RU2731415C2 - Method of producing alpha-fetoprotein immobilized on pet microparticles - Google Patents

Abstract

FIELD: biochemistry; molecular biology; biotechnology.SUBSTANCE: invention relates to a reagent based on alpha-fetoprotein immobilized on the surface of solid microparticles of polyethylene terephthalate (PET microparticles), as well as to a method for site-specific covalent immobilisation of alpha-fetoprotein on the surface of functionalised PET microparticles per a glycoside protein fragment.EFFECT: disclosed is a method of producing alpha-fetoprotein immobilized on PET microparticles.2 cl, 11 ex, 1 dwg

Description

The technical field to which the invention relates

The present invention relates to the field of biochemistry, molecular biology and biotechnology, namely to a reagent based on alpha-fetoprotein immobilized on the surface of solid microparticles of polyethylene terephthalate, as well as to a method of site-specific covalent immobilization of alpha-fetoprotein through a hydrophilic spacer on the surface of functionalized PET for the glycosidic fragment of the protein.

The prior art to which the invention relates

Immobilization of proteins and other biomolecules on solid carriers is an integral part of many techniques in the field of molecular biology and biotechnology. For the immobilization of biomolecules, various methods have been proposed based on physical sorption and covalent binding to a solid carrier.

The authors of the patent [1] proposed the immobilization of proteins on the carrier by the sorption method. The protein that binds to functional biomolecules is adsorbed onto a solid support without being covalently bound to it.

For the covalent immobilization of proteins on a solid polymer carrier, the reaction between the surface primary amino groups of lysine and ornithine, included in the polypeptide chain of the protein, and the active groups of the functionalized polymer carrier with the formation of an amide (peptide) bond is usually used. Activated carboxyl, sometimes isocyanate or isothiocyanate groups are most often used in the support [2]. However, immobilization of the protein onto the carrier is not site specific.

The patent [3] claims the immobilization of antibodies specific to ricin on polymer microspheres MapPlex with surface carboxyl groups using water-soluble carbodiimide (EDC) and N-hydroxysuccinimide. Antibodies bind to microspheres by surface amino groups, while the immobilization of antibodies to the carrier is not site-specific. To increase the specificity of the orientation of the protein molecule relative to the surface of the carrier, it is proposed to add the amino acid lysine to the C-end of the protein polypeptide chain into recombinant antibodies, when they are produced by a genetic engineering method.

Usually on the surface of the protein there are several tens of chemically accessible amino groups, each of which can be immobilized on the surface of a solid support. Immobilization by binding to surface amino groups of the protein occurs statistically heterogeneously. Different protein molecules bind by different amino groups, and sometimes several amino groups at the same time. Amino groups that are part of the composition of lysine and ornithine are often included in the epitopes (antigenic determinants) of this protein. When immobilized for amino groups, some protein epitopes lose their antigenic properties. Direct binding of the surface amino groups that are part of the polypeptide chain of the protein with the solid functionalized surface of the polymeric carrier leads to additional steric (spatial) inaccessibility of some antigenic determinants of the protein. As a result, immobilized proteins become antigenically heterogeneous and antigenically less valence.

For covalent immobilization of glycosides, polysaccharides on a solid support, they are functionalized, and then linked to the functionalized solid support. In the patent [2], glycosides were converted into glycosylhydrazones by treatment with hydrazine. As a result, a reactive amino group appeared on the glycosides, which was used to bind to a polymeric functionalized carrier containing phenyl isothiocyanate groups.

For glycoproteins, that is, proteins that include glycoside, polysaccharide, fragments, methods of immobilization for a carbohydrate fragment without affecting the antigenic determinants of the protein part of the glycoprotein are proposed. The glycan portion of the glycoprotein is pre-functionalized. Oxidation with sodium periodate makes it possible to selectively obtain aldehyde groups in the glycan portion without denaturing the protein portion. In publication [4], site-specific immobilization of antibodies for their glycan groups on solid functionalized polysaccharide and silica gel carriers for the purposes of affinity chromatography is proposed.

The authors of the patent [5] presented a method for site-specific immobilization of human alpha1-acid glycoprotein (1-AGP) on a solid support, which is a functionalized silica gel for chiral affinity chromatography. Epoxy groups were grafted onto the silica gel surface using epoxysilane, then they were hydrolyzed to diol groups, oxidized with periodic acid to aldehyde groups, hydrazones were obtained by reaction with hydrazine, and they, in turn, were reduced with sodium borohydride to hydrazine bound to silica gel. As a result, silica gel was obtained, on the surface of which there are active hydrazine groups. Human alpha1-acid glycoprotein (1-AGP) was obtained from pooled sera and contained 44% F variant 1 AGP, 29% S variant and 27% A variant. The carbohydrate portion of the glycoprotein was oxidized with periodic acid, and 1-AGP glycoprotein with aldehyde groups in the glycan portion was obtained. The oxidized glycoprotein 1-AGP was then linked to silica gel bearing hydrazine groups.

Alpha-fetoprotein, immobilized on solid PET microparticles, is intended for the production of DNA fragments, aptamers, functional analogs of monoclonal antibodies. Alpha-fetoprotein (AFP) is one of the multifunctional diagnostically significant human proteins. The protein appears in the developing mammalian embryo and practically disappears from the blood after birth. During embryonic development, AFP takes part in the regulation of tissue growth, the formation and development of body parts and body systems, including the nervous, cardiovascular, reproductive, etc. In various forms of carcinogenesis, a number of growth factors, their receptors and / or proteins mediating intracellular signaling, which is usually characteristic of oncofetal proteins. It was found that it appears in the blood of adults with the development of cancer of the liver and reproductive organs. [6]. Therefore, alpha-fetoprotein is an important diagnostic marker for the presence of human cancer.

Alpha-fetoprotein is not expressed on the surface of normal somatic cells, but is found on the membranes of tumor cells. The expression level of the AFP receptor in nonproliferating cells is very low, while on the surface of tumor cells of various origins, from several hundred to one million AFP receptors per cell is determined [7]. Therefore, receptor, that is, located on the surface of cells, alpha-fetoprotein is a marker of tumor cells, as well as a target for the targeted transport of drugs to tumor cells.

Alpha-fetoprotein contains 5-6 major epitopes, with a content of less than 10-12 amino acids, which are detected by monoclonal antibodies [8].

Embryonic, fetal, alpha-fetoprotein is a glycoprotein with a molecular weight of 69 kilodaltons, consists of 3 peptide domains with a total content of 590 amino acid fragments, includes 1 glycosidic site with a total carbohydrate content of 3.0-5.0% [9].

When immobilizing a protein on a solid carrier, structural and functional epitopes (antigenic determinants) must be preserved, which provide specific binding of the native protein to the corresponding monoclonal antibodies. These epitopes are also needed for specific interaction with sequences from a DNA library during aptamer selection procedures. When chemically aggressive immobilization methods are used, leading to partial denaturation of the protein, only linear, sequential epitopes remain available [10].

Short single-stranded fragments of nucleic acids capable of specifically recognizing proteins and other molecular targets are called aptamers. Aptamers are obtained by experimental selection from DNA libraries according to the parameter of specific binding to a molecular target. The exponential enrichment method is used, based on the use of various approaches to enzymatic amplification of enriched oligonucleotide libraries [11]. Aptamers, by the nature of their interaction with a protein target, are similar to monoclonal antibodies, although they belong to a different class of chemical compounds. The SELEX procedure, selection, involves the specific selection (extraction) of a protein target of single-stranded DNA fragments from a plurality of nucleotide sequences (DNA libraries). The procedure for separating protein molecules with oligonucleotides bound to them is effectively performed if the protein molecules are immobilized on microparticles of a solid carrier. Solid microparticles with bound oligonucleotides can be washed to remove unbound oligonucleotides. The type of solid microparticles and the method of immobilization have a significant impact on the procedure and results of selection of nucleic acids for binding to a molecular protein target.

Aptamers to alpha-fetoprotein, as functional analogs of monoclonal antibodies, are of significant practical interest and can be used as a replacement for monoclonal antibodies in areas traditionally using antibodies. Aptamers to alpha-fetoprotein, being resistant to the action of proteases, can be used for therapeutic purposes and various sensory systems and devices.

For a successful SELEX procedure, the immobilization of alpha-fetoprotein on the carrier microparticles is required, which ensures the maximum availability of oligonucleotides to antigenic determinants and their maximum preservation. When performing the procedure for selecting oligonucleotides for an antigenically heterogeneous immobilized protein, it will be obviously difficult to obtain oligonucleotides that specifically bind to the native protein.

The claimed invention differs from the above-described analogs in that to obtain a reagent based on alpha-fetoprotein immobilized on solid microparticles, site-specific covalent immobilization for the glycosidic fragment is used. Binding to the carrier occurs via a 13-atom hydrophilic spacer. Microparticles of polymeric material polyethylene terephthalate (PET) with a functionalized surface are used as a carrier. Alpha-fetoprotein is selectively oxidized under mild conditions by sodium periodate with the formation of aldehyde groups in the glycosidic part. Reactive carboxyl groups are formed on the surface of PET microparticles by alkaline hydrolysis. After chemical activation at the carboxyl groups, it is bound to a spencer bearing the terminal amino group. The PET microparticles obtained in this way bind via the amino group to the aldehyde groups of the alpha-fetoprotein.

Disclosure of invention

The present invention implements site-specific covalent immobilization of alpha-fetoprotein on the surface of solid polyethylene terephthalate microparticles through a hydrophilic spacer. Polyethylene terephthalate is a linear polycondensation product of terephthalic acid and ethylene glycol, contains ester groups, which can be transformed into reactive carboxyl groups as a result of alkaline hydrolysis. Carboxyl groups can be used for covalent bonding to biomolecules with primary amino groups [12].

The claimed invention provides alkaline hydrolysis of the surface of PET microparticles with the formation of surface carboxyl groups (Scheme 1). To control the specific concentration of carboxyl groups, they were pre-activated with bis- (4-nitrophenyl) carbonate and treated with an amine-containing indodicarbocyanine dye.

To obtain hydrophilic linkers on the surface of PET microparticles, surface activated carboxyl groups were linked with 4,7,10-trioxa-1,13-tridecanediamine. As a result of this treatment, the surface of PET microparticles acquired immobilized spacers containing a primary amino group (Scheme 2). To control the specific concentration of amino groups, the surface was treated with an activated derivative of indodicarbocyanine dye.

To functionalize alpha-fetoprotein, it was selectively oxidized with sodium periodate under mild conditions with the formation of aldehyde groups in the glycosidic portion (Fig. 3). The functional properties of oxidized alpha-fetoprotein were monitored by enzyme immunoassay using an AFP-ELISA kit (Hema, www.xema-medica.com) in accordance with the manufacturer's instructions. In the ELISA method, monoclonal antibodies labeled with horseradish peroxidase bind to the antigenic determinants of oxidized alpha-fetoprotein. To confirm the functional properties of the initial alpha-fetoprotein and the efficiency of the AFP-ELISA kit, the concentration of alpha-fetoprotein in the commercial preparation was additionally monitored. The measured and calculated concentration of alpha-fetoprotein coincided within the margin of error, which indicates the reliability of the data obtained using the AFP-ELISA kit.

The binding of alpha-fetoprotein, which contains aldehyde groups in its structure in the glycosidic part, with PET microparticles having a hydrophilic linker with a terminal amino group on their surface, was carried out according to the Schiff reaction in a sodium carbonate buffer solution at pH 9.5. The azomethine bond formed as a result of the reaction was reduced with sodium borohydride (Scheme 3). The functional properties of alpha-fetoprotein immobilized on the surface of PET microparticles were monitored by enzyme immunoassay using an AFP-ELISA kit. The control results indicate the presence of functional alpha-fetoprotein immobilized on the surface of PET microparticles.

Brief description of circuits

Scheme 1. Scheme of chemical functionalization, carboxylation, surface of PET microparticles and binding with an amine-containing dye.

Scheme 2. Scheme of chemical functionalization of the surface of PET microparticles, amination, and binding with an activated carboxyl-containing dye.

Scheme 3. Scheme of alpha-fetoprotein periodate oxidation and binding to aminated PET microparticles.

Implementation of the invention

The preparation of a commercially available polyethylene terephthalate powder is important for carrying out the invention. Using sieves, a fraction of PET microparticles with a diameter of 40-63 μm was seeded. Small dust-like particles were separated by sedimentation in water. The PET microparticles were washed with acetone with centrifugation and dried in a vacuum desiccator. Received fine polyethylene terephthalate with a particle size of 40-63 microns, free of fine dust particles.

Carboxylated PET microparticles were prepared by the action of an alcoholic solution of potassium hydroxide (Scheme 1). During the destruction of polyethylene terephthalate, the ester bonds of macromolecules are broken with the formation of carboxyl and hydroxyl groups on the polymer surface. The subsequent activation of carboxyl groups on the surface of PET microparticles was carried out in DMSO with bis (4-nitrophenyl) carbonate in the presence of diisopropylethylamine (DIPEA). The activation reaction is carried out for 16 hours at room temperature.

To determine the concentration of 4-nitrophenyloxycarbonyl groups on the surface of PET microparticles, fluorescent labeling was used with an amine-containing indodicarbocyanine dye, which fluoresces in the near-IR region of the spectrum. The condensation reaction of the active 4-nitrophenyloxycarbonyl groups of PET microparticles and cyanine dye was carried out in DMSO in the presence of DIPEA. The amount of the dye bound to the carboxyl groups of PET microparticles was determined spectrophotometrically by determining the concentration of the solution before and after the reaction. The specific concentration of 4-nitrophenyloxycarbonyl groups was 0.60 nanomoles per 1 mg of PET microparticles.

To obtain hydrophilic linkers with a primary amino group on PET microparticles, surface 4-nitrophenyloxycarbonyl groups were treated with a solution of 4,7,10-trioxa-1,13-tridecanediamine in DMSO in the presence of DIPEA (Scheme 2). To control the specific concentration of amino groups, fluorescent labeling with an activated derivative of indodicarbocyanine dye was used. The condensation reaction of the amino groups of PET microparticles and cyanine dye was carried out in DMSO in the presence of DIPEA. The amount of the dye bound to the amino groups of PET microparticles was determined spectrophotometrically by determining the concentration of the solution before and after the reaction. The specific concentration of 4-nitrophenyloxycarbonyl groups was 0.51 nanomoles per 1 mg of PET microparticles.

To immobilize alpha-fetoprotein on the aminated surface of PET microparticles, it was selectively oxidized with sodium periodate to form aldehyde groups in the glycosidic portion (Scheme 3). The functional properties of the oxidized alpha-fetoprotein were monitored by the enzyme immunoassay using an AFP-ELISA kit in accordance with the manufacturer's instructions. The measured concentration of oxidized AFP was 450 ng / ml (450 picomole / μl) and fully coincided with the calculated value (500 ng / ml).

The immobilization of alpha-fetoprotein containing aldehyde groups in the glycosidic part on the surface of PET microparticles with a hydrophilic linker with an amino end group on their surface was carried out using the Schiff reaction in a sodium carbonate buffer solution at pH 9.5 (Scheme 3). As a result of the reaction, an azomethine bond was formed, which was reduced with sodium borohydride. The functional properties of alpha-fetoprotein immobilized on the surface of PET microparticles were monitored by enzyme immunoassay using an AFP-ELISA kit. According to the results of ELISA, PET microparticles with a specific concentration of immobilized alpha-fetoprotein of 100 picomole (100 * 10 ^-12 mol) AFP per 1 mg of PET microparticles are obtained.

Examples of compounds and their applications

Used polyethylene terephthalate powder manufactured by Goodfellow Cambridge Ltd, Enghland grade ES306030, catalog number 264-007-76 (www.goodfellow.com). Natural color material with a density of 1.39 g / cm ³ , polydisperse with a maximum microparticle size of 300 microns.

Alpha-fetoprotein (LEE Biosolution (www.leebio.com) No 105-11-1, 1 mg (2.0 mg / ml, Tris buffer, saline, pH 7.5) was used.

The amine-containing indodicarbocyanine dye was synthesized according to the scheme presented in the publication [12].

The active (4-nitrophenyloxycarbonyl) derivative of the indodicarbocyanine dye was obtained according to the procedure presented in the publication [13].

Used 4,7,10-trioxa-1,13-tridecanediamine, CAS 4246-51-9, Aldrich # 369519-100G.

AFP concentration was performed by ELISA using an AFP-ELISA kit (Hema, www.xema-medica.com) in accordance with the manufacturer's instructions.

Example 1. Preparation of PET microparticles.

With the help of sieves, a fraction of PET microparticles with a diameter of 40-63 μm is sown. Small dust-like particles are separated by sedimentation in water. The PET microparticles are washed with acetone by centrifugation and the supernatant removed. Dry in a vacuum desiccator over P ₂ O ₅ . Finely dispersed polyethylene terephthalate is obtained with a particle size of 40-63 microns, free of fine dust particles.

Example 2. Obtaining carboxylated PET microparticles.

A KOH solution in 96% ethyl alcohol (0.5 M, 1.5 ml) is added to a weighed portion of PET microparticles (100 mg) and mixed by shaking on a shaker (Vortex) for 60 min. Then washed with water, 0.05 M HCl, water. Dry and store in a vacuum desiccator over P ₂ O ₅ .

Example 3. Activation of carboxyl groups on the surface of PET microparticles.

To carboxylated PET microparticles (100 mg), 1.5 ml of a solution of bis (4-nitrophenyl) carbonate (30 nmol / μL) and diisopropylethylamine (60 nmol / μL) in DMSO are added. The mixture is stirred by shaking on a shaker (Vortex) at room temperature for 16 hours. The precipitate is separated by centrifugation and washed with anhydrous acetone (3 × 1 ml) with centrifugation. Dry and store in a vacuum desiccator over P ₂ O ₅ .

Example 4. Determination of the concentration of 4-nitrophenyloxycarbonyl groups on the surface of PET microparticles.

To a weighed portion of PET microparticles with active 4-nitrophenyloxycarbonyl groups (100 mg), add 1 ml of a solution of an amine-containing dye (0.1 mmol / μL) and diisopropylethylamine (0.25 mmol / μL) in DMSO. The mixture is stirred by shaking on a shaker (Vortex) at room temperature for 4 hours. The mixture is centrifuged, the supernatant is separated and the precipitate is washed with 50% acetonitrile in 50 mM triethylammonium bicarbonate buffer solution (TEAN) (5 × 100 μl) with stirring on a shaker ( Vortex) and centrifugation. The supernatant and washings are combined and a 50% solution of acetonitrile in 50 mM TEAN is added to a volume of 1 ml. The concentration of the dye is determined spectrophotometrically. For this, 60 μl of the solution is added to 3 ml of 50% acetonitrile in 50 mM TEAN. Dilution 51 times. The molar extinction coefficient of the amine-containing dye at a wavelength of 647 nm is 2.5 * 10 ⁵ l * M ^-1 * cm ^-1 . The optical density of the solution is measured at a wavelength of 647 nm. The measured optical density with a light path length of 1 cm is 0.196, which corresponds to a concentration of 0.196 * 51 * 1 / 2.5 * 10 ⁵ = 3.998 * 10 ^-5 M / L = 0.3998 * 10 ^-4 M / L = 0.04 mM.

When measuring the initial concentration of the dye in DMSO, a sample of 30 μl is taken and added to 3 ml of 50% acetonitrile in 50 mM TEAN. Dilution 101 times. The molar extinction coefficient of the amine-containing dye at a wavelength of 647 nm is 2.5 * 10 ⁵ l * M ^-1 * cm ^-1 . The measured optical density at a light path length of 1 cm is 0.248, which corresponds to a concentration of 0.248 * 101 * 1 / 2.5 * 10 ⁵ = 10 * 10 ^-5 M / L = 1 * 10 ^-4 M = 0.1 mM.

The amount of the dye bound to PET microparticles is calculated from the difference between the dye taken in the reaction and not bound to PET microparticles in a volume of 1 ml (0.1-0.04) mM / L * 10 ^-3 L = 6 * 10 ^-8 m = 60 nanomole. The specific concentration of 4-nitrophenyloxycarbonyl groups is 60/100 = 0.60 nanomoles per 1 mg of PET microparticles.

Example 5. Amination of the surface of PET microparticles.

To a weighed portion of PET microparticles with surface 4-nitrophenyloxycarbonyl groups (100 mg) add 0.5 ml of a solution of 4,7,10-trioxa-1,13-tridecanediamine (1.0 mmol / μL) and diisopropylethylamine (1.0 mmol / μL) in DMSO. The mixture is stirred by shaking on a shaker (Vortex) at a temperature of + 4 ° C for 4 hours.The mixture is centrifuged, the supernatant is separated and the precipitate is washed with 50% acetonitrile in 50 mM triethylammonium bicarbonate buffer solution (TEAN) (5 × 100 μl) with stirring on a shaker (Vortex) and centrifugation. Dry and store in a vacuum desiccator over P ₂ O ₅ .

Example 6. Determination of the concentration of amino groups on the surface of PET microparticles.

To a weighed portion of PET microparticles (100 mg) containing surface amino groups, add 1 ml of a solution of a 4-nitrophenyloxycarbonyl derivative of indodicarbocyanine dye (0.1 mmol / μL) and DIPEA (0.25 mmol / μL) in DMSO. The mixture is stirred by shaking on a shaker (Vortex) at room temperature for 4 hours. The mixture is centrifuged, the supernatant is separated and the precipitate is washed with 50% acetonitrile in 50 mM triethylammonium bicarbonate buffer solution (TEAN) (5 × 100 μl) with stirring on a shaker ( Vortex) and centrifugation. The supernatant and washings are combined and a 50% solution of acetonitrile in 50 mM TEAN is added to a volume of 1 ml.

During the washing procedures, the 4-nitrophenyloxycarbonyl group of the dye is hydrolyzed to the carboxyl group. The released 4-nitrophenol does not interfere with the spectrophotometric control of the dye. The dye concentration is determined spectrophotometrically. For this, 60 μl of the solution is added to 3 ml of 50% acetonitrile in 50 mM TEAN. Dilution 51 times. The molar extinction coefficient of the carboxyl-containing dye at a wavelength of 647 nm is 2.5 * 10 ⁵ l * M ^-1 * cm ^-1 . The optical density of the solution is measured at a wavelength of 647 nm. The measured optical density with a light path length of 1 cm is 0.239, which corresponds to a concentration of 0.239 * 51 * 1 / 2.5 * 10 ⁵ = 4.876 * 10 ^-5 M / L = 0.488 * 10 ^-4 M / L = 0.049 mM.)

When measuring the initial concentration of the carboxyl-containing dye in DMSO, a sample of 30 μl is taken and added to 3 ml of 50% acetonitrile in 50 mM TEAN. Dilution 101 times. The molar extinction coefficient of the carboxyl-containing dye at a wavelength of 647 nm is 2.5 * 10 ⁵ l * M ^-1 * cm ^-1 . The measured optical density at a light path length of 1 cm is 0.248, which corresponds to a concentration of 0.248 * 101 * 1 / 2.5 * 10 ⁵ = 10 * 10 ^-5 M / L = 1 * 10 ^-4 M = 0.1 mM.

The amount of dye bound to 100 mg of PET microparticles is calculated from the difference between the dye taken in the reaction and not bound to PET microparticles in a volume of 1 ml (0.1-0.049) mM / L * 10 ^-3 L = 5.1 * 10 ^-8 m = 51 nanomoles. Get aminated PET microparticles with a specific concentration of amino groups 51/100 = 0.51 nanomole per 1 mg (510 picomole per 1 mg) of PET microparticles.

Example 7. Selective oxidation of alpha-fetoprotein with the formation of aldehyde groups in the glycosidic part.

A solution of alpha-fetoprotein in Tris-buffered saline (100 μl) at a concentration of 2.0 mg / ml was dialyzed against 20 mM sodium acetate and 0.15 M sodium chloride, pH 5.0 at 4 ° C overnight. Get 200 μl of a solution of alpha-fetoprotein with a concentration of 1 mg / ml in 20 mm sodium acetate and 0.15 M sodium chloride, pH 5.0. To 100 μl of a solution of alpha-fetoprotein with a concentration of 1 mg / ml in 20 mM sodium acetate and 0.15 M sodium chloride, pH 5.0, add 20 μl of a freshly prepared 0.1 M solution of NaIO ₄ in water. The mixture is stirred for 20 minutes at room temperature. The resulting solution was dialyzed against 1 mM sodium acetate, pH 5.0 at 4 ° C overnight. Get 200 μl of a solution of alpha-fetoprotein containing aldehyde groups in the glycosidic part, with a calculated concentration of 0.5 mg / ml (500 μg / μl) in 1 mM sodium acetate, pH 5.0.

Example 8. Control of the initial alpha-fetoprotein by enzyme immunoassay.

The concentration of alpha-fetoprotein in the original stock Tris-buffered saline solution with a concentration of 2.0 mg / ml (according to the manufacturer's data) is determined by ELISA using an AFP-ELISA kit (Hema, www.xema-medica.com).

According to the recommendation of the manufacturer of the AFP-ELISA kit, the concentration of alpha-fetoprotein in the test solution should not exceed 500 IU / ml, 605 ng / ml (1 IU / ml = 1.21 ng / ml). A test AFP solution with a calculated concentration of 500 ng / ml in 20 mM Tris and 0.15 M sodium chloride, pH 7.5 is prepared from the initial stock solution of AFP with a concentration of 2.0 mg / ml (2000 μg / μL).

Conjugate of horseradish peroxidase with mouse monoclonal antibodies (100 μl), the analyzed AFP solution (50 μl) and calibration samples (50 μl) are added to the wells of the plate. Incubated at 37 ° C for 60 minutes. Wash the wells of the plate with washing solution (1x included in the kit, 5 × 200 μl). Add a substrate solution (tetramethylbenzidine), 100 μl, to the wells of the plate and incubate for 20 minutes in a place protected from light. Stop the reaction by adding a stop reagent (100 μl, sulfuric acid solution, included in the kit). A photometric measurement of the optical density of the solution in the well of the plate is carried out at 450 nm. A calibration graph is plotted using calibration samples. The calibration graph is used to determine the concentration of AFP in the test solution. The measured concentration of AFP in the test solution is 495 ng / ml (calculated concentration 500 ng / ml).

Example 9. Control of oxidized alpha-fetoprotein by enzyme immunoassay.

To 1 ml of a solution of 20 mM Tris and 0.15 M sodium chloride, pH 7.5, add 1 μl of a solution of oxidized AFP with a concentration of 0.5 mg / ml (500 μg / μl) in 1 mM sodium acetate, pH 5.0. A solution is obtained with a calculated content of 500 ng / ml of oxidized AFP in 20 mM Tris and 0.15 M sodium chloride, pH 7.5.

Conjugate of horseradish peroxidase with mouse monoclonal antibodies (100 μl) and the test solution of oxidized AFP (50 μl) and calibration samples (50 μl) are added to the wells of the plate. Incubated at 37 ° C for 60 minutes. The wells of the plate are washed with wash solution (1x included in the kit, 5 × 200 μl). Then, a substrate solution (tetramethylbenzidine), 100 μl, is added to the wells of the plate and incubated for 20 minutes in a place protected from light. Stop the reaction by adding a stop reagent (100 μl, sulfuric acid solution, included in the kit). A photometric measurement of the optical density of the solution in the well of the plate is carried out at 450 nm. A calibration graph is plotted using calibration samples. The calibration graph is used to determine the concentration of AFP in the test solution. The measured concentration of oxidized AFP is 450 ng / ml (450 pmol / μl). The calculated concentration is 500 ng / ml.

Example 10 Binding of oxidized AFP to aminated PET microparticles.

To a weighed amount of aminated PET microparticles (20 mg, specific concentration of amino groups 0.51 nanomole per 1 mg, 510 pmol per 1 mg of PET microparticles, 10.2 nanomoles of amino groups) add 100 μl of 0.2 M sodium carbonate buffer solution pH 9.5 and 10 μl of a solution of oxidized AFP in 1 mM sodium acetate, pH 5.0 with a concentration of 450 pmol / μl (4500 pmol = 4.5 nanomolar). The reaction mixture is stirred for 2 h at room temperature. Then add 2 μl of a freshly prepared 0.1 M aqueous solution of NaBH ₄ (4 mg / ml, 4 g / L, M.V. 38 g / m, 0.1 M, 1 μl = 0.1 μm = 100 nanomoles, 2 μl = 200 nanomoles) and stirred for 2 h at 4 ° C.

The PET microparticles are washed with a solution of 20 mM Tris and 0.15 M sodium chloride, pH 7.5 (5 × 200 μl) using centrifugation. Get PET-microparticles with immobilized alpha-fetoprotein. Store PET microparticles with immobilized alpha-fetoprotein under a layer of 20 mM Tris and 0.15 M sodium chloride, pH 7.5 at a temperature of 5 ° C.

Example 11. Control of alpha-fetoprotein immobilized on PET microparticles by enzyme immunoassay.

A portion of PET microparticles with immobilized AFP, obtained from 20 mg of aminated PET microparticles, is suspended in a solution of horseradish peroxidase conjugate with mouse monoclonal antibodies to human AFP (100 μL) and incubated at 37 ° C for 1 hour. The PET microparticles are washed with PET microparticles. solution (1x included in the kit, 5 × 200 μl) using centrifugation. Add a substrate solution (tetramethylbenzidine) (100 μl) and incubate for 20 min protected from light. The reaction is stopped by adding a stop reagent (sulfuric acid solution, included in the kit, 100 μl). A photometric measurement of the optical density of the solution in the well of the plate is carried out at 450 nm. The calibration curve is used to determine 2000 picomole AFP immobilized on PET microparticles. Get PET-microparticles with a specific concentration of immobilized alpha-fetoprotein 100 picomole (100 * 10 ^-12 mol) AFP per 1 mg of PET microparticles.

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Claims (2)

1. Reagent for obtaining DNA fragments, aptamers, functional analogs of monoclonal antibodies, including alpha-fetoprotein immobilized on solid polyethylene terephthalate microparticles (PET microparticles) through a hydrophilic spacer. 2. A method for producing a reagent according to claim 1, including carrying out alkaline hydrolysis of the surface of PET microparticles with the formation of surface carboxyl groups, binding of surface activated carboxyl groups with 4,7,10-trioxa-1,13-tridecanediamine to form immobilized spacers with a primary amino group , oxidation of alpha-fetoprotein with sodium periodate with the formation of aldehyde groups in the glycosidic part, binding of alpha-fetoprotein containing aldehyde groups in the glycosidic part, with PET microparticles having on their surface spacers with a terminal amino group, according to the Schiff reaction in sodium carbonate buffer pH 9.5, while not less than 100 picomole of alpha-fetoprotein is immobilized on 1 mg of polyethylene terephthalate microparticles.

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