RU2138813C1 - Method of immunosorbent producing (variants) - Google Patents

Abstract

FIELD: medicine, immunology. SUBSTANCE: invention involves modifying a carrier with 0.3-0.4% of polyglucin solution. Sorbent is dried, activated with secondary sodium alkyl sulfate and activated sorbent is bound covalently with the protein ligand. By the first variant alumosilicate is used as a carrier and by the second variant - a mixture of magnetic powder and alumosilicate. Of protein ligands immunoglobulins against plague, cholera or tularemia pathogens can be used. Invention is used for production of immunosorbents for detection of specific reaction antigen-antibody. EFFECT: improved method of producing. 3 cl, 6 ex

Description

 The invention relates to immunochemistry, microbiology and can be used in the production of immunosorbents to detect a specific antigen-antibody reaction.

 A known method of producing an immunosorbent based on cellulose, comprising covalent binding of a protein ligand to a carrier. The disadvantage of this method is the low level of specificity of the sorbent, in addition, the organic carrier is unstable, subject to destruction when exposed to microorganisms (KL Shakhanina et al. Preparation of various immunosorbents and their use for the isolation of antigens and antibodies. J. Questions of medical chemistry, N 6 , 1976. - S. 127-136).

 Solid-phase carriers, including various granular sorbents, and more recently, sorbents with magnetic properties, are promising for use in immunoassay methods.

 The main requirements for such sorbents are their inertness with respect to biological preparations, stability of properties, mechanical strength, sufficient sorption capacity when immobilizing ligands. The most durable attachment of ligands to the surface of sorbents is provided by a covalent bond, which is carried out by preliminary activation of the surface of sorbents, for example, glutaraldehyde (V.I. Efremenko, V.G. Pushkar, etc. Production and use of magnetic sorbents in immunoassay methods of microorganisms. ZhMEI, N 12 1989, p. 100-104).

 A known method of producing an immunosorbent, including the activation of aminopropylsilochrome by glutaraldehyde at a concentration of 12.0-13.0% for 2 hours at room temperature and pH 8.0, followed by covalent binding of protein A from Staphylococcus aureus and washing (a.c. USSR N 1517545, G 01 N 33/53, 12/15/93, Bull. N 45-46).

 The aminopropylsilochrome used as a carrier (manufactured by the Olainen Chemical Reagents Plant) is deficient, the activating agent is not firmly fixed to the surface of the carrier, since glutaraldehyde is hydrolyzed in an aqueous medium, which leads to non-specific sorption and a decrease in stability in biospecific processes.

 In addition, the immunosorbent obtained by this method has insufficient specific capacity and sensitivity in immunoassay. The method does not provide for immunosorbent with magnetic properties.

 Known methods for producing immunosorbents with magnetic properties.

 A method of emulsion polymerization, in which a magnetic powder and a ligand (immunoglobulins) are included in the reaction mixture of polyacrylamide comonomers and a catalyst. In this case, the biologically active substances are mechanically incorporated into the cellular structure of the polyacrylamide gel (Actual issues of immunodiagnostics of especially dangerous infections. Abstracts of the All-Union Scientific and Practical Conference, Stavropol, 1986, p. 50-53).

 The disadvantage of this method is the high consumption of immunoglobulins with a concentration of 40 mg / ml for protein, which increases the cost of the drug and limits the possibility of organizing industrial production.

 According to another method, first get the magnetic sorbent by pre-processing the magnetic powder with giving it corrosion resistance and hydrophilicity, then emulsion polymerization of a mixture of polyacrylamide comonomers and a catalyst with treated magnetic powder is carried out. The prepared magnetosorbent is activated with 5% glutaraldehyde for 18-20 hours and immobilized with specific immunoglobulins in a FSB solution during incubation for 18-20 hours. To block unbound aldehyde groups, the magneto-immunosorbents are additionally treated with an albumin solution for 1-3 hours (US Pat. RF N 2068703, A 61 K 39/385, C 12 N 11/00 // G 01 N 33/53, op. 10.11.96; Bull. N 31).

 The disadvantages of the method are the duration and multi-stage process of its preparation, the use of expensive imported toxic reagents, which limits the organization of industrial production and worsens the environmental conditions of the process.

 The immunosorbents obtained by this method have insufficient specific capacity and sensitivity in immunoassays.

 A known method for producing immobilized aminoacylase, including covalent binding of an enzyme to activated silochrome (A.S. USSR N 1060676, C 12 N 11/14; C 12 N 9/78; op. 15.12.83, Bull. N 46).

 The silochrome is treated with aminopropyltriethoxysilane and activated with n-benzoquinone. After the ligand is turned on, the finished product is additionally treated with an aqueous glyoxal solution in order to prevent acylase dissociation and increase the stability of the immunosorbent.

 The disadvantage of this method is the use of scarce expensive drugs, which complicates the organization of industrial production. The method does not provide for immunosorbent with magnetic properties.

 The aim of the invention is the organization of industrial production of immunosorbents, including those with magnetic properties, with increased specific capacity and sensitivity due to the simplification of the process, the use of affordable and inexpensive drugs.

 The goal is achieved in two ways.

 According to the first embodiment, the method of producing immunosorbents includes modifying the support, which is used as an aluminosilicate, with a 0.3-0.4% solution of polyglucin, drying the sorbent, activating it with secondary sodium alkyl sulfate, followed by covalent binding to a protein ligand.

 According to the second variant, the method for producing immunosorbents involves modifying the carrier, which is used as a mixture of aluminosilicate and magnetic powder, with a 0.3-0.4% polyglucin solution, drying the sorbent, activating it with secondary sodium alkyl sulfate, followed by covalent binding to a protein ligand.

 In relation to the prototype, which is selected as the method of producing the immunosorbent according to a. with. USSR N 1517545, G 01 N 33/53, 12/15/93, Bull. N 45-46, the inventive method has the following distinctive features.

 Use as a carrier aluminosilicate, which is an affordable finely divided filler, which is a complex anion of aluminum and silicon with an excess negative charge, which is compensated by alkaline earth metals. The carrier has improved adhesive properties compared to polyacrylamide and silochrome, which allows it to be mixed with magnetic powder without first preparing the latter.

 Modification of the carrier with polyglucin, which is a polysaccharide, a linear polymer based on the cyclic form of glucose, where the units are linked by a 1,6 - glycosidic bond. When the carrier is treated, a polymer film is formed on its surface, which provides an increase in the intensity of subsequent activation of the carrier and an increase in the specific capacity of the sorbent, and, accordingly, its sensitivity in immunoassays.

 Activation with sodium secondary alkyl sulfate, which is an anionic surfactant, creates active groups on the modified surface of the sorbent that are capable of chemical interaction with the residues of various amino acids of the protein ligand, which helps to increase the specific capacity and sensitivity of the immunosorbent.

 The choice of the claimed concentration limits of polyglucin is optimal, so, a decrease in its concentration below 0.3% leads to a decrease in the specific capacity and sensitivity of the immunosorbent, and an increase above 0.4% is no longer practical.

 All drugs used according to the claimed method are available and inexpensive, the method provides a simple and environmentally friendly technology for the production of immunosorbents, including those with magnetic properties.

 The possibility of practical application of the invention is illustrated by examples of its specific implementation using the full totality of the claimed features.

Example 1. (option 1). 2.5 g of the aluminosilicate carrier are suspended in 40 ml of a 0.4% aqueous solution of polyglucin and kept at a temperature of 24 ^° C. for 1 hour. The resulting sorbent is dried at 100-110 ^° C. for 30 minutes. To 1 g of the obtained preparation, add 12 ml of a 24% solution of sodium chloride and 1 ml of secondary sodium alkyl sulfate, incubate the mixture for 1-2 hours at a temperature of (37 ± 1) ^o C. Pour 1 ml of plague immunoglobulins with a concentration of 1 ml of activated sorbent protein 3.5 mg / ml. The mixture is left at (37 ± 1) ^o C for 1-2 hours, then the supernatant is removed, and the immunosorbent is washed with 0.9% sodium chloride solution to “0” extinction on a spectrophotometer. The duration of the immunosorbent is 3-4 hours.

The results of determining the specific capacity of the immunosorbent are carried out by the method of quantitative immunofluorescence (Vladimtseva I.V., Efremenko V.I., Klimova I.M. et al. Laboratory case No. 7, 1988, p. 53-55). To 0.1 ml of a 10% solution of the immunosorbent, 0.2 ml of the plague microbe is added at a concentration of 10 ⁹ -10 ² m.k. / ml and the mixture is incubated for 1 h at 24 ^o C. Next, the immunosorbent is washed with 50 ml of 0.9% sodium chloride solution. Contribute 0.2 ml of plague luminescent immunoglobulins in working dilution and incubate for 30 minutes. The sorbent is washed with a 0.9% sodium chloride solution and the results are recorded in a Lumam R-2 luminescent microscope using the conventional DASS system. In parallel with the experimental sample, the luminescence of the control sample (immunosorbent without contact with the plague microbe) is measured. The specific capacity of the immunosorbent is expressed in arbitrary units (cu), which are determined by the ratio of the luminous intensity indicators of the experimental and control samples. The immunosorbent capacity measured in this way is 10 cu The sensitivity of the method is 10 ² MK / ml of the plague microbe.

Example 2 (option 2). A mixture consisting of 2.5 g of aluminosilicate filler and 0.5 g of magnetic powder is suspended in 40 ml of a 0.4% aqueous solution of polyglucin and then kept at a temperature of 24 ^o C for 1 h. The resulting sorbent is dried at 100-110 ^o C for 30 minutes To 1 g of the obtained preparation, add 12 ml of a 24% solution of sodium chloride and 1 ml of secondary sodium alkyl sulfate, incubate the mixture for 1-2 hours at a temperature of (37 ± 1) ^o C. Pour 1 ml of plague immunoglobulins with a concentration of 1 ml of activated sorbent protein 3.5 mg / ml. The mixture is left at (37 ± 1) ^o C for 1-2 hours, then the supernatant is removed, and the immunosorbent is washed with 0.9% sodium chloride solution to “0” extinction on a spectrophotometer. The duration of the immunosorbent is 3-4 hours.

The specific capacity of the immunosorbent, measured by immunofluorescence, is 10 cu The sensitivity of the method is 10 ² MK / ml of the plague microbe.

Example 3. The method of obtaining immunosorbent is carried out according to the method of example 1, but to modify the sorbent using a 0.4% solution of polyglucin. The specific capacity is 10 oz. e. The sensitivity of the method is 10 ² MK / ml of the plague microbe.

Example 4. A method of producing an immunosorbent is carried out according to the method of example 1, but to modify the sorbent using 0.3% solution of polyglucin. The specific capacity is 9 cu The sensitivity of the method is 10 ³ MK / ml of the plague microbe.

Example 5. A method for producing an immunosorbent is carried out according to the procedure of Example 1, but at the last stage of synthesis, 1 ml of tularemia immunoglobulins with a protein concentration of 3.5 mg / ml is poured into 1 ml of activated carrier. The specific capacity of the immunosorbent is 10 cu The sensitivity of the method is 10 ² MK / ml tularemia microbe.

Example 6. A method for producing an immunosorbent is carried out according to the procedure of Example 2, but at the last stage of synthesis, 1 ml of cholera immunoglobulins with a protein concentration of 3.5 mg / ml are poured into 1 ml of activated carrier. The specific capacity of the immunosorbent is 10 cu The sensitivity of the method is 10 ² MK / ml cholera vibrio.

 Thus, the invention is practicable, its use will allow you to organize a simple, environmentally friendly technology for the production of immunosorbents, including those with magnetic properties, which are 3 times more specific in sensitivity and 100 times more sensitive than similar products obtained by known methods.

Claims (2)

 1. A method of producing an immunosorbent, including activation of a carrier and covalent binding of a protein ligand to it, characterized in that an aluminosilicate is used as a carrier, which is modified with a 0.3-0.4% polyglucin solution and, after drying, it is activated with sodium sodium alkyl sulfate.  2. A method of producing an immunosorbent, including activation of a carrier and covalent binding of a protein ligand to it, characterized in that the carrier is a mixture of aluminosilicate with magnetic powder, which is modified with a 0.3-0.4% polyglucin solution and activated after drying secondary sodium alkyl sulfate.