CS221747B1 - A method for preparing immunosorbents from polysaccharides isolated from yeast - Google Patents

Abstract

The invention relates to chemistry, biochemistry, immunology and microbiology. The purpose of the invention is to prepare an undissolved immunosorbent from polysaccharides isolated from yeast, which has the property of being able to sorbly bind to its surface protein antibodies against rash fungal diseases. This is achieved by crosslinking the polysaccharide isolated from the yeast cells in an alkaline medium with a bifunctional reagent most preferably 15 to 30% by weight with epichlorohydrin. to the weight of the dry polysaoharide at temperatures up to 50 ° C. After crosslinking, the insoluble polysaccharide gel is suspended in a water-swirl mixer in water to a particle size of 80-250 microns, which are then filtered, washed well with water and. water is displaced by organic solvent, preferably ethanol or acetone. The cross-linked dried polysaccharide immunosorbent has a swelling volume in water of 4 to 8 ml / g and is a suitable carrier for isolation and purification of antibodies effective against rash fungal diseases. The invention has utility in both human and veterinary medicine for the isolation of antibodies against rhinorrhagic diseases.

Description

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The present invention relates to a process for preparing immunosorbents from polysaccharides isolated from yeast.

The major immunologically active component of yeast and cyanobacterial microorganisms are Bananas, or mannose containing heteropolysaccharides such as galactomannans, glucomannans, gluconoxylOmanans, which are found on the cell surface of these microorganisms. The mannans of the pathogenic Candida species are the high-branched poles of the carbohydrate having in the main chain "(1-6), in the divine chains and (1-2), and (1-3) glycosidic bonds. The immunodominant group of these antigens are the de-chain consisting of 9-6 mannitose units lukewarm (1-2) glycosidic bonds (S. Suzuki and H. Sunayama: Jap. J. Uicrobiol. 13, 95 (1969)).

Immunologically active polysaccharides of microorganisms are isolated from the nutrient medium by non-multiple precipitation with ethanol, from yeast biomass by extraction with potassium hydroxide at a concentration of 2% by weight. (PAJ Gorin and JFT Spencer: Adv. Appl. Uicrobiol., 13, 25/1970) or with 0.2 U sodium chloride (O. Sikl et al. Coll. Czech. Chem. Commun, 35, 2,969 / 1970)) at elevated temperature. pure mananes are obtained by subsequent precipitation of soluble polysaccharides with Fehling's solution. However, the extracts of the immunologically active mananes are very low and do not exceed 5% by weight of the biomass feedstock.

Uanins of pathogenic yeast microorganisms are soluble in water and not directly used for the preparation of solid sorbents. In order to obtain suitable insoluble sorbents. it is desirable to modify the polysaccharides from yeast microorganisms by slagging. Kuniak, Η. H. Uarchesault: Starch 24, 110 (1972)) so that immunodominant groups of polysaccharide are retained for immunosorption of specific antibodies from blood serum.

The molecular weight of the mannans of the yeast microorganism polysaccharides is relatively low and ranges from 15,000 to 30,000. Several determinant groups are found on one polysaccharide molecule, but only six of them are specific to the specific antigenic antibody-antibody response. (J. Šandula and U. Vojtková: Folia Microbioiogica, 19, 94 (1974)). By cross-linking mannan, there is a denser distribution of determinant groups in the mannan macromolecule, so that the junction of the active immunosorbent centers does not interfere with the antigen-antibody interaction from spatial reasons.

Immunologically active mannans are found in the cell walls of yeast complexed with proteins and immunologically inactive glucans (C. E. Ballau: Adv. Uicrobiol. Phys. 14,93 (1976)). Complete removal of these accompanying biopolymers interferes with the difficulty and requires a complex purification process. We have found that it is not necessary to completely purify manan prior to the preparation of the infertile immunosorbent, the small amount of protein and glucan is not defective. The degree of diffusion of the polysaccharide decreases, the molecular weight decreases, and the number of immuno-dominant groups capable of reducing the polysaccharide decreases. therefore, it is preferable to use polysaccharide only partially purified with higher molecular weight.

The present invention provides that the polysaccharide isolated from the yeast microorganisms preferably Candida albicans or Candida tropicalis is crosslinked in a first stage by a multifunctional crosslinking reagent, preferably 1-chloro-2,3-epoxypropane in an amount of 5 to 30% by weight. on the weight of the polysaccharide in the alkaline medium, and in the second step, the cross-linked polysaccharide gel is subjected to aqueous dispersion in a high shear mixer under aggressive neutralization of the free alkali, and in the third step the insoluble polysaccharide particles are washed on the filter with alcohol and dried at up to 60 ° C.

The insoluble polysaccharide thus prepared with a particle size of 60 to 250 µm is a suitable immunosorbent for the isolation of antibodies against various fungal diseases.

The most common cause of fungal diseases in Poodle is Candida albicans, which accounts for 80% of pathological microbiological findings. The residue consists of microorganisms such as 3 221747

Candida tropicalis, C. stellatiodea, C. guilliermondi, C. parapsilosis, C. famata and others. Many Candida species have the same or similar 'antigenic composition as C. albicans, serologically difficult to distinguish and up to 90 microorganisms of this serological group % of all candidias. Since many Candida albicans are strongly pathogenic, cultivation to produce immunologically active polysaccharides must be made strictly sterile, increasing the cost of preparing biomass.

Therefore, the C. tropicalis strain, which is used industrially for the production of yeast, is non-pathogenic or only slightly pathogenic and, moreover, well-cultured on various waste lignocellulosic materials. C. tropicalis mannan used as an immunosorbent captures serum antibodies of homologous species as well as specific substances produced by microorganisms against mannan of said serogroup.

Crosslinking of polysaccharide from yeast microorganism cells is advantageous in such reaction conditions that optimally the amount of crosslinking reagent is 10 to 30% by weight. to the weight of the dry polysaccharide, at a concentration of 5 to 8% sodium hydroxide as the alkali catalyst, and the concentration of polysaccharide in the reaction medium to be 15 to 30% by weight. to obtain a gel and a non-dosing volume of 4.5 to 8.0 ml / g polysaccharide. The crosslinking is preferably carried out at 20 to 30 ° C for 1 to 2 hours, and at the second stage the reaction temperature is increased to 50 ° C at which temperature the reaction is completed in 1 hour. The crosslinked polysaccharide is subjected to wet defibrillation in a high speed mixer. In order to achieve a normal particle size (80-250 microns), a period of 30-60 seconds of defibrator turnover is sufficient. Before defibrating, it is expedient to add the acid acetic acid to the aqueous medium to neutralize the free alkali. Without neutralization, a very fine gel would be obtained which poorly filters and is not suitable for column operation.

However, the greatest advantage of the novel process is the fact that it allows for the preparation of a new high-activity immunosorbent, by means of which it is possible to isolate, in principle, simple desired antibodies from biological materials, the isolation of which by other means is more substantial and complex.

A new approach to my advantage in that it uses an unpurified polysaccharide that has ameliorated my ample free immunologically active groups capable of effectively binding antibodies from biological materials. the more significant is the fact that it can also be based on non-pathogenic strains which, by way of industrial use, feed yeast production, which considerably improves the economic process. the more violent advantages and details of the new process are apparent from the exemplary procedure, which is not exhaustive, but merely demonstrates the essential features of a novel process for preparing an immunororbent suitable for isolating antibodies against fungal diseases caused by Candida microbial microorganisms. EXAMPLE 1 10 g of a crude, dry polysaccharide isolated from Candida tropicalis microorganism cells are moistened with 20 ml of distilled water and allowed to warm for 5 to 10 minutes. Then, 4 ml of 40% by weight sodium hydroxide solution are added. it is good to mix and is allowed to dissolve until the solution is homogeneous with no apparent insoluble particles, typically no more than 10 to 15 minutes. 3 ml of 1-chloro-2-3 epoxipropane (epichlorohydrin) is then added and mixed well so that the crosslinking reagent is homogeneously distributed throughout the reaction mixture. Close the reaction flask and allow to react for 1 to 2 hours at room temperature, then increase the temperature gradually to 50 ° C and allow the reaction mixture to react without stirring for 1 hour, then the cross-linked polysaccharide gel mixes.

Claims (4)

221747 4 me in water (to which acetic acid is added to neutralize free alkali) about 30 to 60 seconds in a high-speed mixer, to 80 to 250 µl particles · Insoluble polysaccharide gel is decanted several times from the finest particles that will increase resistance to work in a column. Decanted and thoroughly washed gel particles are filtered by dewatering sequentially with ethanol, respectively. acetone and dried at up to 60 ° C. The swelling volume of the prepared immunosorbent is about 6 to 7 ml / g and is suitable for immunosorbency of antibodies from biological fluids. Example 2. The procedure of Example 1, except that we use semi-aqueous amounts of sodium hydroxide, sodium hydroxide and crosslinking reagent, while obtaining a polysaccharide gel with a dispensing volume of 4-5 ml / g. The gel has better hydrodynamic properties when working on a column with unchanged antibody absorption capacity. 4 Example 3 The procedure of Example 1 except that the polysaccharide isolated from Candida albicans is used as the immunologically active polysaccharide. Crosslinked polysaccharide has similar properties to crosslinked polysaccharide isolated from Candida tropicalis. Example Procedure as in Example 1 except that a 3-fold pre-crosslinked polysaccharide from Candida tropicalis was used for crosslinking. The polysaccharide solution was poorly viscous at the same concentration and was very poorly milled, had a high swelling volume after crosslinking, and was column mounted to partially dissolve. This example serves as a comparative example which demonstrates that raw, unpurified polysaccharide from yeast microorganisms is more preferred for immunosorbents. Popi sleigh. the method of preparing immunosorbents from microbial polysaccharides is universally applicable, not only to candida but to any immunologically active polysaccharide. The invention has utility in the treatment, isolation, and purification of effective antibodies from biological material in fungal diseases as well as in allergology. BACKGROUND OF THE INVENTION A method of preparing immunosorbents (immunoaffinants) from polysaccharides isolated from gums, characterized in that the polysaccharide isolated from the yeast microorganisms with the preferred Candida albicans or Candida tropicalis species is crosslinked in the first step by a bifunctional titrant, preferably 1-chloro-2-3 epoxypropane 15 to 30 wt. the weight of the polysaccharide in the alkali medium and, in the second step, the cross-linked polysaccharide gel is subjected to aqueous dispersion in a high shear mixer while neutralizing the free alkali, and in the third step the washed insoluble polysaccharide particles are filtered off with ethanol and dried at up to 60 ° C. Severografia, n. P., MOST Price 2,40 Kcs