TW202227816A - Sensitizer and measurement method for immunochromatography - Google Patents

Abstract

The present invention provides a sensitizer for immunochromatography capable of detecting novel coronavirus (SARS-CoV-2) IgM antibodies with high sensitivity, and an measurement method using the sensitizer. Specifically, provided is a sensitizer for immunochromatography, which contains a copolymer represented by the following general formula [1] and uses a novel coronavirus (SARS-CoV-2) IgM antibody as a target substance for detection.

In the formula, n Rs each independently represent a hydrogen atom or a cation; m and n each represent the number of constituent units; and m: n is 99 to 20: 1 to 80.

Description

Sensitizers and assays for immunochromatographic assays

The present invention relates to a sensitizer for an immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as a measurement object, and a novel coronavirus ( SARS-CoV-2) IgM antibody is an immunochromatographic assay method of the target substance.

Immunochromatography is a method combining a chromatographic method using membrane capillary phenomenon and an immunological method using antigen-antibody reaction. Immunochromatography is widely used as a clinical diagnostic method because it has high specificity due to antigen-antibody reaction, is easy to operate, can be inspected on the spot without requiring a site or special equipment, and can visually determine the results (Patent Document 1). ).

Although the immunochromatographic method has the advantage of being easy to measure, due to the low precision of detection sensitivity, even if a positive sample is used, it may still be judged as a false negative. Therefore, immunochromatographic assay with higher sensitivity is expected. Law.

Various methods have been examined for the purpose of establishing a highly sensitive immunochromatographic assay. For example, in Patent Document 2, it is disclosed by changing the addition position of the reagent for immunochromatography And the method of high sensitivity. In addition, Patent Document 3 discloses a method of increasing the sensitivity by improving the material of the film.

Furthermore, a method for achieving high sensitivity is known by adding an additive to a sample diluent used in conventional immunochromatography. For example, in Patent Documents 4, 5 and 6, it is disclosed that bovine serum albumin (BSA), a polymer having a phosphorocholine group, and hyaluronic acid are contained in the sample dilution solution. In these disclosures, there is no need to change the addition position of the reagent, and there is an advantage that a conventional immunochromatographic membrane can be used.

However, the above-mentioned patent documents do not disclose or teach the sensitizer used in the method for measuring the novel coronavirus (SARS-CoV-2) IgM antibody with high sensitivity.

In particular, the novel coronavirus (SARS-CoV-2) IgM antibody is an antibody produced in the early stage of infection with the novel coronavirus (SARS-CoV-2). As long as the IgM antibody can be detected with high sensitivity, it can be isolated in the early stage of infection. Infected persons, thereby inhibiting the spread of infection. In addition, since the treatment can be started early and the therapeutic effect can be improved, a sensitizer capable of detecting IgM antibodies with high sensitivity is considered to be highly useful.

[Prior Art Literature]

[Patent Literature]

[Patent Document 1] Japanese Patent Application Laid-Open No. 01-063865

[Patent Document 2] Japanese Patent Laid-Open No. 2014-66674

[Patent Document 3] Japanese Patent Laid-Open No. 2014-62820

[Patent Document 4] Japanese Patent Laid-Open No. 2008-292326

[Patent Document 5] Japanese Patent Laid-Open No. 2008-058334

[Patent Document 6] Japanese Patent Laid-Open No. 2003-344413

In the present invention, a sensitizer for immunochromatographic assays capable of detecting novel coronavirus (SARS-CoV-2) IgM antibodies with high sensitivity and an assay method using the sensitizer are provided.

The inventors of the present invention have made active studies to find a compound that can detect IgM antibodies against novel coronavirus (SARS-CoV-2) with high sensitivity. As a result, they found that the compound represented by the following general formula [1] has a phosphorylcholine-like compound in the side chain. The present invention has been completed by obtaining a copolymer based on a base having the target properties.

That is, the present invention is described below.

[1] A sensitizer for an immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as a measurement target substance, the sensitizer for an immunochromatographic assay comprising the following general formula [1] ] shown in the copolymer,

In the formula, n R's each independently represent a hydrogen atom or a cation, m and n each represent the number of constituent units, and m:n is 99 to 20:1 to 80.

[2] The sensitizer for an immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as a substance to be measured according to [1], wherein the copolymer has the following general formula [ 2] A copolymer based on the monomer as a constituent unit and a constituent unit based on methacrylic acid or a salt thereof.

[3] The sensitizer for an immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as the substance to be measured according to [1] or [2], wherein the copolymer is a A copolymer in which the ratio (m:n) of the number of constitutional units based on the monomer represented by the formula [2] to the number of constitutional units based on the methacrylic acid or its salt is 99 to 20:1 to 80.

[4] The sensitizer for immunochromatographic assay using the novel coronavirus (SARS-CoV-2) IgM antibody as the substance to be measured according to [1] or [2], wherein the copolymer is a A copolymer in which the ratio (m:n) of the number of constitutional units based on the monomer represented by the formula [2] to the number of constitutional units based on the methacrylic acid or its salt is 90 to 30:10 to 70.

[5] The sensitizer for an immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as a measurement target substance according to any one of [1] to [4], wherein the copolymer It is a water-soluble copolymer with a weight average molecular weight of 10,000 to 5,000,000.

[6] A sample diluent for an immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as a measurement target substance, comprising the immune system described in any one of [1] to [5] Sensitizers for chromatographic assays.

[7] An immunochromatographic assay method using a novel coronavirus (SARS-CoV-2) IgM antibody as a measurement target substance, which is the immunochromatographic assay method described in any one of [1] to [5] The antigen-antibody reaction is carried out in the presence of a sensitizer.

[8] An instrument for immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as a substance to be measured, using the immunochromatographic assay method described in any one of [1] to [5] The development membrane, sample pad, or binding pad is supported with a sensitizer.

[9] A test kit for novel coronavirus infection (COVID-19), comprising the immunochromatographic assay sensitizer described in any one of [1] to [5] and a specific binding agent Substances in IgM antibodies against novel coronavirus (SARS-CoV-2).

[10] A method for examining the possibility that a subject is infected with novel coronavirus infection (COVID-19), comprising the immunochromatographic assay described in any one of [1] to [5] An antigen-antibody reaction is performed in the presence of a sensitizer, thereby determining the novel coronavirus (SARS-CoV-2) IgM antibody in the biological sample from the subject.

[11] A method for treating novel coronavirus infection (COVID-19), comprising the steps of (1) and (2) below:

(1) Determination of novel coronavirus (SARS-CoV-2) with antigen-antibody reaction in the presence of a sensitizer for immunochromatographic assay comprising a copolymer represented by the following general formula [1] steps; and

(2) According to the measurement results of the above (1), the steps of administering the novel coronavirus infection (COVID-19) treatment to the patient.

In the formula, n R's each independently represent a hydrogen atom or a cation, m and n each represent the number of constituent units, and m:n is 99 to 20:1 to 80.

[12] A kit for the treatment of novel coronavirus infection (COVID-19), comprising: a sensitizer for immunochromatographic assay comprising a copolymer represented by the following general formula [1], and Therapeutic agent for novel coronavirus infection (COVID-19).

In the formula, n R's each independently represent a hydrogen atom or a cation, m and n each represent the number of constituent units, and m:n is 99 to 20:1 to 80.

In the assay method of the present invention using the sensitizer for immunochromatographic assay using the novel coronavirus (SARS-CoV-2) IgM antibody as the measurement target substance, compared with the conventional assay The new coronavirus (SARS-CoV-2) IgM antibody immunochromatographic assay can detect the new coronavirus (SARS-CoV-2) IgM antibody with high sensitivity.

The sensitizer for immunochromatographic assays using the novel coronavirus IgM antibody as the measurement target substance of the present invention, and the sample diluent for the immunochromatographic assay using the antibody as the measurement target substance containing the sensitizer will be described below. , an immunochromatographic assay method using the above-mentioned antibody as a measurement object substance by reacting an antigen-antibody in the presence of the sensitizer, an immunochromatographic measurement apparatus containing the sensitizer and using the above-mentioned antibody as the measurement object substance, and a method for treating novel coronavirus infection (COVID-19) comprising the step of making an antigen-antibody reaction in the presence of the sensitizer.

In addition, in the measurement method using the sensitizer for immunochromatographic measurement of the present invention, although the IgM antibody is used as the measurement target substance, other measurement target substances can also be detected. However, the sensitizer for an immunochromatographic assay of the present invention is characterized in that the antibody can be detected with high sensitivity.

In this specification, the so-called novel coronavirus refers to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and refers to the novel coronavirus infection that causes respiratory disease and causes the COVID-19 pandemic ( COVID-19) (also known as coronavirus disease 2019) strains of the coronavirus. The diameter of each SARS-CoV-2 virus particle is 50 to 200 nanometers, and like other coronaviruses, SARS-CoV-2 has known S (spine), E (envelope, also known as 4 structural proteins of M (membrane), M (membrane), and N (nucleo sheath) proteins. The N protein maintains the RNA genome, and the S, E, and M proteins together form the viral envelope. Spike proteins are proteins that allow viruses to attach and fuse to host cell membranes. SARS-CoV-2 is a human-to-human infection, Initially, infection was presumed to be primarily caused by a range of respiratory droplets from coughs or sneezes. In addition, since aerosol may cause virus infection, it is also taught that the virus also has the possibility of being suspended in the air.

The copolymer used as a sensitizer for an immunochromatographic assay using the novel coronavirus IgM antibody as a measurement object of the present invention is represented by the following general formula [1].

In the above formula, each of the n Rs independently represents a hydrogen atom or a cation. Here, as a cation, sodium ion, potassium ion, ammonium ion, etc. are mentioned, and n R may be the same or different. R is preferably a hydrogen atom.

In the above formula, m and n each represent the number of constituent units, and m:n is 99 to 20:1 to 80, preferably 90 to 30:10 to 70.

The copolymer represented by the above formula is preferably a water-soluble copolymer having a weight average molecular weight of 10,000 to 5,000,000, more preferably 50,000 to 2,000,000.

The copolymer represented by the general formula [1] of the present invention is obtained by having a monomer represented by the above general formula [2] {MPC: 2-(methacryloyloxy)ethyl phosphate 2′-( It consists of a copolymer based on a constituent unit based on trimethylammonio)ethyl ester} and a constituent unit based on methacrylic acid or its salt.

Here, the type of the copolymer is not particularly limited, and may be a random copolymer or a block copolymer, but is preferably a random copolymer.

As an example of methacrylic acid or its salt, methacrylic acid, sodium methacrylate, potassium methacrylate, ammonium methacrylate, etc. can be illustrated. Among them, methacrylic acid is preferred.

The copolymers used as sensitizers for immunochromatographic assays are preferably exemplified by those described in the following examples, but are not particularly limited.

As a specific production example of the water-soluble copolymer represented by the general formula [1], the method described in Japanese Patent Laid-Open No. 08-333421 and Patent Document 6 can be exemplified, but it is not particularly limited.

The copolymer used as the sensitizer for immunochromatographic assay of the present invention can be dissolved in various reagents or samples used in immunochromatographic assay and used, or it can be carried in a kit for immunochromatographic assay. It is used as a group, but it is preferably used as a sample diluent.

In addition, the so-called immunochromatographic assay of the present invention generally includes a sample dilution solution and a test strip as a kit for an immunochromatographic assay to be used.

The so-called sample diluent refers to a suitable diluent when assaying by immunochromatography, which can include Tris buffer, phosphate buffer, veronal buffer, boric acid buffer, Good's ( Good's) buffers and other buffer components; albumin, globulin, casein, serum, water-soluble gelatin, surfactants, carbohydrates, chelating agents and other stabilizing components; salicylic acid, benzoic acid, sodium azide and other preservative components .

When the sensitizer of the present invention is used by dissolving the sample diluent, its concentration is not particularly limited in terms of the concentration at which the antigen-antibody reaction is carried out, but it is usually 0.1 to 5.0 w/v%. It is preferable to use it so as to be 0.5 to 1.0 w/v%. In one form, The concentration of the sensitizer of the present invention in the specimen diluent is usually 0.1 to 5.0% by weight, preferably 0.5 to 1.0% by weight.

The so-called test strip, generally speaking, has at least a first substance that can undergo antigen-antibody reaction with the first epitope of the specimen, and a labeled second substance that can undergo antigen-antibody reaction with the second epitope of the specimen. Two substances, and a membrane support, wherein the first substance is previously immobilized on a predetermined position of the membrane support to form a capture site, and the second substance is in a position isolated from the capture site to develop chromatography on the support. configured in a manner.

In one aspect, when the specimen is an antibody (including an IgM antibody), the first substance and/or the second substance may be an antigen to the antibody.

Typical configurations of test strips for immunochromatography include a sample supply part for supplying a sample (hereinafter, sometimes also referred to as a sample pad), a binding pad for arranging a conjugate, and a capture reagent such as an antibody. The detection part is fixed in a linear shape and the above-mentioned immune complex is developed and captured by the mobile phase, and the development film is composed of an absorption pad for absorbing the sample developed on the development film downstream of the detection part.

Specific examples of materials suitable for the sample pad include, but are not limited to, glass fibers, acrylic fibers, hydrophilic polyethylene, drying paper, pulp, and textiles. In addition, the sample pad can also contain commonly used blocking agents and buffer components according to requirements within the scope that does not depart from the purpose of the present invention and does not affect the reaction system. When the sample is blood, it can also contain blood coagulation. agent, etc. In this case, it may be included in at least a part of the sample pad, and may be included in the whole.

In one aspect, the binding pad is composed of a pad-like porous material that can spread the sample that has passed through the sample pad and retain the binding material, and the binding material is held in part or all of it. As the porous material constituting the bonding pad, a pad composed of nonwoven fibers such as paper, cellulose mixture, nitrocellulose, polyester, acrylonitrile copolymer, glass, and rayon can be mentioned.

As for the test strip for immunochromatographic measurement, a commercially available one can be used, or one produced by a conventional method can be used. When produced by a conventional method, as the base material used for the above-mentioned unfolded film, those commonly used in this field may be used, and preferred examples include cellulose, nitrocellulose, nylon, and the like.

Examples of the immunochromatographic measurement device comprising the sensitizer of the present invention and using the novel coronavirus IgM antibody as the measurement target substance include: (1) a development membrane; (2) a specimen labeling portion and The film is developed and formed in such a way that the specimen labeling portion and the development film can move by capillary action; (3) the specimen labeling portion, the unfolding film, and the liquid absorption portion are included, and the specimen labeling portion is formed such that , The unfolding film and the liquid absorbing part can be formed by capillary movement in sequence; (4) It includes a sample dropping part, a sample marking part, a unfolding film and a liquid absorbing part, and the sample is dropped The portion, the specimen labeling portion, the unfolding film and the liquid absorbing portion can be formed in sequence by capillary phenomenon, etc.; however, there is no particular limitation.

When the sensitizer of the present invention is supported on the instrument for immunochromatographic measurement and used, the amount thereof depends on the place of loading, the type of sensitizer to be used, the type of measurement target substance to be used, the labeling substance to be used, and the like. The variation, for example, can be carried on a developing membrane, a sample pad, or a binding pad of an immunochromatographic measurement device. When the sensitizer of the present invention is supported on the development membrane and the specimen labeling portion of the instrument for immunochromatography, the amount of the sensitizer per unit area (cm ² ) is usually 0.01 μg to 10 mg , preferably 0.1 μg to 4 mg, more preferably 1 to 800 μg. When the sensitizer of the present invention is supported on the drop portion of the specimen, and the drop portion of the specimen is a sample pad, the sensitizer of the present invention to be supported is usually 0.01 μg to 50 mg, preferably 100 μg to 20 mg , more preferably 1 to 10 mg. When the drop portion of the specimen is a binding pad, the sensitizer of the present invention to be carried is usually 0.01 μg to 10 mg, preferably 0.1 μg to 4 mg, and more preferably 1 to 800 μg. In addition, the supporting area varies depending on the type and size of the instrument for immunochromatography used, and the amount of the sample for measurement, but in the case of the developed membrane of the instrument for immunochromatography, it is usually 10 to 60% of the total area. , preferably 5 to 15%, when it is the lower part of the specimen drop, usually 5 to 40% of the total area, preferably 10 to 20%.

The present invention provides a kit for testing novel coronavirus infection (COVID-19), which comprises the sensitizer for immunochromatographic assay of the present invention and an IgM antibody that specifically binds to novel coronavirus (SARS-CoV-2) substance.

The so-called "specificity" means that the affinity of the IgM antibody to the new coronavirus (SARS-CoV-2) is higher than that of other substances (in one aspect, to the new coronavirus (SARS-CoV-2) . IgM antibodies to antigens other than proteins of ) are not included in the "other substances"). The specific binding substance binds to the novel coronavirus (SARS-CoV-2) IgM antibody with a KD of, for example, about 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 or less. In addition, the specific binding substance binds to the novel coronavirus (SARS-CoV-2) IgM antibody with a KD of 10 ^-14 , 10 ^-13 , 10 ^-12 or more.

The aforementioned substances are preferably isolated or purified. The so-called "isolation or purification" means an operation to remove components other than the target component from the natural state. The purity of the isolated or purified aforementioned substances (weight ratio of the aforementioned substances relative to the weight of the whole protein) is usually 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 95% or more ( such as substantially 100%).

The aforementioned substances can be labelled directly or indirectly by means of labelling substances. The labeling substances include fluorescent substances (eg: FITC, rhodamine), radioactive substances (eg: ¹⁴ C, ³ H, ¹²⁵ I), enzymes (eg: alkaline phosphatase, peroxidase) ), colored particles (eg: metal colloid particles, colored latex), biotin, etc.

"Substances that specifically bind to novel coronavirus (SARS-CoV-2) IgM antibodies" in the present invention include antigens that specifically bind to novel coronavirus (SARS-CoV-2) IgM antibodies, Novel coronavirus (SARS-CoV-2) IgM antibody antibody, nucleic acid (for example: aptamer), etc., but preferably the new coronavirus (SARS-CoV-2) IgM antibody antigen, anti-novel coronavirus (SARS-CoV-2) IgM antibody -CoV-2) IgM antibody.

As for the aforementioned antigens, as long as it is not limited to the anti-principle of the novel coronavirus (SARS-CoV-2) IgM antibody to be measured in the present invention, for example, the novel coronavirus S (spike) protein, E (envelope) protein can be mentioned. , M (membrane) proteins, and N (nucleo sheath) proteins.

When a recombinant novel coronavirus (SARS-CoV-2) protein is used as an antigen, the recombinant novel coronavirus (SARS-CoV-2) protein can be produced, for example, by the following method. Insert the polynucleotide encoding the amino acid sequence of the new coronavirus (SARS-CoV-2) protein into an appropriate expression vector, insert the vector into an appropriate host and transform it, from which the disrupted cells of the transformed cells can be obtained Targeted recombinant novel coronavirus (SARS-CoV-2) protein. The above-mentioned host cells are not particularly limited, and various host cells used in conventional genetic engineering methods, such as Escherichia coli, Bacillus subtilis, yeast, plant or animal cells, can be used.

Regarding the new coronavirus (SARS-CoV-2) protein, in addition to the recombinant protein produced from the above-mentioned transformants, it can also be the natural new coronavirus (SARS-CoV-2) that can produce this protein by the known protein separation and purification technology. CoV-2) isolated or refined. In addition, it can also be a protein synthesized chemically or biochemically synthesized by a cell-free translation system.

In one aspect, the above-mentioned "anti-novel coronavirus (SARS-CoV-2) IgM antibody" comprises an anti-IgM antibody of an animal of the same species as the animal that produces the IgM antibody.

Antibodies can be of any isotype, such as IgA, IgD, IgE, IgG (eg, IgGl, IgG2, IgG3, or IgG4), IgM, and the like. In addition to polyclonal antibodies and monoclonal antibodies, antibodies can also be chimeric antibodies and single-chain antibodies. Also, it may be a part of an antibody (antibody fragment) having antigen-binding properties such as a Fab fragment or a fragment produced by a Fab expression library. Examples of antibody fragments include: (i) a Fab fragment comprising monovalent fragments of the VL, VH, CL and CH1 regions; (ii) two Fab fragments comprising two Fab fragments linked by disulfide bonds in the hinge region The F(ab') ₂ fragment of the bivalent fragment; (iii) the Fv fragment comprising the VL and VH regions of the antibody one-arm; (iv) the disulfide bond of the F(ab') ₂ fragment is destroyed by mild reducing conditions The resulting Fab'fragments; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) regions of a single variable region (VH or VL) polypeptide of an antibody that binds specifically to an antigen (dAb) ); but not limited to such.

The anti-novel coronavirus (SARS-CoV-2) IgM antibody used in the method of the present invention can be produced by mice, guinea pigs, hamsters, goats or rabbits. In addition, a commercial item can be used for anti-novel coronavirus (SARS-CoV-2) IgM antibody.

The test kit of the present invention contains the above-mentioned sensitizer for immunochromatographic assay of the present invention and a substance that specifically binds to the novel coronavirus (SARS-CoV-2) IgM antibody in a separate container.

Furthermore, the test kit of the present invention may also include substances that describe the sensitizer for immunochromatographic assay of the present invention and the IgM antibody that specifically binds to the novel coronavirus (SARS-CoV-2) can be used or Instructions that should be used for the inspection of the novel coronavirus infection (COVID-19).

The above-mentioned novel coronavirus infection disease (COVID-19) test kit may include any carrier such as a pharmaceutically acceptable carrier, stabilizer or buffer components, other therapeutic drugs or supplements, etc. Examples of pharmaceutically acceptable carriers include diluents such as water and physiological saline, but are not limited thereto. In addition, the kit for examining the novel coronavirus infection (COVID-19) of the present invention may include a sample dilution solution, a test strip for immunochromatography, and an instrument for immunochromatography.

The present invention provides a method for examining the possibility that a subject is being infected with novel coronavirus infection (COVID-19), the method comprising performing an antigen test in the presence of the sensitizer for immunochromatographic assay of the present invention Antibody response, and determination of novel coronavirus (SARS-CoV-2) IgM antibodies in biological samples from the subject.

As the biological sample that can be used for this method, blood can be mentioned, but there is no particular limitation as long as the novel coronavirus (SARS-CoV-2) IgM antibody can be detected. As for "blood", blood from any tissue can be assumed, but in view of convenience of collection, peripheral blood is usually used. As for the method of collecting blood, a method known per se can be appropriately used. In addition, the collected blood can be directly used in this method, or can also be used as a liquid component (plasma) after separating cellular components (erythrocytes, leukocytes, platelets, etc.) by conventional methods such as centrifugation, filtration, etc. method. Moreover, it can also be used for this method as a liquid component (serum) after coagulating blood and separating platelets or coagulation factors.

When carrying out the test method of the present invention, for example, a biological sample of a subject such as blood is added to the extraction solution to suspend, and the antigen is extracted from the biological sample solution. Next, for example, when the instrument for immunochromatography measurement of the above (4) is used, the biological sample liquid subjected to the extraction solution is dropped on the drop portion of the specimen. Thereby, in the immune layer containing the sensitizer for immunochromatographic assay of the present invention In the presence of the sample diluent for analytical measurement, the biological sample liquid is dropped from the sample to the sample labeling portion by capillary action, and spreads on a membrane.

When the target antigen in the biological sample solution (eg: novel coronavirus (SARS-CoV-2) IgM antibody) passes through the specimen labeling part, it will interact with the labeling substance (eg: anti-novel coronavirus) carried on the specimen labeling part. The virus (SARS-CoV-2, IgM antibody) forms an immune complex that unfolds directly to one end of the unfolded membrane. Then, the complex is bound by immunochemical reaction with the substance (for example: the antigen of IgM antibody to the novel coronavirus (SARS-CoV-2)) existing on the detection line of the unfolded membrane (which has been coated on the detection line). From the color, visible lines appear on the unfolded film. When the color is displayed in this way, the specimen is positive, and it can be determined that the subject is infected with the novel coronavirus infection (COVID-19). In addition, the intensity of the positive can be determined by observing the degree of coloration visually or with a suitable analyzer (eg, an immunochromatographic reader, a spectrophotometer). In addition, as for the detection method of labeled antibody, for example, when fluorescent dye is used, the detection line is irradiated with ultraviolet light, and whether or not it is positive can be determined according to the presence or degree of coloration caused by this.

On the other hand, there is a control line after the detection line, and the antibodies present here (which have been coated there) and the labeling substances that do not form immune complexes (eg: anti-novel coronavirus (SARS-CoV-2) IgM antibody) are produced It is combined by immunochemical reaction, and it can be confirmed that the progress of immunochromatography is no problem by the coloration.

After the specimen sample liquid is developed and passed on the development membrane, unreacted labeled antibodies and the like are absorbed through the absorption pad. In addition, when the target antigen (eg: novel coronavirus (SARS-CoV-2) IgM antibody) is not contained in the specimen, the detection line is not colored, and only the control line is colored. At this time, the specimen is negative, and the subject is not determined to be infected with the novel coronavirus infection (COVID-19).

The biological sample to be used in the test method of the present invention is a sample derived from a mammalian subject. The mammal is not particularly limited as long as it is a mammal that may be infected with the novel coronavirus infection (COVID-19), and among them, mice, rats, hamsters, and guinea pigs are preferred. Laboratory animals such as rodents or rabbits, livestock such as pigs, cows, goats, horses, sheep, minks, pets such as dogs and cats, humans, monkeys, Malay monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees Other primates, especially good people.

The method for treating novel coronavirus infection (COVID-19) of the present invention at least includes the steps (1) and (2) below.

(1) A step of measuring a novel coronavirus (SARS-CoV-2) with an antigen-antibody reaction in the presence of a sensitizer for an immunochromatographic assay comprising the aforementioned copolymer.

(2) According to the measurement results of the above (1), the steps of administering the novel coronavirus infection (COVID-19) treatment to the patient.

In addition, patients also include those who are at risk of contracting the novel coronavirus (SARS-CoV-2). In addition, the so-called assay means to detect the presence or absence of novel coronavirus (SARS-CoV-2) IgM antibodies in a specimen from a patient.

In addition, the so-called steps of administering novel coronavirus infection treatment to patients include not only the administration of conventional therapeutic drugs, therapeutic drugs to be marketed in the future, and candidate therapeutic drugs in the clinical trial stage, but also symptomatic therapy (administration of a Spirin, administration of rehydration/infusion, etc.).

Since the treatment method for novel coronavirus infection (COVID-19) of the present invention can detect novel coronavirus (SARS-CoV-2) IgM antibody with high sensitivity, it can be used in patients with novel coronavirus infection disease (COVID-19). Treat for the novel coronavirus (COVID-19) promptly.

Symptoms of novel coronavirus infection (COVID-19) vary from asymptomatic to severe pneumonia and death. Specifically, there are fever, dry cough, fatigue/fatigue, phlegm, smell disturbance/ Taste disturbance, dyspnea, muscle pain/arthralgia, sore throat, headache, chills, nausea/vomiting, nasal congestion, diarrhea, hemoptysis, conjunctival congestion, etc.

In other aspects, the method for treating novel coronavirus infection (COVID-19) of the present invention at least comprises the following steps (1) and (2).

(1) A step of measuring a novel coronavirus (SARS-CoV-2) with an antigen-antibody reaction in the presence of a sensitizer for an immunochromatographic assay comprising the aforementioned copolymer.

(2) The procedure of administering novel coronavirus infection (COVID-19) treatment to subjects other than humans based on the measurement results of (1) above.

Subjects other than humans to be supplied to the treatment method of the present invention are not limited, and preferably refer to mammals. As for the mammal, it is more preferably a mammal that may be infected with the novel coronavirus infection (COVID-19), and among them, rods such as mice, rats, hamsters, guinea pigs, etc., or experiments such as rabbits are preferred. Animals, pigs, cattle, goats, horses, sheep, minks and other livestock, dogs, cats and other pets, monkeys, Malay monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees and other primates.

The present invention provides a treatment kit for novel coronavirus infection (COVID-19), the treatment kit includes a sensitizer for immunochromatographic assay comprising the copolymer represented by the above-mentioned general formula [1], and therapeutic agents for novel coronavirus infection (COVID-19).

The therapeutic agent includes, for example, a therapeutic drug for novel coronavirus infection (COVID-19), and the therapeutic drug is not limited, but includes: remdesivir, dexamethasone, Favipiravir (Avigan), ciclesonide (ciclesonide), nafamostat (nafamostat), camostat (camostat), ivermectin (ivermectin), nafinavir (nelfinavir) et al. The therapeutic drug may also be tocilizumab, baricitinib, acalabrutinib, ravulizumab, eritoran, ibudilast, LY3127804, otilimab, HLCM051, ADR-001, icatibant, apremilast, Sunny Vero (cenicriviroc). It can also be a virus-neutralizing antibody (casirivimab), idelizumab ( imdevimab) and/or mixtures of these. It can also be a therapeutic drug to be marketed in the future and a candidate therapeutic drug in the clinical trial stage. Therapeutic agents may also include aspirin for symptomatic therapy, fluid replacement/infusion, and the like.

In the therapeutic kit of the present invention, the above-mentioned sensitizer for immunochromatographic assay of the present invention and the therapeutic agent for novel coronavirus infection (COVID-19) are contained in individual containers.

Furthermore, the therapeutic kit of the present invention may also include a description that the sensitizer for immunochromatographic assay of the present invention and the therapeutic agent for novel coronavirus infection (COVID-19) can be or should be used for the novel coronavirus. Instructions for the treatment of viral infection (COVID-19).

The above-mentioned novel coronavirus infectious disease (COVID-19) treatment kit may include any carrier such as a pharmaceutically acceptable carrier, stabilizer or buffer components, other therapeutic drugs or supplements, etc. Examples of pharmaceutically acceptable carriers include diluents such as water and physiological saline, but are not limited thereto. In addition, the kit for treating novel coronavirus infection (COVID-19) of the present invention may include a sample dilution solution, a test strip for immunochromatography, and an instrument for immunochromatography.

The therapeutic kit of the present invention may further comprise a substance specifically binding to the novel coronavirus (SARS-CoV-2) IgM antibody.

Since the novel coronavirus infection (COVID-19) treatment kit of the present invention can detect the novel coronavirus (SARS-CoV-2) IgM antibody with high sensitivity, it can detect the novel coronavirus infection (COVID-19) The subjects (patients) were promptly treated for the novel coronavirus infection (COVID-19).

Hereinafter, the present invention will be described in more detail by means of the present embodiment, but the present invention is not limited to these examples.

[Example]

Manufacturing example

(Synthesis of Copolymers of the Invention and Comparative Examples)

The copolymer used as the sensitizer for immunochromatographic assay of the present invention and the copolymer of the comparative example were synthesized. Details are as follows.

The copolymer used in the present invention and the comparative example is a random copolymer synthesized from 2-(methacryloyloxy)ethyl phosphate 2'-(trimethylammonio)ethyl ester (MPC) and methacrylic acid copolymer.

(Copolymer 1)

Weight average molecular weight: 220×10 ³ , the ratio of the number of structural units based on MPC monomer and the number of structural units based on methacrylic acid=90:10

(Copolymer 2)

Weight average molecular weight: 550×10 ³ , the ratio of the number of structural units based on MPC monomer and the number of structural units based on methacrylic acid=70:30

(Copolymer 3)

Weight average molecular weight: 1100×10 ³ , the ratio of the number of structural units based on MPC monomer and the number of structural units based on methacrylic acid=50:50

(Copolymer 4)

Weight average molecular weight: 680×10 ³ , the ratio of the number of structural units based on MPC monomer and the number of structural units based on methacrylic acid=30:70

Example

(Confirmation of detection sensitivity using conventional immunochromatographic instruments)

In this example, it was confirmed whether the above-mentioned copolymer can improve the detection sensitivity (with or without sensitization effect) of the conventional novel coronavirus (SARS-CoV-2) IgM antibody detection kit. Details are as follows.

(Comparative example)

Evaluation was performed with a commercially available novel coronavirus (SARS-CoV-2) IgM antibody detection kit (manufactured by RayBiotec.). More specifically, 25 μL of the new coronavirus-positive patient serum (CoV-PosM-S-100, RayBiotec.) was added to 245 μL of the diluent of the SARS-CoV-2 IgM antibody detection kit, and the diluent was subjected to immunochromatography. The set expands.

(Example 1)

Copolymers 1, 2, 3, and 4 were added to the diluent of the set so that the final concentration would be 0.5% by weight, and further, the serum of a novel coronavirus-positive patient was diluted 100 times compared to the comparative example. A solution was prepared and developed, and the detection sensitivity was compared.

(Example 2)

Copolymers 1, 2, 3, and 4 were loaded on the sample pad of the set at a loading amount of 5 mg, and further, the serum of the new coronavirus positive patient was prepared by diluting 100 times compared to the comparative example to obtain a solution , develop the solution, and compare the detection sensitivity.

(Example 3)

The copolymers 1, 2, 3, and 4 were loaded on the binding pad of the set at a loading amount of 500 μg, and the serum of the new coronavirus-positive patient was prepared by diluting 100 times compared with the comparative example to obtain a solution , develop the solution, and compare the detection sensitivity.

(Confirmation results of detection sensitivity using immunochromatographic instruments)

Table 1 shows the improvement results of the detection sensitivity when the copolymer is added to the diluent of the conventional novel coronavirus (SARS-CoV-2) IgM antibody detection kit.

[Table 1]

When 0.5% by weight of copolymers 1, 2, 3, and 4 were added, it was confirmed that IgM was detected from a detection line that was the same as or darker, or slightly lighter than the control group of the comparative example. The sensitivity was improved by the addition of the copolymers 1, 2, 3, and 4.

Table 2 shows the improvement results of the detection sensitivity when the polymer of the conventional novel coronavirus (SARS-CoV-2) IgM antibody detection kit is supported on the sample pad.

[Table 2]

When the copolymers 1, 2, 3, and 4 were supported on the sample pads, regarding IgM, it was confirmed from the detection line of the same or slightly lighter shades than the control group of the comparative example. Copolymers 1, 2, 3, and 4 were supported on the sample pad to improve sensitivity.

Table 3 shows the improvement results of the detection sensitivity when the polymer of the conventional novel coronavirus (SARS-CoV-2) IgM antibody detection kit is supported on the binding pad.

[table 3]

When the copolymers 1, 2, 3, and 4 were supported on the binding pad, regarding IgM, it was confirmed from the detection line that was the same as or darker or slightly shallower than the control group of the comparative example. Copolymers 1, 2, 3, and 4 are supported on the bonding pad to improve sensitivity.

According to the above embodiment, compared with the conventional immunochromatographic assay using novel coronavirus (SARS-CoV-2) IgM antibody as the measurement object, the present invention can provide an immunochromatographic assay with excellent and significant sensitizing effect Law.

As can be seen from the above, compared with the conventional immunochromatographic assay method using novel coronavirus (SARS-CoV-2) IgM antibody as the measurement object, the present invention can provide an immunochromatographic assay method with excellent and significant sensitizing effect .

Furthermore, since the treatment method for novel coronavirus infection (COVID-19) of the present invention can detect novel coronavirus (SARS-CoV-2) with high sensitivity, it can be used to treat patients with novel coronavirus infection or those infected with novel coronavirus. Patients with concerns about the coronavirus (SARS-CoV-2) are rapidly treated for novel coronavirus infection, and thus have a high therapeutic effect.

[Industrial Availability]

The present invention can provide a sensitizer for an immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as a measurement target substance.

This application is based on Japanese Patent Application No. 2020-181484 (filing date: October 29, 2020) filed in Japan, and all the contents thereof are incorporated in this specification.

Claims (12)

A sensitizer for an immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as a measurement target substance, the sensitizer for an immunochromatographic assay comprises the following general formula [1] of copolymers,

In the formula, n R's each independently represent a hydrogen atom or a cation, m and n each represent the number of constituent units, and m:n is 99 to 20:1 to 80. The sensitizer for immunochromatographic assay using novel coronavirus (SARS-CoV-2) IgM antibody as the substance to be measured according to claim 1, wherein the copolymer is a compound having the following general formula [2] A copolymer based on the monomer shown as a constituent unit and a constituent unit based on methacrylic acid or its salt,

The sensitizer for an immunochromatographic assay using the novel coronavirus (SARS-CoV-2) IgM antibody as the substance to be measured according to claim 1 or 2, wherein the copolymer is represented by the general formula [2] A copolymer in which the ratio (m:n) of the number of constituent units based on the monomer to the number of constituent units based on methacrylic acid or its salt is 99 to 20: 1 to 80. The sensitizer for an immunochromatographic assay using the novel coronavirus (SARS-CoV-2) IgM antibody as the substance to be measured according to claim 1 or 2, wherein the copolymer is represented by the general formula [2] A copolymer in which the ratio (m:n) of the number of constituent units based on the monomer to the number of constituent units based on methacrylic acid or its salt is 90 to 30: 10 to 70. The sensitizer for immunochromatographic assay using novel coronavirus (SARS-CoV-2) IgM antibody as the measurement object according to any one of claims 1 to 4, wherein the copolymer is a weight-average molecular weight It is a water-soluble copolymer of 10,000 to 5,000,000. A sample diluent for an immunochromatographic assay using a novel coronavirus (SARS-CoV-2) IgM antibody as a measurement object substance, comprising the immunochromatographic assay described in any one of claims 1 to 5. sensitizer. An immunochromatographic assay method using a novel coronavirus (SARS-CoV-2) IgM antibody as a measurement object substance, which is a sensitizer for immunochromatographic assay described in any one of claims 1 to 5. Antigen-antibody reaction was carried out in the presence of. An instrument for immunochromatographic assay using novel coronavirus (SARS-CoV-2) IgM antibody as a measurement object substance, which is loaded with the sensitizer for immunochromatographic assay described in any one of claims 1 to 5. Loaded on a spreading membrane, sample pad, or binding pad. A test kit for novel coronavirus infection (COVID-19), comprising the immunochromatographic assay sensitizer described in any one of claims 1 to 5 and specifically binding to the novel coronavirus (SARS) -CoV-2) IgM antibody substance. A method for examining the possibility that a subject is infected with novel coronavirus infection (COVID-19), comprising the sensitizer for immunochromatographic assay described in any one of claims 1 to 5. antigen-antibody reaction in the presence of Novel coronavirus (SARS-CoV-2) IgM antibody in biological samples. A method for treating a novel coronavirus infection (COVID-19), comprising the steps of the following (1) and (2): (1) Determination of novel coronavirus (SARS-CoV-2) with antigen-antibody reaction in the presence of a sensitizer for immunochromatographic assay comprising a copolymer represented by the following general formula [1] steps, and (2) According to the measurement results of (1) above, the steps of administering the novel coronavirus infection (COVID-19) treatment to the patient,

In the formula, n R's each independently represent a hydrogen atom or a cation, m and n each represent the number of constituent units, and m:n is 99 to 20:1 to 80. A therapeutic kit for novel coronavirus infection (COVID-19), comprising: a sensitizer for immunochromatographic assays comprising a copolymer represented by the following general formula [1], and a novel coronavirus Therapeutic agent for infectious disease (COVID-19),

In the formula, each of n R independently represents a hydrogen atom or a cation, and m and n each represent a constituent unit. Arity, m:n is 99 to 20: 1 to 80.