Endotoxin assay

Summary

Endotoxin is a component of lipopolysaccharide (LPS) in the cell wall of Gram-negative bacteria, and lipid A is the active part. Individual Gram-positive bacteria also contain endotoxin; spirochetes, chlamydia, and rickettsiae also contain LPS, which is endotoxically active. Endotoxin is not released into the environment when the bacteria are living, but only released after autolysis of the bacteria or lysis of the bacteria by artificial methods. Its nature is more stable, heat-resistant, antigenic is not strong, the relative molecular mass is more than 100,000, the endotoxin of different bacteria cause the body to produce the same symptoms, such as fever, leukopenia, microcirculatory lesions and shock. In the past, endotoxin was detected by rabbit fever method, which has been replaced by horseshoe crab test due to its susceptibility to the influence of individual animals and environmental factors. Source: Microbiology Laboratory (Third Edition)

Operation method

gelation

Principle

Limulus amebocyte lysate (LAL) is a marine arthropod whose nucleated metastatic cells in the blood contain coagulase proteins and coagulable proteins. After freeze-thawing and lysing of these cells, horseshoe crab limulus amebocyte lysate (LAL) is made. If this lysate meets with endotoxin in the specimen to be examined, the endotoxin activates the coagulase of LAL to become coagulase, and then acts on the coagulable protein to make it coagulate into a gel state, which makes the horseshoe crab test one of the most sensitive methods to detect endotoxin, and is 10~100 times more sensitive than rabbit thermogenic test, and can detect 0.01~1 ng/g. Horseshoe crab test is one of the most sensitive methods to detect endotoxin, which is 10-100 times more sensitive than rabbit pyrogen test, and can detect trace endotoxin of 0.01-1 ng/ml. Horseshoe crab test is clinically used for the early diagnosis of endotoxemia caused by gram-negative bacterial infections, gram-negative bacterial meningitis, as well as for the detection of pyrogens in pharmaceuticals and biological products, and it is a rapid, simple and sensitive method. There are three main methods of horseshoe crab test: gel method, precipitated protein method and color-producing substrate method. The latter two methods are improved from the gel method, and their sensitivity is 5-10 times higher than that of the gel method, which can quantitatively determine the content of endotoxin, but the operation is more cumbersome. The following is a description of the commonly used method - gel method.

Materials and Instruments

Samples to be examined
Horseshoe crab reagent Standard endotoxin Sterile distilled water without pyrogens
Small test tubes Straws Constant temperature water bath

Move

1. Take one piece of horseshoe crab reagent and add the specified amount of pyrogen-free sterile distilled water to dissolve it according to the instructions.

2. Take the endotoxin standard and dissolve it in sterile distilled water without pyrogen.

The concentration used depends on the sensitivity of each batch of solubilized material.

Generally dilute to 20 Eu/ml (Eu is the unit of endotoxin). 3.

3. Take 3 small test tubes, number them individually, add the components according to the table below and proceed. 4.

4. At the end of incubation, gently remove the tubes from the water bath.

Do not shake the tubes to prevent gel disruption and false negatives.

Slowly invert the tube 180°. If the gel forms a solid clot, it is positive; if it is semi-fluid, it is weakly positive; if only an increase in turbidity is seen, or a small amount of flocculent juns, or if there is no change, it is negative.

Caveat

Because a very small amount of endotoxin can lead to LAL gelation, test tubes and pipettes used in this test should be de-thermogenized beforehand. Heat-resistant articles such as glass can be dry-heated above 180 ℃ for 2 h or 250 ℃ for 30 min to completely destroy the endotoxin; non-heat-resistant articles can be soaked in hydrogen peroxide.

Common Problems

Although the horseshoe crab test is sensitive for the determination of endotoxin, the following factors may affect its sensitivity in the test. 1:

1. the quality of LAL reagents, the sensitivity of each batch of reagents may be different, and this difference is related to the source, preparation method, etc. 2. pH has a significant effect on the gelation of LAL.

2. pH has a significant effect on LAL gelation, pH should be controlled within 6-8, the optimal pH is 7.0 earth 0.2, therefore, in the detection of drug pyrogen, the acidic or Pian drug, must be adjusted to its pH.

3. Incubation temperature and time. Generally 37 ℃, 45 ~ 60 min, can also be incubated for 4 ~ 24 h, to extend the observation time, can increase the positive rate. 4.

4. The nature of the object to be detected, such as the detection of blood endotoxin concentration of less than 10 ug / L, can only be measured in the platelet fraction.

When using this method. Platelet-rich plasma should be used.

If diluted with vaporized water. Not only does platelet lysis release endotoxin, and can dilute the inhibitory factor in plasma, so it can significantly enhance endotoxin activity. In order to remove the inhibitory factors in the blood, the diluted plasma can be heated and then test the long-snout, or chloroform extraction and other methods.

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