may cause harmful symptoms such as fever and septic shock, which may be fatal in severe cases. Therefore, any drug product entering the human body, including parenteral drugs and injectable devices, must be tested for endotoxins before being placed on the market.
Figure 1. LPS stimulate the innate immunity. (Harm S, et al., 2021)
Bacterial endotoxin inspection can be done in six different ways:
Limulus reagent is a biological reagent obtained by extracting amoeba cell lysate from the blue blood of the arthropod "Limulus" living in the ocean and then freeze-drying at low temperatures. It is specially used for the detection of bacterial endotoxin.
In the gel method, within a specific pH range, endotoxin molecules will coagulate in the gel to form a solid gel. High endotoxin levels will result in large gels, while low endotoxin levels will result in small gel spots. Therefore, by observing the distribution of the gel over a specific pH range, the level of endotoxin can be quantitatively determined. This method is relatively simple to operate, economical and does not require special measuring equipment.
The nephelometric method is a method of optical detection of turbidity increase, which can be divided into the endpoint turbidimetric method and dynamic turbidimetric method according to different measurement principles.
The principle of endpoint turbidimetry is to mix endotoxin with a specific pigment and use spectroscopic technology to detect endotoxin levels. In addition, the level of endotoxin can also be judged by observing the color change of the pigment.
The principle of dynamic turbidimetry is that when the endotoxin level is high, the host cell will secrete endotoxin, thereby inhibiting the growth of its cells. In this case, the experiment can be performed by measuring the turbidity of the cells. The method is simple, fast, highly sensitive, and wide detection range, but requires sophisticated special equipment.
The chromogenic method is a method for determining the content of endotoxin by detecting the coagulation enzyme produced when the Limulus reagent reacts with endotoxin and detecting the amount of chromophore released from a specific substrate. According to the detection principle, it can be divided into the endpoint chromogenic method and the dynamic chromogenic method.
The endpoint chromogenic method is a method for determining the endotoxin content based on the quantitative relationship between the endotoxin concentration in the reaction mixture and the amount of chromophore released at the end of incubation.
The dynamic chromogenic method is a method for detecting the reaction time required for the chromaticity of the reaction mixture to reach a preset absorbance or detecting the growth rate of the chromaticity. The method is simple, rapid, sensitive, and reproducible.
ELISA uses a microwell plate coated with anti-endotoxin antibody to absorb endotoxin in the sample, then develops color with enzyme-labeled antibody and substrate, measures its activity by absorbance, and calculates the amount of endotoxin quantitatively. The method is highly sensitive and can detect very low doses of endotoxin.
In addition, there is the rocket immunoelectrophoresis limulus test (TALRIE), L-polylysine ELISA method, double antibody sandwich ELISA method, etc. These methods have the characteristics of strong specificity and high accuracy, but their application has yet to be clinically verified. And the operation needs to be further simplified.
By utilizing the characteristics of LPS to stimulate immune cells to produce IL-1 and TNF-α, standardized cells are used as target cells to detect the content of cytokines in the cell culture supernatant and calculate the content of cytokines and lipopolysaccharide in the test sample.
Taking the neutrophil oxidative reaction induced by CR1 and CR3 receptors as the reaction platform, the content of endotoxin was detected by measuring the biological effect of endotoxin on neutrophils.
After the endotoxin is fluorescently labeled with a monoclonal antibody against the endotoxin surface epitope, it is detected by flow cytometry.
After derivatization of the lipid A moiety of endotoxin, detection was performed by high-performance liquid chromatography (HPLC) and gas chromatography (GC-MS).
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