Method for noncovalent immobilisation of lipopolysaccharide shigella flexneri 2a on solid hydrophobic carrier

FIELD: chemistry.

SUBSTANCE: method involves passing a solution of lipopolysaccharide Shigella flexneri 2a in a 0.05 M triethylammonium acetate buffer at pH 4.7, which contains 10% n-propanol, through a column with a hydrophobic carrier which is Octyl Sepharose CL-4B, and then washing the column with the same buffer solution.

EFFECT: method can be used to produce carriers which enable extraction and purification of specific antibodies to lipopolysaccharides using affinity chromatography.

1 ex



The invention relates to the field of immunopharmacology and can be used to create media for affinity chromatography.


Affinity chromatography is one of the most effective methods of isolation and purification of specific immunoglobulins (antibodies) [1]. For this purpose usually carry out the following sequence of operations: activation of the solid carrier, chemical immobilization on it antigenic ligand, the processing of the thus obtained carrier mixture containing the specific antibodies, formation of a strong immune complex immobilized ligand with specific immunoglobulin complexes (antigen-antibody), delete all not contacting the ligand of the components of the mixture, the dissociation of the complex antigen-antibody, accompanied by the release of specific immunoglobulins purified.

One of the few drawbacks of this method is the need to create "links" (often apparently numerous) when immobilization of the antigen molecule on activated carrier that can lead to distortion of its spatial structure. When using affinity chromatography for isolation of antibodies against lipopolysaccharides (LPS) of gram-negative microorganisms often have extra for is rudnany, due to the formation of a strong micelles, which may include up to several thousand LPS molecules [2].

LPS is the major polysaccharide antigen of gram-negative bacteria, localized on the outer surface of the outer membrane of the cell wall and is a family of structurally related amphiphilic macromolecules. Polysaccharide fragment FSC presents the O-specific polysaccharide (OPS), built of oligosaccharide repeating units, the number of which may vary from 1 to several dozen. OPS is attached to the oligosaccharide bark, which, in turn, is associated with the lipid component of the lipid A. Lipid And serves as an "anchor"that holds the LPS molecule in the membrane of the bacterial cell, and the polysaccharide fragment is directed outward toward the environment.

In the LPS micelles formed in aqueous solutions, lipid And is located inside the micelles, and OPS, built in most cases of hydrophilic monosaccharide residues, as in bacterial cells, pointing towards the aqueous phase. When immobilization of LPS on the activated carrier the formation of covalent bonds ("links") can occur only with spatial affordable LPS molecules in the micelles, as well as with the individual, not part of the micelles LPS molecules, the number of which in aqueous solution is x small. Thus, despite the fact that quantification can show the desired level of immobilized LPS, the number of LPS molecules irreversibly covalently coupled to a carrier, may be low. So often at the stage of immobilization and subsequent stages of affinity chromatography in the eluates there is a noticeable level of LPS, which leads to contamination of the target product.


We propose a method of immobilization of the LPS of gram-negative bacteria on solid hydrophobic matrix without the formation of covalent bonds between the matrix and the LPS molecules. Received thus activated by lipopolysaccharide media can be used for isolation and purification of specific antibodies to LPS using affinity chromatography. A strong Association between LPS and hydrophobic matrix fixation of LPS on the surface of the matrix as in the formation of an immune complex, and the process of dissociation. The proposed method includes processing a hydrophobic matrix solution chemicalromance LPS in aqueous buffer with a pH of 3.0 to 8.0, containing the required number n-propanol. The introduction of n-propanol is achieved domiciliaria LPS that provides for effective adsorption of individual molecules of LPS on the surface of the matrix. The tendency LPS to domiciliario depends on solargeneration components in OPS (N-acetylamino-, deoxy-, detoxifier, O-acetyl groups and the like), as well as the charge OPS. So OPS, part of the FSC can be both neutral and contain acid fragments (residues of uronic acids, phosphate), positively charged components (residues of amino sugars, ethanolamine), as well as to have zwitter-ionic character, i.e. it may bear, for example, alternating carboxyl and amino groups. The degree of domicilliary LPS is significantly influenced by the composition, ionic strength and pH of the buffer. Therefore, when immobilization of specific FSC is necessary to determine the optimal qualitative and quantitative composition of the buffer, providing a full digitalnirvana LPS. Control of the completeness of domicilliary can be carried out using penetrating gel-chromatography on a column of Separate CL-4B or other similar characteristics of the separation gel with elution buffer intended for immobilization. Retention time chemicalromance form LPS is almost identical to the retention time of the OPS, which can be selected from the given LPS using mild acid hydrolysis (1%acetic acid, 100°C, 1 hour)accompanied by cleavage of lipid A.


As a solid carrier for immobilization of LPS can be used phenyl-, methyl-, ethyl-, butyl-, amyl-, hexyl-, ecil or domiciliaries (4% crosslinking), and octyl-. or phenyl-Sepharose CL-4B, and the column Supersoy 4 or 6 (Fast Flow) column with Toyopearl Resins butyl-650 and phenyl-650. The concentration of n-propanol in the buffer is determined experimentally, but usually does not exceed 10-15%. The salt concentration of the buffer components does not exceed 0.2 M Temperature in the process of immobilization is maintained in the range of 10-20°C, the temperature at which is affinity chromatography, typically no greater than the temperature maintained in the process of immobilization of LPS on the media. The immobilization time is usually 0.5 to 20 hours. The hydrophobic carrier is mixed with a solution of LPS in the buffer at a speed of 20-50 rpm using an orbital shaker and then washed with buffer for affinity chromatography, to lack of components immobilization buffer and LPS. All stages of immobilization can be carried out directly in the column for affinity chromatography. To determine the number sorbirovannoe on the media FSC measured volume of the gel is washed 2-3-fold volume of immobilization buffer containing 40-45% n-propanol, followed by dialysis against distilled water and lyophilization.


The following example is given to detail and illustrate the invention, is not intended to limit the claims of the present invention.

Through a column (1.5 x 1cm) with 10 ml octyl-Sepharose CL-4B miss 30 ml aqueous 0.05 M triethylammonium buffer, pH of 4.7, containing 45% n-propanol, at a speed of 5 ml/hour, and then balance column with the same buffer containing 10% n-propanol (about./about.) (buffer A). Next to the column attached to a flow-through UV detector (wavelength 226 and 276 nm), put a solution of 10 mg LPS Shigella flexneri 2a (obtained water-phenol extraction method of Westphal and Desription) in 3 ml of buffer A. After washing with 30 ml of buffer A. the column was washed with 30 ml fishriver and put on her 1 ml of hyperimmune rabbit serum. The column was washed with fistarol to absorption at 226 nm of less than 0.05 AU and then a 3.5 m solution of magnesium chloride for the destruction of the immune complex. The fractions containing (according to UV-detector) protein components, unite, dialist against fishriver (4 sessions, 1 hour) at a temperature of 8-10°C and lyophilized. A comparative analysis of the original serum and the resulting product using a response inhibition passive haemagglutination assays, enzyme immunoassay and Western blot turns showed that the affinity chromatography quantitatively selected mixture of specific antibodies against LPS Shigella flexneri 2a.

Sources of information

1. Affinity chromatography. Methods. Editors Pdin, Ugandan, Fmil. TRANS. from English. Kida. chem. Sciences Bagladeshi. M.: Mir, 1988.

2. High-molecular Weight Components in Lipopolysaccharides of Salmonella typhimurium, Salmonella minesota, and Escherichia coli. Peterson, A.A., and E.J.Mcgroarty. J.Bacterioloy, 1985, 162:738-745.

The method of immobilization of the lipopolysaccharide of Shigella exneri 2a on a hydrophobic solid media due to the formation of non-covalent bonds between the carrier and the lipopolysaccharide molecules, characterized in that through a column of hydrophobic media, representing octyl-Sepharose CL-4B miss the solution of lipopolysaccharide of Shigella flexneri 2a in 0.05 M triethylammonium buffer, pH 4,7, containing 10% n-propanol, followed by washing the column with the same buffer.


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