JP7631666B2 - Methods for suppressing non-specific reactions - Google Patents

Description

The present invention relates to a method for preventing inaccurate measurement results caused by viscous mucin in immunoassays that use mucin-containing specimens as samples.

Immunoassays are widely used in clinical testing to measure trace substances in biological samples, such as various body fluids such as blood and cerebrospinal fluid, excretions such as stool and urine, and extracts of various tissues. Many immunoassay methods are known, including RIA, EIA, immunoturbidimetry, latex agglutination, metal colloid agglutination, and immunochromatography.

In addition to serum and plasma, various biological samples such as nasal swabs, nasal aspirates, nasal washings, nasal blows, and throat swabs are used in immunoassays. However, samples other than serum and plasma contain viscous components such as mucin, which can lead to inaccurate measurement results, such as false positives and false negatives.

To date, many techniques for suppressing non-specific reactions have been known in immunoassays that utilize antigen-antibody reactions. Examples include a method for suppressing non-specific binding of non-target substances in a sample to a solid-phase surface (Patent Document 1) and a method for suppressing non-specific reactions by treating a sample with a sample treatment solution that contains a compound with two or more sulfate groups (Patent Document 2).

However, the above studies have not revealed all the causes of non-specific reactions, and even when the above methods are used, there are still cases where measured values differ from the true values. Therefore, there is a need to develop further useful means to avoid non-specific reactions that can occur when using biological samples that contain mucin, a viscous component.

JP 2008-216237 A Patent No. 6116268

The present invention has been made in consideration of the above-mentioned conventional techniques, and the object of the present invention is to suppress the occurrence of non-specific reactions (especially false positives) that are caused by mucin in a specimen in an immunoassay method that uses a specimen containing mucin as a sample.

As a result of intensive research conducted by the inventors to achieve the above object, they have succeeded in effectively suppressing the occurrence of non-specific reactions (particularly false positives) that may be caused by mucin in a specimen by using a solution containing a buffer component having a carboxyl group as a solution for use in an immunoassay using a biological sample containing mucin, thereby completing the present invention.

That is, the present invention includes the following aspects.
[Item 1] A method for suppressing non-specific reactions in an immunoassay using a mucin-containing biological sample, the method comprising using a solution containing a buffer component having a carboxy group.
[Item 2] The method according to Item 1, wherein the buffer component having a carboxy group is at least one selected from the group consisting of N,N-bis(2-hydroxyethyl)glycine, N-[tris(hydroxymethyl)methyl]glycine, N-(2-acetamido)iminodiacetic acid, acetic acid, citric acid, and tartaric acid.
[Item 3] The method according to item 1 or 2, wherein the mucin-containing biological sample is a nasal swab, a nasal aspirate, a nasal wash, a nasal blown fluid, or a pharyngeal swab.
[Item 4] The method according to any one of Items 1 to 3, wherein the immunoassay is a method for immunoassaying influenza virus nucleoprotein.
[Item 5] The method according to any one of Items 1 to 4, wherein the immunoassay is a method for immunoassaying influenza B virus nucleoprotein.
[Item 6] The method according to any one of Items 1 to 5, wherein the solution containing a buffer component having a carboxy group is any one of a washing solution, a developing solution, a specimen treatment solution, a specimen dilution solution, and an antibody solution.
[Item 7] The method according to any one of Items 1 to 6, wherein the solution containing a buffer component having a carboxy group contains a buffer component having a carboxy group at a concentration of 1 to 1000 mM.
[Item 8] The method according to any one of Items 1 to 7, wherein the solution containing a buffer component having a carboxy group further contains at least one selected from the group consisting of surfactants, proteins, salts, pH adjusters, and preservatives.
[Item 9] The method according to any one of Items 1 to 8, wherein the immunoassay comprises a step of performing detection on a solid phase.
[Item 10] A composition for suppressing non-specific reactions in an immunoassay using a mucin-containing biological sample, comprising a buffer component having a carboxy group.
[Item 11] The composition according to Item 10, wherein the buffer component having a carboxy group is at least one selected from the group consisting of N,N-bis(2-hydroxyethyl)glycine, N-[tris(hydroxymethyl)methyl]glycine, N-(2-acetamido)iminodiacetic acid, acetic acid, citric acid, and tartaric acid.
[Item 12] The composition according to Item 10 or 11, wherein the mucin-containing biological sample is a nasal swab, a nasal aspirate, a nasal wash, a nasal blown fluid, or a pharyngeal swab.
[Item 13] The composition according to any one of Items 10 to 12, which is used in a method for immunoassaying influenza virus nucleoprotein.
[Item 14] The composition according to any one of Items 10 to 13, which is used in a method for immunoassaying influenza B virus nucleoprotein.
[Item 15] The composition according to any one of Items 10 to 14, which is any one of a washing solution, a developing solution, a specimen treatment solution, a specimen dilution solution, and an antibody solution.
[Item 16] The composition according to any one of Items 10 to 15, comprising a buffer component having a carboxy group at a concentration of 1 to 1000 mM.
[Item 17] The composition according to any one of Items 10 to 16, further comprising at least one selected from the group consisting of surfactants, proteins, salts, pH adjusters, and preservatives.
[Item 18] The composition according to any one of Items 10 to 17, which is used in an immunoassay comprising a step of detecting on a solid phase.
[Item 19] A kit for use in an immunoassay, comprising the composition according to any one of Items 10 to 18.

The present invention makes it possible to highly suppress non-specific reactions (particularly the occurrence of false positives) that may occur due to mucin in immunoassays that use mucin-containing specimens as samples. Therefore, it is possible to provide highly reliable test results in immunoassays using mucin-containing specimens.

FIG. 1 shows the results in Example 1.

The following describes in detail the embodiments of the present invention, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference. In addition, "~" in this specification means "more than or equal to, less than or equal to", for example, if the specification states "X~Y", it means "more than or equal to X, less than or equal to Y". In addition, "and/or" in this specification means either one or both.

In one embodiment, the present invention provides a method for suppressing non-specific reactions (particularly the occurrence of false positives) in an immunoassay using a mucin-containing biological sample (also referred to herein as a "mucin-containing biological sample"), the method being characterized by using a solution containing a buffer component having a carboxyl group.

Mucin, also known as mucus glycoprotein, is well known as the main component of mucus secreted from epithelial cells in animals. For example, it is known that mucous membranes such as the nasal cavity and oral cavity are covered with mucin. Therefore, nasal swabs, nasal aspirates, nasal washings, nasal blows, pharyngeal swabs, and pharyngeal aspirates, which are used as samples in immunoassays, often contain mucin secreted from these mucous membranes and exhibit viscosity. It is also known that mucin is contained in nasal mucus, saliva, tears, gastric juice, intestinal juice, and other fluids secreted from the living body.

Such mucin-containing biological samples are prone to nonspecific reactions, for example, by remaining at the detection site in an immunoassay or affecting the antigen-antibody reaction, resulting in false positives (sometimes referred to as "false positives" in this specification), or by inhibiting the antigen-antibody reaction or reducing the fluidity of the solution, resulting in false negatives. For example, a nonspecific reaction between mucin, an antibody, and a solid phase can cause false positives by leaving unreacted labeled antibodies on the solid phase where detection is performed, or in the case of immunochromatography, a false negative can occur due to a decrease in the migration efficiency of immune complexes, making it difficult for them to reach the detection site. In particular, false positives are likely to occur when viscous mucin-containing biological samples remain at the detection site in an immunoassay, and unreacted antigens, antibodies, etc. contained therein remain.

In one preferred embodiment, for example, the present invention is a method for suppressing false positives when detecting a target substance in a biological sample containing mucin by immunoassay, characterized in that antibodies or antigens (particularly antibodies) that do not bind to the target substance are treated with a solution containing a buffer component having a carboxy group (e.g., washed with a washing solution). Since the occurrence of false positives leads to inaccurate measurement results, this method can also be said to be a method for reducing the effect of mucin on the measurement results of immunoassay, or a method for improving the accuracy of the measurement results of immunoassay.

One of the features of the present invention is the use of a solution containing a buffer component having a carboxy group in order to suppress the occurrence of non-specific reactions in immunoassays. The buffer component having a carboxy group is not particularly limited as long as it is a compound that contains a carboxy group in its structure and can exhibit buffering ability. For example, examples of buffer components having a carboxy group include, but are not limited to, N,N-bis(2-hydroxyethyl)glycine (known as "Bicine"), N-[Tris(hydroxymethyl)methyl]glycine (known as "Tricine"), N-(2-acetamido)iminodiacetic acid (known as "ADA"), acetic acid, citric acid, tartaric acid, and the like. These buffer components having a carboxy group may be used alone or in combination of two or more. In immunoassays using biological samples containing mucin, from the viewpoint of more reliably obtaining a high false positive suppression effect, in the present invention, bicine, tricine, and/or ADA are preferred as buffer components having a carboxy group, bicine and/or tricine are more preferred, and tricine is particularly preferred. As shown in the results of the test examples described below, when a solution containing these buffer components having a carboxy group is used, a high false positive prevention effect has been confirmed.

The solution containing a buffer component having a carboxy group used in the present invention may be a solution in which the above-mentioned buffer component having a carboxy group is dissolved in any solvent. The solvent used here is not particularly limited as long as it can exert the effects of the present invention, and may be any liquid such as water or an organic solvent. From the viewpoint of being less likely to inhibit the antigen-antibody reaction in the immunoassay method and being easy to dissolve the buffer component having a carboxy group, it is preferable to use water and/or a water-soluble organic solvent, and of these, water is preferable.

The concentration of the buffer component having a carboxy group in the solution is not particularly limited as long as the effect of the present invention is achieved, but as an example, it can be about 1 to 1000 mM, preferably about 5 to 500 mM, and more preferably about 5 to 100 mM. By using a solution containing a buffer component having a carboxy group in such a concentration range, the false positive suppression effect can be more reliably achieved.

The pH of the solution containing a buffer component having a carboxy group is not particularly limited as long as the buffer component having a carboxy group used has a buffering capacity and is within a range in which the effects of the invention can be exerted. For example, the pH of the solution may be 3 to 12. As an example, when a solution containing a buffer component having a carboxy group is used as a washing solution in an immunoassay, the washing solution may preferably have a pH of about 4 to 11, more preferably about 5 to 10, and even more preferably about 6 to 9.

The solution containing the buffer component having a carboxy group used in the present invention may further contain any other components as long as the effects of the present invention can be exhibited. Examples of such further components that may be contained include, but are not limited to, additives such as surfactants, proteins, salts, pH adjusters, and preservatives. Such additives may be blended in a concentration of, for example, about 0.001 to 1000 mM relative to the entire solution.

In a particular embodiment, the solution containing a buffer component having a carboxyl group used in the present invention preferably further contains a surfactant. By further containing a surfactant in this way, false positives caused by a biological sample containing mucin can be suppressed to a higher degree. This effect is particularly noticeable when a solution containing a buffer component having a carboxyl group is used as a washing solution in an immunoassay. As the surfactant that can be used in the present invention, any surfactant such as a nonionic surfactant, an anionic surfactant, a cationic surfactant, or a zwitterionic surfactant can be used, and preferably, a nonionic surfactant and/or an anionic surfactant is used. As the nonionic surfactant, any nonionic surfactant that can be commonly used in an immunoassay can be used, and for example, polyoxyethylene sorbitan monolaurate (e.g., Tween 20, Tween 80), octylphenol ethoxylate (e.g., Triton X100), etc. can be used to achieve a high false positive suppression effect. As the anionic surfactant, any anionic surfactant that can be commonly used in an immunoassay can be used, and for example, sodium dodecyl sulfate, etc. can be used.

In a further embodiment, the solution containing a buffer component having a carboxy group used in the present invention preferably contains a protein that can function as a blocking agent or the like. The effect of the present invention can be more easily achieved by further containing such a protein. Such a protein is preferably a protein that can act as a blocking agent in an immunological measurement method, and examples of such a protein include bovine serum albumin (BSA), casein, skim milk, gelatin, blood proteins, plant proteins, and the like. Also, rabbit blood components containing blood proteins may be used as they are.

In a further embodiment, the solution containing a buffer component having a carboxy group used in the present invention preferably further contains a salt. As the salt, any salt that can be commonly used in immunological assays can be used. The salt may be an inorganic salt or an organic salt, but is preferably an inorganic salt, more preferably a sodium salt, and even more preferably sodium chloride.

The immunoassay to which the method of the present invention is applied is not particularly limited as long as it is a method for detecting a target substance in a biological sample containing mucin by immunological analysis. In particular, mucin is likely to remain at the detection site (also called the reaction site, determination site, etc.) of an antigen-antibody reaction, causing false positives, and the present invention can effectively suppress the occurrence of such false positives. Therefore, a preferred immunoassay method is an immunoassay method that includes a step of performing detection on a solid phase, such as a flow-through or lateral flow immunoassay method in which determination is performed at the detection site of the solid phase. From the viewpoint of including a washing step for reducing the remaining mucin at the detection site of the solid phase, the immunoassay method performed in the present invention is preferably a flow-through immunoassay.

In one preferred embodiment, when the present invention is carried out using a flow-through immunoassay, it is preferable to include, for example, a step of removing antibodies or antigens that are not bound to the substance to be detected using a washing solution. Examples of such immunoassays include the RIA method and the EIA method.

In the case of the EIA method, for example, the method described in the following procedure (A) or (B) can be mentioned.
(A)
Step (1): A primary antibody modified so as to be able to bind to the solid-phase carrier described below, an enzyme-labeled secondary antibody, and a sample containing the substance to be measured as a component are mixed and incubated to form a sandwich complex with the substance to be measured.
Step (2): The solution containing the complex formed in step (1) is added to a reaction layer of a carrier to which an antibody capable of binding to the modified primary antibody has been immobilized and to which a blocking agent has been added, thereby binding the complex to the solid phase.
Step (3): The reaction layer is washed with a washing solution to remove unreacted labeled antibodies and the like.
Step (4): A substrate solution is added to the reaction layer, and the amount of color and luminescence is monitored.

(B)
Step (1): A sample containing a substance to be measured as a component is added to a reaction layer on which a primary antibody has been immobilized, and the sample is incubated to allow the substance to be measured to be captured by the primary antibody.
Step (2): The reaction layer is washed with a washing solution to remove sample components other than the substance to be measured that has been captured by the primary antibody.
Step (3): A solution containing an enzyme-labeled secondary antibody is added to the reaction layer, and the labeled antibody is allowed to bind to the substance to be measured that has been captured by the primary antibody.
Step (4): The reaction layer is washed with a washing solution to remove unreacted labeled antibodies and the like.
Step (5): A substrate solution is added to the reaction layer, and the amount of color and luminescence is monitored.

The present invention will be described below taking the above procedure (A) as an example, but the present invention is not limited thereto.
There are various variations of the EIA immunoassay method in addition to the above examples, and each method has already been established in the art. Therefore, the knowledge can be applied to the present invention to measure the amount or concentration of a biological component in various samples, and the mode is not particularly limited.

In the present invention, a solution containing a buffer component having a carboxyl group is used as any solution used in the above-mentioned immunoassay. The solution used in the immunoassay may be, from the viewpoint of being easily acted on the detection site of the solid phase where mucin remains and false positives are likely to occur, preferably a washing solution for washing the detection site of the solid phase (e.g., B/F separation washing solution, etc.), but is not particularly limited. For example, a pretreatment solution (e.g., specimen treatment solution, etc.) used before applying a mucin-containing biological sample to the immunoassay, or a developing solution used to develop an antigen-antibody reactant in an immunochromatography method, may affect the fluidity of the solution on the solid phase or reach and act on the detection site on the solid phase together with the mucin contained in the biological sample, and therefore may exert the effect of the present invention. Therefore, the solution containing a buffer component having a carboxyl group used in the present invention may be, for example, a washing solution, a developing solution, a specimen treatment solution, a specimen diluting solution, an antibody solution, etc. used in the immunoassay. From the viewpoint of being able to easily obtain a higher false positive suppression effect, the solution containing a buffer component having a carboxy group used in the present invention is preferably a cleaning solution, more preferably a B/F separation cleaning solution.

One preferred embodiment of the present invention is a method for suppressing false positives in an immunological analysis method in which an antigen-antibody complex is formed on a solid phase, and then unbound antigen or antibody is washed away with a solution (washing solution) containing a buffer component having a carboxy group. One embodiment of such a method is, for example, a method for measuring a substance to be measured in a sample containing mucin, which is carried out according to the following steps (1) to (4).
Step (1): A sample containing a substance to be measured and a viscous component is mixed with a solution containing an antibody (primary antibody) capable of binding to the substance to be measured and modified with a ligand, and a solution containing an antibody (secondary antibody) capable of binding to the substance to be measured and labeled with an enzyme, to form a sandwich complex of the primary antibody, the substance to be measured, and the secondary antibody through an immune reaction.
Step (2): A solution containing the sandwich complex is brought into contact with a carrier on which a substance capable of binding to the ligand has been immobilized in advance, thereby binding the complex to the antibody on the immobilized carrier.
Step (3): The solid-phase carrier is washed with a washing solution containing a buffer component having a carboxy group to remove the primary antibody and the secondary antibody that are not bound to the antibody on the solid-phase carrier.
Step (4): A liquid containing a substrate for the enzyme is added to the immobilized carrier, and the amount of chemical change in the substrate caused by the enzyme is monitored on the immobilized carrier, thereby carrying out the measurement.
According to the above-mentioned method, it is preferable that in step (3), a viscous biological sample containing mucin and any remaining unreacted antigens or antibodies that are entangled with the biological sample can be thoroughly washed away using a solution containing a buffer component having a carboxy group.

For example, various solid phases such as beads, magnetic particles, microtiter plates, tubes, and membranes can be used. From the viewpoint of making it easier to obtain a high level of the false positive suppression effect of the present invention, the solid phase is preferably a glass filter. The solid phase may be one in which a substance capable of binding to the ligand is immobilized in advance. When immobilizing, the method of immobilization is not particularly limited. When a microtiter plate or a tube is used as the solid phase carrier, the solid phase carrier itself can be used as a reaction vessel, but various reaction vessels can also be used separately from the solid phase carrier.

As a substance (preferably an antibody or antigen) that specifically binds to the substance to be measured, various substances can be used that can be selected depending on the substance to be measured in the sample. These may be commercially available products, or can be obtained by known methods.

In addition, methods are also known in which these antibodies, etc. are modified or labeled according to purpose with ligands such as enzymes activated by introducing glutaraldehyde or maleimide groups or biotin-binding proteins (avidin) targeting primary amines or free sulfhydryl groups in the proteins, and are not particularly limited. With such methods, for example, it is possible to prepare antibodies (primary antibodies) modified with ligands capable of binding to the substance immobilized on the solid phase, or anti-(secondary antibodies) labeled with enzymes or the like. These modified or labeled antibodies, etc. are usually held in aqueous solution, and the composition is not particularly limited as long as it does not impair the respective functions.

The method of binding the solid phase to the substance to be measured is not particularly limited. The binding between the solid phase and the substance to be measured is performed, for example, via a ligand that modifies the primary antibody and the binding between the ligand and the solid-phase substance. The principle of binding between the ligand and the solid-phase substance is not particularly limited, and in addition to the above, a combination of immobilized streptavidin and a biotin-labeled antibody can be used. A known spacer may be inserted into these bonds as appropriate.

The immunological analysis method of the present invention preferably further includes a step of blocking the solid phase. By blocking the solid phase in this manner, non-specific reactions are easily suppressed, and the false positive suppression effect is more easily exerted. The blocking step may be performed at any time before step (2), may be performed before step (1), or may be performed after step (1).
The blocking method is not particularly limited. For example, a blocking agent may be added in advance to the carrier used in step (2) of procedure (A). The blocking agent used in the present invention is not particularly limited. Examples include casein, skim milk, bovine serum albumin (BSA), gelatin, and the like, as well as those containing blood proteins or plant proteins as active ingredients, rabbit blood components, and the like. Among these, casein is preferred.

After the immune reaction, it is preferable to perform B/F separation to remove the primary and secondary antibodies that are not bound to the antibodies on the solid-phase carrier. In the present invention, the washing solution used for B/F separation may be a solution containing a buffer component having a carboxy group.

In the immunoassay method according to the present invention, there is no particular limitation on the enzyme-substrate system used for detection. For example, there are systems using peroxidase or alkaline phosphatase. There is also no particular limitation on the substrate used for detecting enzyme activity. Commercially available products can be used for these. There is no particular limitation on the composition of the substrate solution containing the substrate, so long as its function is not impaired.

In the present invention, the specimen containing the substance to be measured as a component (also referred to as "biological sample" or simply "sample" in the present specification) is not particularly limited as long as it contains mucin, and examples thereof include various body fluids such as saliva, gastric juice, and nasal mucus, excrement such as urine, and diluted products such as stool from which solids have been removed, and extracts of various tissues. Although not particularly limited, mucin-containing biological samples that can be used in the method of the present invention are preferably nasal swabs, nasal aspirates, nasal washes, nasal blows, and pharyngeal swabs. Mucin-containing biological samples obtained by diluting or pretreating these can also be used. Note that the specimen does not necessarily need to contain the substance to be measured as a component, and may be a specimen that is assumed to possibly contain the substance to be measured as a component.

The measurement targets to which the method of the present invention is applicable include proteins, lipids, and sugars, including, for example, various antigens, antibodies, receptors, and enzymes. The measurement target is preferably a viral protein, and may be, for example, a protein of an enveloped virus, preferably a protein of an Orthomyxoviridae virus, more preferably an influenza virus protein, even more preferably an influenza virus nucleoprotein, and particularly preferably an influenza B virus nucleoprotein. As shown in the results of the test example described below, it has been confirmed that the present invention can significantly suppress false positives when detecting a mucin-containing sample containing an antigen of influenza B virus nucleoprotein by immunoassay.

In the method of the present invention, steps can be added or omitted as appropriate to the steps exemplified in step (A).

The immunoassay to which the present invention is applied may be divided into several steps. For example, in the case of procedure (A), the step of antigen-antibody reaction between the "primary antibody modified to be able to bind to a solid phase" and the substance to be measured and the step of antigen-antibody reaction between the "enzyme-labeled secondary antibody" and the substance to be measured may be carried out separately. In this case, either step may be carried out first.

In yet another embodiment, the present invention can be practiced in a lateral flow immunoassay in which immunochromatography is performed using an immunostrip. Immunochromatography allows immunoassay to be performed in a simple manner. When the method for suppressing non-specific reactions of the present invention is performed in a lateral flow immunoassay, it is preferable, although not particularly limited, to use a solution containing a buffer component having a carboxy group as a specimen treatment liquid and/or developing liquid. For example, the present invention can be practiced by adding a buffer component having a carboxy group to a specimen treatment liquid for diluting a collected mucin-containing biological sample, or by adding a buffer component having a carboxy group to a developing liquid for mixing with a mucin-containing biological sample or a sample that has been previously treated with a specimen treatment liquid.

From another perspective, the present invention also provides a composition for suppressing non-specific reactions in an immunoassay using a mucin-containing biological sample, characterized in that it contains a buffer component having a carboxy group. The type and amount of the buffer component having a carboxy group, the type of mucin-containing biological sample, the type of immunoassay, etc. used here can be the same as those described above.

The effects of the present invention are shown below in the form of examples, but these examples do not limit the present invention in any way.

(Example: Inhibitory effect of non-specific reactions when using a solution containing various components)
The following reagents and devices were used to evaluate the effect of suppressing false positives when treated with solutions containing various components. In this test example, a solution of mucin (manufactured by SIGMA) diluted with POCube pretreatment solution (manufactured by Toyobo Co., Ltd.) was used as a pseudo-viscous specimen (mucin-containing biological sample: containing 1% mucin).
Specifically, when measuring a mucin-containing biological sample by a flow-through immunoassay, a solution containing various components was used as a washing solution (B/F separation washing solution). As buffer components, MOPS, trishydroxymethylaminomethane, BES, TES, HEPES, DIPSO, TAPS, Bicine (N,N-bis(2-hydroxyethyl)glycine), and Tricine (N-[tris(hydroxymethyl)methyl]glycine) were used, and washing solutions containing 10 mM of any of these were prepared according to a conventional method. In addition to the buffer components, the washing solution contained 0.5% polyoxyethylenesorbitan monolaurate and 150 mM sodium chloride (pH approximately 7.2).
The measurement was carried out using a small chemiluminescence immunoanalyzer POCube (manufactured by Toyobo Co., Ltd.) according to the following procedure.
(Operation Procedure)
(1) 70 μl each of 5 ng/ml biotin-labeled anti-type B influenza virus nucleoprotein antibody solution A and 0.2 ng/ml alkaline phosphatase-labeled anti-type B influenza virus nucleoprotein antibody solution B was added to the reagent wells of a 13-well container dedicated to POCube.
(2) 30 μl of antibody solution A and 40 μl of antibody solution B were aspirated from the reagent well to which they had been added in (1), and added to another reagent well and mixed.
(3) 150 μl of a standard sample or diluted antigen solution was added to the sample wells of the 13-series container. The standard sample used was inactivated influenza B virus Hong Kong 5/72 strain (manufactured by Fitzgerald).
(4) 100 μl of the antigen solution (or standard sample) prepared in (3) was added to a well containing the mixture of antibody solution A and antibody solution B prepared in (2), mixed, and reacted at 40° C. for 4 minutes to form an antigen-antibody sandwich complex.
(5) 50 μl of a blocking agent containing 1% by weight of casein was added to a reaction vessel for POCube (a vessel containing a porous filter (a glass filter solid phase to which an anti-biotin antibody is bound) to which a ligand capture agent that specifically recognizes the ligand bound to the first antibody is bound), and then
150 μl of the mixture prepared in (4) was added to the POCube dedicated reaction vessel, and the antigen-antibody sandwich complex was bound to the reaction vessel.
(6) The POCube reaction vessel was washed with a washing solution for B/F separation containing different buffer components. Furthermore, 30 μl of a color-developing substrate dispensed into the reagent wells of the 13-tube vessel was added, and the luminescence intensity was measured.
(7) The antigen dilution solution measurement value was converted to a C.O.I value (cutoff index value) according to the following (Formula I). Samples with a C.O.I of 1 or more were judged to be positive, and samples with a C.O.I of less than 1 were judged to be negative.
(Formula I)
C.O.I = S (luminescence intensity of sample) / C (cutoff value)
C: luminescence intensity of standard sample × 0.05

The results are shown in Table 1 below and in FIG.

As can be seen from these results, it has become clear that when a washing solution containing bicine (N,N-bis(2-hydroxyethyl)glycine) or tricine (N-[tris(hydroxymethyl)methyl]glycine) is used, false positives that tend to occur in immunoassays using samples containing mucin can be highly suppressed. Bicine and tricine share common properties as buffer components with carboxy groups. Therefore, the results of this example show that the occurrence of false positives caused by mucin-containing biological samples can be avoided by the simple measure of using a solution containing a buffer component with a carboxy group. In particular, it has become clear that the use of tricine as a buffer component with a carboxy group provides a particularly high effect of suppressing false positives.

The present invention makes it possible to avoid non-specific reactions when performing immunoassays using mucin-containing biological samples such as nasal swabs, nasal aspirates, nasal washes, nasal blows, or pharyngeal swabs, and to provide accurate and reliable test results.

Claims (15)

A method for suppressing non-specific reactions in a flow-through immunoassay using a mucin-containing biological sample, comprising using a solution containing a buffer component having a carboxy group,
the buffer component having a carboxy group is at least one selected from the group consisting of N,N-bis(2-hydroxyethyl)glycine, N-[tris(hydroxymethyl)methyl]glycine, N-(2-acetamido)iminodiacetic acid, acetic acid, citric acid, and tartaric acid, and the solution containing the buffer component having a carboxy group is a B/F separation washing solution. The method according to claim 1, wherein the mucin-containing biological sample is a nasal swab, a nasal aspirate, a nasal wash, a nasal blow, or a pharyngeal swab. The method according to claim 1 or 2, wherein the flow-through immunoassay is a method for immunoassaying influenza virus nucleoprotein. The method according to any one of claims 1 to 3, wherein the flow-through immunoassay is a method for immunoassaying influenza B virus nucleoprotein. The method according to any one of claims 1 to 4, wherein the solution containing a buffer component having a carboxy group contains a buffer component having a carboxy group at a concentration of 1 to 1000 mM. The method according to any one of claims 1 to 5, wherein the solution containing a buffer component having a carboxy group further contains at least one selected from the group consisting of surfactants, proteins, salts, pH adjusters, and preservatives. The method according to any one of claims 1 to 6, wherein the flow-through immunoassay is a flow-through immunoassay comprising a step of performing detection on a solid phase. A composition for suppressing non-specific reactions in a flow-through immunoassay using a mucin-containing biological sample, comprising a buffer component having a carboxy group,
the buffer component having a carboxy group is at least one selected from the group consisting of N,N-bis(2-hydroxyethyl)glycine, N-[tris(hydroxymethyl)methyl]glycine, N-(2-acetamido)iminodiacetic acid, acetic acid, citric acid, and tartaric acid, and the solution containing the buffer component having a carboxy group is a B/F separation washing solution. The composition according to claim 8, wherein the mucin-containing biological sample is a nasal swab, a nasal aspirate, a nasal wash, a nasal blow, or a pharyngeal swab. The composition according to claim 8 or 9, which is used in a method for immunoassaying influenza virus nucleoprotein. The composition according to any one of claims 8 to 10, which is used in a method for immunoassaying influenza B virus nucleoprotein. The composition according to any one of claims 8 to 11, comprising a buffer component having a carboxy group at a concentration of 1 to 1000 mM. The composition according to any one of claims 8 to 12, further comprising at least one selected from the group consisting of surfactants, proteins, salts, pH adjusters, and preservatives. The composition according to any one of claims 8 to 13, which is used in a flow-through immunoassay comprising a step of performing detection on a solid phase. A kit for use in a flow-through immunoassay, comprising the composition according to any one of claims 8 to 14.