From the 30s of the 20th century to the present, the technical methods for separating and identifying endotoxins designed according to the physical and chemical properties of endotoxins are mainly as follows:
1. Separation and extraction
(1) Trichloroacetic acid method
Boivin and Messrobeanu in 1935 combined live bacteria or freeze-dried E. coli under 4 conditions with 025N trichloroacetic acid is shaken together or vigorously stirred to lyse bacterial cells, allowing LPS molecules to upstream detach from the ruptured extracellular membrane. After centrifugation, the supernatant was concentrated, 2 times the volume of frozen ethanol was added, and the generated precipitate was dissolved, concentrated and freeze-dried to obtain LPS dry powder. The LPS extracted by this method contains about 10% bacterial proteins, which can affect the physicochemical properties and biological activity of LPS.
(2) Phenol-water method
The method established by Wesphal and Luederitz in 1952 for the isolation of LPS with low protein content has since been widely used for the extraction of water-soluble S-LPS. The specific process is as follows: in the aqueous solution of bacteria, the same amount of 90% phenol is added to lyse the cells, so that the endotoxin molecules are separated from the ruptured extracellular membrane. 68, after shaking for 10 15min, cooling, centrifugation, collecting the LPS-rich aqueous phase, and repeatedly extracting with water 3 5 times, then concentrating and freezing the aqueous phase in drying, to obtain crude LPS, dissolving the crude product in water, high-speed centrifugation (100000g, 2 3h), obtaining granular LPS, and then dissolving in water freeze-drying. The LPS extracted by this method has a high purity, and the protein content is 1% 3%.
(3) Phenol-chloroform-petroleum ether method
A method designed by Galanos for the extraction of R-LPS. The desiccating bacteria after degreasing with ethanol, acetone and ether in advance were extracted 2 3 times with 90% phenol chloroform and petroleum ether according to the mixed extract composed of 2 5 8, and the phenolic phase was taken to obtain LPS and porin (porin) extract, the solvent was removed by reducing pressure, water was added to form a precipitate, and washed 3 5 times, and then reconstituted with 80% phenol, dried under reduced pressure, and the obtained LPS crude product was dissolved in water, and the precipitation was obtained by ultracentrifugation, that is, the pure LPS product was obtained. The LPS purified by this method does not contain nucleic acids and polysaccharides, and the protein content can be controlled below 1%.
(4) Water-butanol method
Designed by Morrison equal to 1975. The water-butanol method is simpler and milder than the phenol method, and is suitable for the extraction of LPS of Escherichia coli and Salmonella. The resulting product contains about 1% protein. However, there are still some shortcomings in this method, because butanol cannot inactivate the enzymes in the product, resulting in the degradation of lipid A during preparation and storage.
2. Purification
(1) Ion exchange chromatography
Taking advantage of the non-specific adsorption of negatively charged LPS by cationic resins, LPS were separated and purified from the mobile phase.
(2) Affinity chromatography
Taking advantage of the characteristics of polymyxin B specifically binding to LPS, polymyxin B was coated on the surface of polymers such as polyacrylamine resin to make a chromatography column that specifically binds to LPS, and the LPS could be purified by affinity chromatography.
(3) Removal of metal ions and small molecular weight polyamines
Under normal circumstances, there are a large number of small molecule charged substances in the unpurified LPS solution, such as Mg2+, Ca2+ and a small amount of Fe2+, A13+, Zn2+, Na+ plasma and small molecular weight polyamines such as ethanolamine, putrescine spermine, spermidine and cadaverine. They neutralize the negatively charged phosphate groups on the LPS molecule, making it significantly less effective to remove LPS with charged adsorbents. Electrodialysis is the method of ion exchange for LPS purification. The principle is that a lot of anion exchange membranes are staggered in series, and the electrolyte solution flows between the membranes, and the cations (such as inorganic cations, alkaloids, etc.) in the solution move to the cathode after direct current is applied on both sides, and the anions (such as inorganic anions, organic acids, etc.) move to the anode. The anions can pass through the anion membrane smoothly, but when they move further, they are blocked by the adjacent cation membrane. Similarly, cations can pass through the cation membrane smoothly but not through the anion membrane. Neutral compounds and polymer compounds remain in the dialysate to obtain purified extracts (Figures 2-6).
Due to the fact that the extracted LPS contains many positively charged impurities, the pH value of the cathode can be found to increase during the electrodialysis of LPS, and the pH value of the anode does not change significantly. During the electrodialysis process, the pH value of LPS solution often decreases to a certain extent, and at the same time, due to the removal of Mg2+ and Ca2+ plasma components with stable LPS polymer function, the LPS polymer becomes a monomer and precipitates. To avoid this, neutralize it to pH 7 with NaOH or triethylamine0, so that it forms a neutral, highly water-soluble LPS sodium salt and LPS triethylamine salt and redissolves. At the end of electrodialysis, LPS is generally an acidic product, which has very low solubility in water, and can be directly lyophilized, stored at -4 -20 cryogenics, and can be neutralized and converted into LPS salts with different bases before each use.
(4) Removal of nucleic acid components
According to the difference in molecular weight between LPS and nucleic acid fragments, they can be removed by ultracentrifugation, electrophoresis and other methods.