RU2246968C2 - Method for preparing magnetic immunosorbent for detection of bacterial antigens - Google Patents

Abstract

FIELD: medicine, immunology.

SUBSTANCE: method involves synthesis of magnetic latex in the presence of colloidal-dispersed ferromagnetic particles of iron oxide wherein iron oxide is added into microspheres of polyacroleinic sorbents after their synthesis. Method provides simplifying the technology in preparing the serological test-system.

EFFECT: improved preparing method.

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Description

The invention relates to immunochemistry, microbiology and biotechnology, and can be used in the preparation of magneto-immunosorbents for highly sensitive serological test systems when a specific antigen-antibody reaction is detected when bacterial antigens are detected with high stability, demonstrability, reproducibility and sensitivity of the recorded results.

Solid-phase carriers, including 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 during ligand immobilization, and low non-specific capacity. A more durable attachment of the ligands to the surface of the sorbents is provided by a covalent bond, which is formed due to the preliminary activation of the surface of the sorbents by aldehydes (Efremenko V.I., Pushkar V.G. et al. Preparation and use of magnetic sorbents in methods of immunoassay of microorganisms // ZhMEI. - 1989 - No. 12. - P.100-104; Efremenko V.I., Kalnoy S.M. Method of producing sorbents: Pat. No. 35).

The immunological properties of the test system largely depend on the choice of vehicle on which antibodies are immobilized.

Various methods for producing immunosorbents with magnetic properties are known.

For example, a method of emulsion polymerization, according to which a magnetic powder and a ligand (immunoglobulins) are introduced into a reactive mixture of comonomers - polyacrylamide and catalyst. This involves the mechanical inclusion of particles of magnetic powder and biologically active substances (immunoglobulins) in the cellular structure of a polyacrylamide gel (Pushkar V.G., Trofimov E.N. Enzyme-linked immunosorbent assay of antigenic material fixed on magnetic polyacrylamide sorbents // Actual issues of immunodiagnosis of especially dangerous infections Abstracts of the All-Union Scientific and Practical Conference (May 26-27, 1986). -Stavropol, 1986. - Part II. - P.50-53).

The disadvantage of this method is the increased consumption of immunoglobulins with a concentration of 40 mg / ml protein. This increases the cost of the drug and limits the organization of industrial production of drugs.

A known method of producing magnesorbent by pre-processing a magnetic powder with giving it corrosion resistance and hydrophilicity, followed by emulsion polymerization of a mixture of comonomers - polyacrylamide and a catalyst with processed magnetic powder. The prepared magnetosorbent is activated with 5% glutaraldehyde for 18-20 hours and immobilized with specific immunoglobulins in a solution of phosphate-saline buffer (FSB) during incubation for 18-20 hours. To block unbound aldehyde groups, magnetic immunosorbents (MIS) are additionally treated with an albumin solution for 1-3 hours (Pat. RF No. 2068703, A 61 K 39/385, C 12 N 11/00 // G 01 N 33/53, op. 11/10/96; Bull. No. 31).

The disadvantages of this method are the multi-stage and duration of the process of obtaining a magnetic immunosorbent, the use of expensive and toxic reagents, which worsens the environmental conditions of production and limits the organization of industrial production of drugs.

It is known that magnesorbents (MS) prepared on the basis of aluminosilicate and iron oxide (I.V.Zharnikova, E.N. Afanasyev, I.S. Tyumentseva and others. A method for producing an immunosorbent (Options) can be used as carriers: Pat. RF No. 2138813, G 01 N 33/543, A 61 K 39/385 // G 01 N 33/569, C 12 N 11/14, dated September 27, 1999, Bull. No. 27). Test systems made on their basis have great advantages over traditional solid-phase enzyme-linked immunosorbent assay systems due to increased sensitivity and reduced reaction time (I.V. Zharnikova, A. V. Brykalov, I. S. Tyumentseva. Use of composite immunosorbents in an enzyme-linked immunosorbent assay (ELISA) for the rapid diagnosis of pathogens of OOI // Actual issues for the prevention of plague and other infectious diseases: Materials of an interstate scientific and practical conference dedicated to the 100th anniversary of the discovery of excitations the plague. - Stavropol, 1994. - P.41-42.).

The disadvantage of this type of immunosorbents is the significant dispersion of their carriers in size and shape, which makes it difficult to comply with strictly standard conditions when setting up reactions in experimental and control volumes.

A known method of producing biomagnosorbent (Kalnoy S.M., Goncharov V.I. "Method for producing biomagnosorbent". AS USSR No. 2092854 C1, 6 G 01 N 33/554, 1997, from 10.10.97, Bull. No. 28 ), in which a ferromagnet is introduced into preliminarily sublimated biomaterial due to successive liquid treatments with concentrated solutions of iron (II) sulfate salt and an aqueous solution of ammonia with exposure in each solution for 30-50 hours and with sublimation of the material after each treatment. The main disadvantage of this method is the need to use complex and energy-intensive biotechnological equipment in compliance with the exact ratios of the ingredients used for the synthesis of a ferromagnet.

, стабилизированных олеатом натрия. За счет осмотического насыщения частиц латексов коллоидными растворами оксида железа происходит приобретение последними магнитных свойств.The most acceptable carrier are latex magnetic immunosorbents (Lukin Yu.V., Egorov V.V., Zubov V.P., Gritskova I.A., Kiselev E.M., Voronov S.A., Puchin V.A. Synthesis method functional magnetic latexes. A.S. USSR No. 1249024, op. 10.08.86, Bull. No. 29). For this, the synthesis of magnetic latexes is carried out in the presence of colloidal dispersed ferromagnetic particles of iron oxide Fe ₃ O ₄ with a variable valence of 100-250

stabilized by sodium oleate. Due to the osmotic saturation of the latex particles with colloidal solutions of iron oxide, the latter acquires magnetic properties.

The disadvantage of this method of preparing MIS is that the increase in the content of magnetic material up to 0.33% weight. leads to a significant increase in the diameter of the microspheres from 0.13 to 0.5 μm and the expansion of the zones of particle size distribution. At a higher concentration of colloidal dispersed ferromagnetic particles of iron oxide, latex particles of irregular shape begin to form with varying degrees of their filling with ferromagnetic material (Lukin Yu.V., Bakharev V.N., Zaichenko A.S., Voronov S.A., Zubov V.P., Gritskova I.A., Pravednikov A.N. Polyacrolein latexes: synthesis, introduction of fillers and formation mechanism // Reports of the Academy of Sciences. - 1985. - Volume 285, No. 1. - S.159-161.) .

The aim of the invention is to simplify the technology for biomagnoimmunosorbent based on polyacrolein latex microspheres with ligation of specific antibodies to detect microorganism antigens in the enzyme-linked immunosorbent assay (ELISA).

The technical result of the invention is to increase the stability, reproducibility, demonstrativeness and sensitivity of the results of reactions of serological test systems based on inorganic MIS and enzyme immunoassay conjugates in ELISA.

The specified technical result is achieved by the fact that the proposed method involves setting up a serological enzyme-linked immunosorbent reaction - ELISA with an immunosorbent based on magnetic polyacrolein latex particles containing iron oxide and an enzyme-linked immunosorbent conjugate (as a double antibody sandwich), characterized in that the ferromagnetic material is oxide iron - is introduced into the microspheres of polyacrolein magnetic immunosorbents not at the time of their synthesis, but after its completion. Polyacrolein magnetic latex particles were obtained due to the formation of iron oxide in the microspheres of polyacrolein sorbents after their synthesis by sequentially treating these suspensions with 25-35% solutions of iron (II) sulfate for 48 ± 2 hours and 20-25% aqueous ammonia for 48 ± 2 hours.

The magnetic properties of the latex suspension particles were imparted due to the formation of iron oxide Fe ₃ O ₄ in them and mainly on their surface, with variable valency and magnetic properties. Along with other porous sorbents, which include iron oxide (Neymark IE Synthetic mineral adsorbents and catalyst supports. - Kiev: Naukova Dumka, 1982. - 216 pp.), The latter has high porosity, strength, is stable and adsorbs biomacromolecules well. . Particles of latexes containing iron oxide become magnetically controlled, have good kinetic and aggregative stability. According to the size of the dispersed phase, a suspension of magnetic latexes belongs to a colloidal dispersed group (particle size &λτ; 100, γ 1 μm, dispersoids), has the same size and uniform shape of the dispersed phase (Adamov A.K., Agafonov V.I. Suspension antigens , antibodies and immunosorbents. - M .: Medicine, 1969. - 176 p.).

In relation to the prototype, which is selected as the "Method for the synthesis of functional magnetic latexes" (Lukin Yu.V., Egorov V.V., Zubov V.P., Gritskova I.A., Kiselev E.M., Voronov S. A., Puchin V.A) A.S. USSR No. 1249024, Bull. No. 29, dated 10.08.86), the claimed method has the following distinctive features.

The introduction of ferromagnetic material into polyacrolein latex microspheres formed with a known size and shape, results in the production of monodisperse and similar in size and shape magnetic immunosorbents with a porous surface, which allows them to be more evenly distributed quantitatively among the vessels in the series of experimental and control samples, which accordingly leads to standardize the conditions for the performance of reactions, increases sensitivity, reproducibility, demonstrativeness, reduces the level of fluctuation of the background new values of the reaction results.

The use of polyacrolein MIS for the production of ELISA with imparting magnetic properties due to the ferromagnetic material in the form of colloidal dispersed solutions of iron oxide Fe ₃ O ₄ introduced during the synthesis of latex particles increases the probability of an increase in the level of fluctuation of the background values of the reaction results due to the presence of a significant diagnostic preparation in the suspension the number of heterogeneous in size and polymorphic microspheres of magneto-immunosorbents.

The proposed method improves the reproducibility, stability, demonstrativeness and sensitivity of the results of reactions of serological test systems based on magnetic immunosorbents and enzyme-linked immunosorbent conjugates, performed as a double antibody sandwich due to high porosity, strength, stability, good adsorption properties with respect to biomacromolecules in iron oxide, which increases specific surface of microspheres.

The practical use of the proposed method is confirmed by examples of specific production of polyacrolein magnetic immunosorbents for the detection of bacterial antigens.

Example 1. Polyacrolein magnetic latex particles were obtained due to the formation of iron oxide in the microspheres of polyacrolein sorbents after their synthesis by sequential processing of a 10% suspension of latexes with a 5-fold 25% solution of iron (II) sulfate salt (FeSO ₄ · 7H ₂ O) within 48 ± 2 hours and then after removing the excess salt solution with 20% aqueous ammonia (NH ₄ OH) for 48 ± 2 hours.

Plague, tularemia and brucellosis magnetic immunosorbents were prepared on the basis of the obtained polyacrolein magnetic latexes by sensitizing their 10% suspension for 24 ± 2 hours with species-specific competent immunoglobulins from plague, tularemia or brucellosis sera with an antibody content of 10 mg / ml. Previously, the surface of the magnetic latex microspheres was activated with 5% glutaraldehyde solution for 4 hours. To block unbound aldehyde groups, magneto-immunosorbents (MIS) were further treated with a solution of bovine serum albumin (BSA) for 3 hours. After each treatment (with glutaraldehyde, species-specific immunoglobulins, BSA), MISs were washed 5 times with a 10-fold volume of 0.01 FSB pH 7.2-7.4.

Methodology for the formulation of the reaction.

Samples containing suspensions with a concentration of microbial cells of the plague pathogen 1 × 10 ⁶ were disinfected with 1% formalin at 56 ° C for 30 min. Samples with suspensions of tularemia 1 × 10 ⁶ and brucellosis 1 × 10 ⁶ pathogens were disinfected by boiling for 30–40 min. They were examined in enzyme-linked immunosorbent assays with peroxidase conjugate as a double antibody sandwich. 0.1 ml of phosphate-saline buffer solution (FSB) pH 7.2-7.4 with 0.05% tween - 20 was added to the ranks of experimental and control containers with a volume of at least 0.25 ml. To the contents of the first experimental containers, respectively 0.1 ml of disinfected suspension of plague, brucellosis or tularemia microbial microbial cells was added. The test material was titrated with a ratio of 1: 2, an excess volume of 0.1 ml was removed from the last containers. 0.1 ml of a 0.5-1.0% suspension of plague particles of plague magneto-immunosorbent polyacrolein diagnosticum suspended in 0.01 M FSB pH 7.2-7.4 with 0.05% tween were added to the experimental and control series of containers. - 20.

The experimental and control samples were incubated at 37 ° C for 1 hour. Antigens that did not bind to MIS were separated by 5-fold washing with 0.01 M FSB pH 7.2-7.4 with 0.05% Tween-20.

Plague, brucellosis or tularemia immunoglobulin peroxidase conjugates in working dilution (respectively 1: 300; 1: 400; 1: 300) in the volume of 0.1 ml per 0.01 M FSB pH 7.2-7 were added to the ranks of experimental and control containers , 4 with 0.05% tween - 20 and 1.0% bovine serum albumin (BSA). The experimental and control samples were incubated at 37 ° C. Enzyme-linked immunosorbents that did not bind to antigens adsorbed on magnetic immunosorbents were separated by 5-fold washing with 0.01 M FSB pH 7.2-7.4 with 0.05% Tween-20 .

0.1 ml of the substrate-indicator mixture, each containing a solution containing 10 ml of nitrate buffer, pH 5.0, 5 mg of orthophenylenediamine and 0.05% hydrogen peroxide, was added to all containers. All samples were incubated at room temperature without access of light for 5-10 minutes. The reaction was stopped by adding 50 μl of a 2.0 M sulfuric acid solution to each container. The results were taken into account on a Multiscan instrument at a wavelength of 492 nm. An ELISA was considered a positive excess of the optical density of the test samples over the optical density of the negative control by 2 or more times.

The sensitivity of the method was 1 · 10 ³ -1 · 10 ⁴ m.k. / ml for suspensions of the plague microbe, 1 · 10 ³ -1 · 10 ⁵ m.k. / ml for tularemia and 1 · 10 ³ -1 · 10 ⁴ m.k. / ml - brucellosis.

Example 2. Polyacrolein magnetic latex particles were obtained due to the formation of iron oxide in the microspheres of polyacrolein sorbents after their synthesis by sequential processing of a 10% suspension of latexes with a 5-fold 35% solution of iron (II) sulfate salt (FeSO ₄ · 7H ₂ O) within 48 ± 2 hours and then after removing the excess salt solution with 25% aqueous ammonia (NH ₄ OH) for 48 ± 2 hours.

Plague, tularemia and brucellosis magnetic immunosorbents were prepared on the basis of the obtained polyacrolein magnetic latexes by sensitizing their 10% suspension for 24 ± 2 hours with species-specific competent immunoglobulins from plague, tularemia or brucellosis sera with an antibody content of 10 mg / ml. Previously, the surface of the magnetic latex microspheres was activated with 5% glutaraldehyde solution for 4 hours. To block unbound aldehyde groups, magneto-immunosorbents (MIS) were further treated with a solution of bovine serum albumin (BSA) for 3 hours. After each treatment (with glutaraldehyde, species-specific immunoglobulins, BSA), MISs were washed 5 times with a 10-fold volume of 0.01 FSB pH 7.2-7.4.

Methodology for the formulation of the reaction.

The ELISA is similar to the procedure described in example 1, with the exception of the results of reaction accounting.

The sensitivity of the method was 1 · 10 ² -1 · 10 ³ m.k. / ml for suspensions of the plague microbe, 1 · 10 ² -1 · 10 ⁴ m.k. / ml for tularemia and 1 · 10 ³ -1 · 10 ⁴ mk / ml brucellosis.

The results were taken into account on a Multiscan instrument at a wavelength of 492 nm. An ELISA was considered a positive excess of the optical density of the test samples over the optical density of the negative control by 2 or more times. In each case of ELISA, the optical density of the experimental samples exceeded the optical density of the negative control by 6-8 times, which proves the demonstrative nature of the reactions using the developed MIS. Plague, tularemia and brucellosis liquid MIS were stored in the refrigerator for 2 years (observation period) with a check of their quality every 6 months. The preparations remained stable without loss of specific activity during the entire observation period, while non-fixed “Ig” matrices lost their specific activity under the same storage conditions for 7 days. This proves the stability of the drugs.

The sensitivity of the method was 1 · 10 ² -1 · 10 ³ m.k. / ml for suspensions of the plague microbe, 1 · 10 ² -1 · 10 ⁴ m.k. / ml for tularemia and 1 · 10 ³ -1 · 10 ⁴ MK / ml brucellosis, with high demonstrability, reproducibility and stability of the reaction results. This was due to a significantly smaller fluctuation (40-60%) of the background values of the results of enzyme immunoassay.

As shown by the results of experimental studies, the exposure of polyacrolein latex microspheres in each of the solutions of the ingredients for less than 48 ± 2 hours does not lead to the formation of particles with a satisfactory magnetic moment. An increase in the exposure time of latexes in ingredient solutions of more than 40 ± 2 hours does not improve the magnetic properties of carriers.

The decrease in the concentration of the solutions of the ingredients when processing latexes below 25% for FeSO ₄ · 7H ₂ O and below 20% for NH ₄ OH leads to a decrease in the magnetic moment of the particles. An increase in the concentration of the iron (II) sulfate salt solution above 35% requires an increase in the temperature of the liquid, which complicates the technology.

The ratio of the suspension volumes of latexes and processing fluids is 1: 4-1: 5 and is an essential condition for obtaining high-quality carriers that contribute to their more uniform saturation by solution ions due to the effective equalization of osmotic pressure.

Thus, the claimed method is practicable and has an advantage over the prototype, as it simplifies the biotechnology of obtaining a serological test system for the detection of bacterial antigens with high sensitivity and greater reproducibility, stability and demonstrativeness of the recorded reaction results.

Claims (1)

A method for producing a magnetic immunosorbent for detecting bacterial antigens, including the synthesis of magnetic latexes in the presence of colloidal dispersed ferromagnetic particles of iron oxide, characterized in that the iron oxide is introduced into the microspheres of polyacrolein sorbents after their synthesis by sequential treatment with 25-35% iron sulfate solutions in suspension ( II) for (48 ± 2) hours and 20-25% aqueous ammonia solution for (48 ± 2) hours, while the suspension volumes of latexes and treatment fluids are 1: 4-1: 5.