Method for electrochemical immunoassay for detecting viruses/viral antigens

FIELD: medicine.

SUBSTANCE: invention involves the sandwich-type immune complex formation between antibodies and measles virus/viral antigen followed by attaching an antibody conjugate with a signal-forming label; the signal-forming label represents magnetic nanocomposite particles that are produced by the silicon oxide coating formation on the surface of the magnetic nanoparticles of transition metal oxide, which is supposed to be conjugated with the antibodies; before the stage of the immune complex formation. The immune complex is formed by concentrating it on a solid-phase chemically inert carrier as a result of the magnetic field exposure, which is followed by removing this immune complex from a medium formed on a medium formed on the carrier; the virus/viral antigens and their concentration are detected and measured by a signal generated by transient metal ions formed by the acid treatment of the immune complex.

EFFECT: simplifying the analytical procedure, increasing the rate and reproducibility.

3 cl, 4 dwg, 3 ex

 

The invention relates to biologists and medicine, namely to the immunoassay, in particular to electrochemical methods for determination of virus/virus antigens in various objects and can be used for differential diagnosis of infectious diseases.

The disadvantages of currently used methods of immunoassay are: the need to create special conditions for their implementation, such as methods of analysis based on polymerase chain reaction, and the high cost of the reagents used and the equipment, for example enzyme-linked immunosorbent assay.

The prior art discloses a method mnogoukladnogo immunoassay with the use of microparticles, comprising mixing of different categories of microparticles coated biospecifically reagents for binding a variety of desired analytes and labeled with one or more fluorochromes at different concentrations, emitting long-lived fluorescence, the addition to the mixture of microparticles of the test sample and biospecifics developing reagent labeled detection fluorochrome, conducting the reaction for the formation of biospecifically complexes, the excitation of the fluorochromes and the dimension in the temporary authorization amount of long-lived fluorescence emission to identify the category� of microparticles and the amount of fluorescence emission detector fluorochrome for measuring the amount of desired analytes, thus formed biospecifically complexes carbide is precipitated on a carrier, the emission fluorescence of all fluorochromes excited by sources of radiation in two spectral ranges, for tagging biospecifics showing the detection reagent is used fluorochrome with the short-lived fluorescence, the area of excitation and emission of fluorescence which is outside the regions of excitation and emission fluorescence of fluorochromes with long-lived fluorescence, the ratio of the concentrations of fluorochromes with long-lived fluorescence of the microparticles to determine one type of analyte constantly, and to identify different types of analytes the concentration ratio varies at least twice (see RF patent №2379691 for the invention "Method mnogoukladnogo immunoassay with the use of microparticles", priority date 07.07.2008 G., published 20.01.2010)

The disadvantages of this method are due to the need for a multi-stage process, and the difficulty of obtaining an analytical signal.

The known method mnogoukladnogo immunoassay with the use of microparticles, comprising coating the surface of a porous membrane of the reaction mixture containing the analyte, the first binding molecule associated with the detecting substance and specific to the analyte, explore�th sample and the particles, not able to pass through the membrane pores, coated with the second binding molecules, also specific for the analyte, incubating the mixture for the formation of biospecific complex, the laundering of the reaction mixture from unbound reagents and the detection of the analyte in the sample according to the light signal of the detecting substance associated with biospecifically complex, thus use at least two types specific to the sought analytes of the first and second binding molecules, wherein each species of molecules is specific to only one desired analyte, each of the first binding molecule is associated with at least two long luminescent detection substances the ratio of the concentrations in each of the first binding molecule selected in advance and corresponds to a certain desired analyte is recorded in the temporary permission phosphorescence signals in the spectral ranges of emission detection of substances corresponding to the time constant of decay of these substances, and determine the desired analyte by the ratio of the phosphorescence signal, wherein the spatial resolution of the detection system, Δ (µ2) is determined from the following relationship:

Δ≤S/10Nµ2where

S - the area of adsorption of particles on the membrane, µ2;

N is a specific number of latex� particles in the sample (see RF patent №2339953 for the invention "Method mnogoukladnogo immunoassay with the use of microparticles", the filing date 13.06.2007 G., published 27.11.2008 g).

This method allows you to detect and quantitatively detect low concentrations of biological analytes in samples in research on a solid surface. The need for the use of the membrane makes high demands on the composition of the sample.

The disadvantages of this method are the need for a multi-stage process, and the difficulty of obtaining an analytical signal.

The closest technical solution to the claimed invention, selected as a prototype, is a method, carried out using electrochemical immunosensors developed for the detection of surface antigen of hepatitis B. First, biotinylated antibodies to hepatitis b immobilizers coated with streptavidin on the magnetic nanoparticles. Then add the antigen of hepatitis B virus and after this phase is injected secondary antibodies conjugated to signalosome label, which is used as a horseradish peroxidase. The result is the formation of immunocomplex on a "sandwich". Thereafter, a working substrate, which is used as the buffer solution of Britton-Robinson containing re�ord hydrogen and antoniniana, the horseradish peroxidase catalyzes the oxidation of aminophenol 3-aminophenazone. Then take an aliquot of the sample, transfer it to the electrochemical cell and register a cyclic voltamperometry. The magnitude of the peak current of oxidation is proportional to the number 3 aminophenazone, which, in turn, is proportional to the content labeled with horseradish peroxidase "sandwich"structures, and hence the content of the antigen of hepatitis b virus. This method of immunoassay allows you to define from 0.001 to 0.015 ng/ml of antigen, the detection limit of 0.9 ág/ml at a signal/noise 1/3 (see Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis surface antigen In / Sara Nourani, Hedayatollah Ghourchian, Seyed Mehdi Boutorabi // Analytical Biochemistry, 2013, V. 441, P. 1-7).

The disadvantages of this method include a multistage process analysis (pre biotinylating antibodies, conjugation of antibodies with horseradish peroxidase), high labor-intensive, time-consuming and also high requirements to qualification statements.

The technical result, which is aimed to offer the protection of the technical solution is to simplify the analysis process, the increase of quick testing were and reproducibility.

The specified technical result is achieved in that the proposed method of electrochemical immunogen�Lisa to identify viruses/antigens of viruses, comprising the formation of immunocomplex on a "sandwich" between the antibodies and the virus/virus antigen followed by the addition of conjugate antibodies with signalosome label for the purpose of determining the presence and concentration of virus/virus antigen by obtaining electrochemical response due to the use of signalosome label, which is part of the "sandwich"structure, according to the invention as signalosome label use of magnetic nanocomposite particles, which before the stage of formation of immunocomplex get by creating on the surface of magnetic nanoparticles transition metal oxide oxydrene coating followed by obtaining the conjugate with antibodies, in this case the formation of immunocomplex (antibody - virus/virus antigen - antibody with signalosome magnetic label) is carried out by its concentration on the solid inert carrier by exposure to a magnetic field with the subsequent seizure of immunocomplex from the environment, formed on the carrier, and detecting the presence and concentration of virus/virus antigen is performed on a signal generated by transition metal ions resulting from the acid treatment of immunocomplex, wherein the transition metal is used in iron, and as terdapat�wow chemically inert carrier - thick graphite electrode.

To ensure interaction between the magnetic nanoparticles and antibodies to magnetic nanoparticles can be modified by aminopropyltriethoxysilane, forming, thereby, execrative coverage.

For acid treatment, contributing to the chemical destruction of immunocomplex, use a mixture of nitric and sulfuric acids.

The formation of immunocomplex significantly reduced through the use of magnetic concentration of immunocomplex on a solid phase carrier, which, in turn, allows you to accelerate the procedure for immunoassay. In addition, increases sensitivity.

The concentration of virus/virus antigen is determined by obtaining the electrochemical response recorded from ion Fe3+obtained by acid treatment of the carbon substrate with immobilizerturning the immunological complex.

Technical solutions, which coincides with the set of essential features of the claimed invention, not identified that allows to make a conclusion on compliance of the claimed invention to such a condition of patentability as "novelty".

Declare the essential features that determine the receipt of the indicated technical result that is explicitly not follow from the prior art, which allows to conclude that according�accordance of the present invention, such a condition of patentability, as "inventive step".

The condition of patentability "industrial applicability" is confirmed on the example of a specific implementation.

The claimed invention is illustrated by drawings, where

Fig.1

(a) front view carbon substrates - thick graphite electrode (CGE);

b) is a side view of a carbon substrate - thick graphite electrode (CGE);

Fig.2 - cyclic voltamperometry registered in model solutions containing (a, 4-6) and not containing (b, 4-6) antigen of measles virus (NovO/96), where 4 - voltamperometry of the supporting electrolyte, 5 - voltamperometry the model solution, 6 - voltamperometry registered after the introduction the model solution of the additive ions Fe3+;

Fig.3 - cyclic voltamperometry registered in samples containing (a, 7-9), and not containing (b, 7-9) the measles virus, where 7 - voltamperometry of the supporting electrolyte, 8 - voltamperometry samples, 9 - voltamperometry registered after the introduction of additives to the sample ions Fe3+.

Fig.4 - cyclic voltamperometry registered in samples containing (a, 10-12), and not containing (b, 10-12) the measles virus, where 10 - voltamperometry of the supporting electrolyte, 11 - voltamperometry samples, 12 - voltamperometry registered after the introduction of additives to the sample ions Fe .

Thick graphite electrode - tungsten carbide carrier consists of a carbon substrate 1 (electrode) made of fiberglass, which contained a track of conductive material 2, which is used, for example, graphite composition, carbon ink. Track 2 is coated with the layer of the insulator 3 or cementite.

The claimed invention is confirmed by examples of specific performance.

Example 1.

The model solution with the antigen of measles virus (NovO/96) were incubated for 30 minutes at 37°C with magnetic nano-particles of Fe3O4-SiO2on which surface immobilizerpower polyclonal IgG to measles virus. After incubation the solution is placed the (Fig.1), a modified IgG, and incubated for 20 minutes at 37°C. To accelerate the delivery to the surface of the nanocomposite particles with immobilizerturning on the surface of the immunological complex using a magnetic field. Then the electrode was washed with buffer containing normal horse serum (TLS) and tween 20. Extracted from the analyzed solution, the electrode is placed in a mixture of nitric and sulfuric acids (0.36 M H2SO4and 0.28 M HNO3for the subsequent destruction of the "sandwich" and the dissolution of the nanocomposite particles. As a signal characterizing the content�of virus/virus antigens in the sample using electrochemical response of iron passed into the acid solution at the destruction of the "sandwich." To conduct no-load experience using a phosphate buffer not containing antigen of the virus (NovO/96) (Fig.2). In the model solution discovered 8×10-2mg/ml of antigen.

Example 2.

The sample containing the washings from the nasopharynx of a patient infected with measles virus, incubated for 30 minutes at 37°C with magnetic nano-particles of Fe3SO4-SiO2on which surface immobilizerpower polyclonal IgG to measles virus. After incubation the solution is placed the (Fig.1) a modified IgG, and incubated for 20 minutes at 37°C. To accelerate the delivery to the surface of the nanocomposite particles with immobilizerturning on the surface of the immunological complex using a magnetic field. Then the electrode was washed with buffer containing normal horse serum (TLS) and tween 20. Extracted from the analyzed solution, the electrode is placed in a mixture of nitric and sulfuric acids (0.36 M H2SO4and 0.28 M HNO3) for the destruction of "sandwich" and the dissolution of the nanocomposite particles. As the signal characterizing viruses/virus antigens in a sample, using electrochemical response of iron passed into solution by acid destruction "se�of dicha". To conduct no-load experience using a sample containing the washings from the nasopharynx of a healthy patient (Fig.3). In a sample taken from an infected patient, detected concentrations of virus, corresponding to 3×10-3mg/ml of antigen.

Example 3.

The sample containing the washings from the nasopharynx of a patient infected with measles virus, incubated for 30 minutes at 37°C with magnetic nano-particles of Fe3O4-SiO2on which surface immobilizerpower polyclonal IgG to measles virus. After incubation the solution is placed the (Fig.1) a modified IgG, and incubated for 20 minutes at 37°C. To accelerate the delivery to the surface of the nanocomposite particles with immobilizerturning on the surface of the immunological complex using a magnetic field. Then the electrode was washed with buffer containing normal horse serum (TLS) and tween 20. Extracted from the analyzed solution, the electrode is placed in a mixture of nitric and sulfuric acids (0.36 M H2SO4and 0.28 M HNO3) for the destruction of "sandwich" and the dissolution of the nanocomposite particles. As the signal characterizing viruses/virus antigens in a sample, using electrochemical response of iron passed into the acid solution at the destruction of the "sandwich." For a blank experience used�isout sample containing the washings from the nasopharynx of a healthy patient (Fig.4). In a sample taken from an infected patient, detected concentrations of virus, corresponding to 4×10-4mg/ml of antigen.

The proposed method can significantly reduce material and labor costs required for carrying out the process, increase productivity and reduce cost definition.

In addition, the invention allows to simplify the process of immunoassay, as well as to increase the rapidity and reproducibility, and use it in different environments.

1. Method of electrochemical immunoassay for determination of viruses/antigens of measles, including education immunocomplex on a "sandwich" between the antibodies and the virus/virus antigen followed by the addition of conjugate antibodies with signalosome label for the purpose of determining the presence and concentration of virus/virus antigen by obtaining electrochemical response due to the use of signalosome label, which is part of the "sandwich"structure, characterized in that as signalosome label use of magnetic nanocomposite particles, which before the stage of formation of immunocomplex get by creating on the surface of magnetic nanoparticles transition metal oxide oxydrene coating with subsequent proceneonate with antibodies, in this case the formation of immunocomplex (antibody-virus/virus antigen - antibody with signalosome magnetic label) is carried out by its concentration on the solid inert carrier by exposure to a magnetic field with the subsequent seizure of immunocomplex from the environment, formed on the carrier, and detecting the presence and concentration of virus/virus antigen is performed on a signal generated by transition metal ions resulting from the acid treatment of immunocomplex, wherein the transition metal is used in iron, and as the solid chemically inert carrier - thick graphite electrode.

2. Method of electrochemical immunoassay for determination of viruses/antigens of measles virus according to claim 1, characterized in that to enable interaction between the magnetic nanoparticles and antibodies to magnetic particles coated with aminopropyltriethoxysilane.

3. Method of electrochemical immunoassay for determination of viruses/antigens of measles virus according to claim 1, characterized in that the acid treatment, a mixture of nitric and sulfuric acids.



 

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