JP4668768B2 - Immunoassay method and non-specific reaction suppression method - Google Patents

Description

The present invention relates to a non-specific reaction inhibitor, an immunoassay reagent, an immunoassay method, and a non-specific reaction inhibitor capable of effectively suppressing a non-specific reaction that occurs when performing immunoassay and realizing high detection sensitivity. It relates to the manufacturing method.

Conventionally, immunoassay methods have been widely used for measuring trace substances contained in blood, urine and the like. The immunoassay method is based on the specific strong binding of the antigen-antibody reaction, and it is possible to measure the target substance specifically with high sensitivity even from a sample in which various substances are latent. However, in such an immunoassay method, when reacted with a sample such as serum, it reacts not only with a positive sample containing an antigen as a substance to be measured, but also with a negative sample not containing these antigens, Sometimes judged positive.

The reason why such a nonspecific reaction occurs is not clear, but one of the causes is considered to be the influence of factors such as complement contained in serum. In addition, the Fc part of the antibody modified when the antigen to the measurement substance is immobilized on the insoluble carrier reacts with the rheumatoid factor in the test solution, causing nonspecific aggregation, or a buffer that suspends the insoluble carrier. When an inert protein (such as bovine serum albumin) is added to the solution as a stabilizer, it may contain a lot of impurities or contain certain types of proteolytic enzymes, depending on the grade of the reagent used. In many cases, accurate measurement was not possible, including positive and negative errors.

For the purpose of suppressing non-specific reactions, a method of diluting a specimen with a buffer solution such as glycine and a method called an immobilization treatment for inactivating the complement in serum have been conventionally performed. However, such treatment has problems that it is difficult to sufficiently suppress non-specific reactions and that the operation of the immunoassay method becomes complicated. In addition, methods have been reported to add animal denatured globulins or add protease inhibitors, but all of these methods are sensitive to the setting of degenerative conditions and it is difficult to produce stable denatured globulins. However, there are problems such as high costs, all of which are insufficient in terms of suppressing non-specific reactions.

In order to solve such a problem, Patent Document 1 fixes an antibody or an antigen that does not immunologically react with a substance to be measured in a particle smaller than a particle that specifically reacts for the purpose of suppressing a nonspecific reaction. A method of adding the above is disclosed. Patent Document 2 discloses a method of adding heat-denatured albumin to an immunoassay reagent in order to suppress nonspecific reactions. However, in these techniques, although non-specific reaction is slightly reduced, it has not yet been achieved to achieve high-sensitivity and accurate measurement. Therefore, a better method for suppressing non-specific reaction has been desired.
Japanese Patent No. 3623657 Japanese Patent Laid-Open No. 11-23573

In view of the above situation, the present invention effectively suppresses nonspecific reactions that occur when immunoassays are performed, and provides nonspecific reaction inhibitors, immunoassay reagents, and immunoassays capable of realizing high detection sensitivity. It aims to provide a method.

In the present invention, in the presence of a non-specific reaction inhibitor, by optically measuring or visually observing the degree of aggregation caused by the antigen-antibody reaction of the analyte to be measured and the immunoassay particles in the sample, An immunoassay method for measuring a substance to be measured in a specimen, wherein the immunoassay particles are supported on a carrier having an average particle diameter of 0.05 to 0.5 μm in the presence of an organic solvent in the presence of the organic solvent. The non-specific reaction inhibitor is an antibody or antigen that does not immunologically react with the substance to be measured on an insoluble carrier having an average particle size smaller than that of the carrier. Is an immunoassay method in which is supported in the presence of an organic solvent.
The present invention relates to an immunoassay particle in which a substance to be measured in a specimen and a lipid antigen are supported on a carrier having an average particle diameter of 0.05 to 0.5 μm in the presence of an organic solvent and subjected to a blocking treatment in the presence of the organic solvent. Is a method for suppressing nonspecific reactions that occur in an immunoassay method for measuring a substance to be measured in a specimen by optically measuring or visually observing the degree of aggregation caused by antigen-antibody reaction. In addition, a non-specific reaction is performed by adding a non-specific reaction inhibitor carrying an antibody or antigen that does not immunologically react with the substance to be measured in the presence of an organic solvent to an insoluble carrier having an average particle size smaller than that of the carrier. It is a suppression method.
The present invention is described in detail below.

As a result of intensive investigations, the present inventors have used insoluble organic carrier particles that are smaller than those of immunoassay particles for the purpose of suppressing non-specific reactions, and in the presence of organic solvents. By carrying an antibody or antigen that does not immunologically react with a substance on an insoluble carrier, a nonspecific reaction inhibitor capable of effectively suppressing nonspecific reactions and realizing high detection sensitivity can be obtained. As a result, the present invention has been completed.

The nonspecific reaction inhibitor of the present invention comprises an insoluble carrier carrying an antibody or antigen that does not immunologically react with a substance to be measured in the presence of an organic solvent.

The antibody that does not immunologically react with the substance to be measured refers to an antibody that does not have specific reactivity with the antigen to be measured, and examples of such antibodies include those derived from humans. When immunoassay is performed on a specimen such as blood or urine, antibodies derived from normal animal sera other than humans can be used.

An antibody that does not immunologically react with the substance to be measured may be fragmented by a commonly used method. Examples of the fragmentation method generally include a method of degrading antibody molecules with various enzymes. For example, Fab fragment is obtained by degradation with papain or the like, and F (ab ′) 2 fragment is obtained by degradation with pepsin or the like. Is obtained.

Further, an antibody that does not immunologically react with the substance to be measured and an antibody that immunologically reacts with a substance to be measured used for immunoassay particles used in immunoassay (hereinafter also referred to as a specific antibody). It is preferable to use those fragmented by the same enzyme. For example, as the specific antibody, F (ab ′ ′), which is a rabbit-derived subclass IgG antibody having specific reactivity with a substance to be measured, fragmented by pepsin treatment and purified by column chromatography (gel filtration) and affinity chromatography. ) When two fragments are used, an antibody that does not immunologically react with the analyte to be used for the non-specific reaction inhibitor of the present invention is a rabbit-derived antibody that does not have specific reactivity with the analyte. It is preferable to use F (ab ′) 2 fragments obtained by fragmenting antibodies of subclass IgG by pepsin treatment and purifying by column chromatography (gel filtration).

The antigen that does not immunologically react with the substance to be measured refers to an antigen that does not have specific reactivity with the antibody to be measured, such as bovine serum albumin. (BSA), casein, casamino and the like. Antigens that do not immunologically react with the substance to be measured are the same as or different from the blocking agents used in immunoassay particles described later and those used as stabilizers in immunoassay reagents. May be. Further, as an antigen that does not immunologically react with the substance to be measured, a heat-treated antigen may be used.

The nonspecific reaction inhibitor of the present invention is obtained by supporting the antibody or antigen on an insoluble carrier in the presence of an organic solvent. Specifically, for example, after an organic solvent is added to an aqueous solution of an insoluble carrier, an antibody or antigen that does not react immunologically with the substance to be measured is sensitized to physically adsorb it on the surface of the insoluble carrier. By carrying the antibody or antigen using such a method, the reaction with the non-specific reaction inducing substance in the specimen becomes more prominent, and the effect of suppressing the non-specific reaction can be further improved.

The organic solvent is not particularly limited as long as it does not dissolve or modify the insoluble carrier, and examples thereof include ethanol, methanol, isopropyl alcohol, and the like.
The concentration of the organic solvent is preferably 1% and preferably 50% with respect to the entire reaction solution for carrying. If it is less than 1%, the effect of suppressing nonspecific reaction may be insufficient. If it exceeds 50%, a substance that does not immunoreact with an organic solvent may precipitate, and a nonspecific reaction inhibitor may not be prepared.

The nonspecific reaction inhibitor of the present invention is preferably in a state of being suspended in a suspension medium. As the suspending medium, various buffer solutions are used according to the type of the substance to be measured, the measurement principle used, and the measurement method, and the measurement substance is not inactivated and does not inhibit the antigen-antibody reaction. The buffer solution is not particularly limited as long as it has such an ion concentration and pH, and examples thereof include a phosphate buffer solution, a tris-hydrochloric acid buffer solution, and a glycine buffer solution. Preferably, it belongs to the Good buffer group, and has, for example, tris (hydroxy) aminomethane and a sulfonic acid group. More preferably, 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), N-tris ( And hydroxymethyl) methyl-2-aminopropanesulfonic acid (TES).

An additive may be used in the buffer solution as long as the substance to be measured is not deactivated and the antigen-antibody reaction is not inhibited. Examples of such additives include quaternary ammonium salts such as choline chloride for enhancing specificity; reaction accelerators; stabilizers such as albumin and sucrose; and polyethylene glycols that are expected to increase sensitivity. Water-soluble polysaccharides such as dextran; sodium azide as a preservative; sodium chloride for adjusting the salt concentration can be appropriately added.

The preferable lower limit of the pH of the buffer solution is 7.0, the preferable upper limit is 8.5, the more preferable lower limit is 7.5, and the more preferable upper limit is 8.0.

The average particle size of the insoluble carrier is not particularly limited as long as it is smaller than the average particle size of the carrier used for the immunoassay particles, and the shape, size, detection method, measuring instrument, etc. of the carrier used for the immunoassay particles However, the preferable lower limit is 0.01 μm, and the preferable upper limit is 0.25 μm. When it is less than 0.01 μm, it may take a lot of labor when preparing as a non-specific reaction inhibitor. If it exceeds 0.25 μm, the surface area per unit weight of the insoluble carrier becomes small, and if the number of carriers is not increased, the non-specific reaction suppression effect is not sufficient, or the substance to be measured against the signal indicated by the insoluble carrier As a result, the signal indicating the specific reaction between the antigen or antibody and the immunoassay particle becomes small, and as a result, the detection sensitivity may deteriorate. In addition, when the concentration of the nonspecific reaction inhibitor of the present invention is relatively high, the background may increase when performing immunoassay, and therefore it is preferable to use an insoluble carrier having a smaller particle size. .

The insoluble carrier is preferably in the form of spherical particles because of its ease of preparation and ease of use. In the present invention, an insoluble carrier made of latex is preferable because it has excellent antibody adsorptivity and can maintain biological activity stably for a long period of time. Polystyrene latex particles It is more preferable that

Examples of the material of the insoluble carrier include those conventionally used for fine particle carriers in immunochemical agglutination and agglutination inhibition reactions, and organic fine particles that can be industrially mass-produced. Although preferable, it is not limited to this. Examples of the organic fine particles that can be industrially mass-produced include, for example, a single polymer and a copolymer made by polymerizing vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylic acid ester, and methacrylic acid ester. Fine particles made of a polymer (polystyrene, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, vinyl chloride-acrylic acid ester copolymer, etc.); styrene-butadiene copolymer, methyl methacrylate-butadiene Fine particles made of a butadiene copolymer such as a copolymer, acrylonitrile-butadiene copolymer; fine particles made of an organic polymer such as insoluble agarose, cellulose, insoluble dextran; carboxyl group, primary amino group, carbamoyl as a functional group group (-CONH _2), water Group having an aldehyde group or the like, and the substrate is such as reactive organic fine particles made of the organic fine particles. The material of the insoluble carrier is preferably the same as the material of the carrier used for the immunoassay particles described later.

The substance to be measured that can be measured using the non-specific reaction inhibitor of the present invention is not particularly limited as long as it is a physiologically active substance contained in a biological sample and can be measured using an antigen-antibody reaction. Examples include proteins, glycoproteins, lipid proteins, receptors, enzymes, viral antigens / antibodies, etc., specifically, CRP, human fibrinogen, rheumatoid factor, alphafetoprotein (AFP), HBs antigen, ferritin, anti Examples include streptridine O antibody, syphilis treponema antibody, antibody against syphilis lipid antigen, HBs antibody, HBc antibody, HBe antibody and the like.

As described above, the non-specific reaction inhibitor of the present invention that can effectively suppress non-specific reactions occurring in the case of performing immunoassay and achieve high detection sensitivity is immunological to the substance to be measured. It can be produced by supporting an antibody or antigen that does not react with an insoluble carrier in the presence of an organic solvent. A method for producing such a nonspecific reaction inhibitor is also one aspect of the present invention.

Use of the nonspecific reaction inhibitor of the present invention in combination with immunoassay particles carrying an antibody or antigen that immunologically reacts with a substance to be measured on a carrier having an average particle size of 0.05 to 0.5 μm. It can be used as an immunoassay reagent. Such an immunoassay reagent is also one aspect of the present invention.

The immunoassay particles are those in which an antibody or antigen that reacts immunologically with a substance to be measured (hereinafter also referred to as a specific antibody or a specific antigen) is supported on a carrier having an average particle size of 0.05 to 0.5 μm. It is.

The antibody that reacts immunologically with the substance to be measured may be, for example, a monoclonal antibody, or obtained by inoculating an animal such as a normal rabbit, mouse, horse, or goat with an immunogen. It may be a polyclonal antibody. In the field of cell fusion technology, the above monoclonal antibodies can be produced and obtained by appropriately selecting methods known per se and combining them to form a monoclonal antibody-producing fusion cell line.
The antigen that reacts immunologically with the substance to be measured is not particularly limited, and may be a hapten other than the substance having immunogenicity.

It is preferable that the immunoassay particles are further subjected to a blocking treatment. The blocking treatment refers to an immunological method against a specific antibody or a specific antigen in order to prevent nonspecific adsorption of proteins or the like in a sample after the specific antibody or specific antigen is supported on the carrier. This refers to the further binding of proteins that do not react with.
Examples of the blocking agent used for the blocking treatment include bovine serum albumin (BSA) and casein.

It is preferable that the blocking agent and the immunologically unresponsive antigen used for the nonspecific reaction inhibitor are the same substance.
The substance used as the blocking agent is immunologically inactive, has no specific reactivity with the antigen or antibody to be measured, but reacts with nonspecific reaction-inducing substances in serum There is. In addition, there may be some denaturation due to interactions with insoluble carriers and side reactions of binding reactions, and this denaturation may cause reactions with nonspecific reaction-inducing substances in serum. is there.
However, by making the blocking agent and the immunologically unreactive antigen used for the nonspecific reaction inhibitor the same substance, even in such a case, the antigen is a nonspecific reaction inducing substance in serum. It is thought that nonspecific reaction is inhibited (suppressed).

When bovine serum albumin is used as the blocking agent, it is preferable to use a bovine serum albumin that has been heat-denatured in advance. Here, the degree of modification by heat treatment changes depending on the temperature and modification time during the heat treatment, so it is necessary to lengthen the modification time when the temperature during the heat treatment is relatively low, and the treatment time when the temperature is high. Need to be shortened.

The lower limit of the average particle diameter of the immunoassay particles is 0.05 μm, and the upper limit is 0.5 μm. Within the above range, it is possible to visually or optically detect an aggregate produced as a result of an agglutination reaction caused by an antigen-antibody reaction between a specific antibody or a specific antigen supported on a carrier and a substance to be measured. Become.

The dosage form of the immunoassay reagent of the present invention is selected depending on the addition method from the viewpoints of non-specific reaction inhibitory effect, ease of use and the like. Specifically, for example, the non-specific reaction inhibitor of the present invention and the immunoassay particles may be used as a one-component reagent by dispersing them in the same medium. You may use as a 2 liquid type reagent comprised by the 1st reagent containing an inhibitor and the 2nd reagent which consists of a buffer solution which added the said immunoassay particle to the medium.

Using the immunoassay reagent of the present invention, the substance to be measured in the specimen can be measured by optically measuring or visually observing the degree of aggregation caused by the antigen-antibody reaction with the substance to be measured in the specimen. it can. Such an immunoassay method is also one aspect of the present invention. Specifically, when the immunoassay reagent of the present invention is a one-part reagent, the substance to be measured in the sample is added by causing the antigen-antibody reaction by adding the immunoassay reagent of the present invention to the sample. When the immunoassay reagent of the present invention is a two-component reagent, the first reagent containing the nonspecific reaction inhibitor of the present invention is added to the sample, and then the immunoassay particles are further added. By adding a second reagent composed of a buffer solution added to the medium, an antigen-antibody reaction can be caused to measure the substance to be measured in the specimen.

In the immunoassay method of the present invention, the amount of the nonspecific reaction inhibitor added is the amount of antibody or antigen that does not immunologically react with the analyte supported on the insoluble carrier, the size and type of the insoluble carrier. Average particle size, concentration of immunoassay particles, type and average particle size, detection method, measuring instrument, degree of non-specific reaction suppression effect, presence / absence of interference with antigen-antibody reaction with immunoassay particles, simple use It is appropriately selected from the viewpoint of properties and the like.

When the immunoassay reagent of the present invention is a one-component reagent, the preferable lower limit of the addition amount of the nonspecific reaction inhibitor is 0.001% by weight with respect to the whole antigen-antibody reaction solution, and the preferable upper limit is 20% by weight. . If it is less than 0.001% by weight, the effect of suppressing nonspecific reaction may be insufficient. If it exceeds 20% by weight, the signal indicating the specific reaction between the antigen or antibody as the substance to be measured and the insoluble carrier on which the specific antibody or specific antigen is immobilized becomes small with respect to the signal indicated by the insoluble carrier. Detection sensitivity may deteriorate.

When the immunoassay reagent of the present invention is a two-component reagent, the amount of the nonspecific reaction inhibitor added is measured in the detection system together with the concentration of the latex solution added when the antigen-antibody reaction is performed later. There is no particular limitation as long as it does not exceed the upper limit. For example, when detection is performed using a spectrophotometer, the absorbance after completion of the reaction may be in a range not exceeding 3.0. There is no particular limitation when the naked eye is judged on the plate.

The measurement conditions for performing the immunoassay method of the present invention are not particularly limited as long as the antigen-antibody reaction between the immunoassay particles and a sample containing an antigen or antibody as a substance to be measured can occur. About measurement temperature, it is preferable to carry out at constant temperature, and it is especially preferable to carry out at 25 to 37 degreeC. The measurement time is not particularly limited, but is preferably 5 seconds to 15 minutes.

The method for measuring the degree of aggregation of the immunoassay particles is not particularly limited, and a known method can be used. When quantitatively measuring the above-mentioned aggregation, it is preferable to measure, for example, optically from the viewpoint of simplicity and accuracy. Specifically, it depends on the size of the insoluble carrier, selection of its concentration, and setting of the reaction time. It is preferable to measure the scattered light intensity, absorbance or transmitted light intensity using an optical instrument. The wavelength of light used for the measurement is preferably 300 to 1200 nm.

According to the present invention, there are provided a nonspecific reaction inhibitor, an immunoassay reagent, and an immunoassay method capable of effectively suppressing a nonspecific reaction that occurs when performing immunoassay and realizing high detection sensitivity. can do.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Example 1
(1) Buffer solution (first reagent)
(1-1) Preparation of Nonspecific Reaction Inhibitor 3 mL of ethanol was added to 1.0 mL of polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.05 μm and stirred at room temperature for 30 minutes. . Subsequently, phosphate buffered saline (hereinafter referred to as phosphate buffered saline is also referred to as PBS) containing 5.0% by weight of bovine serum albumin (hereinafter referred to as bovine serum albumin as BSA) in this solution. After stirring at room temperature for 120 minutes, the mixture was washed by centrifugation at 30,000 rpm. 10 ml of PBS with a phosphate concentration of 25 mM was added to the resulting precipitate to suspend the latex particles to prepare a nonspecific reaction inhibitor.

(1-2) Preparation of Buffer Solution (First Reagent) PBS (pH 6.5) containing 1.0 (W / V)% pullulan (manufactured by Hayashibara), 1.0 (W / V)% BSA Non-specificity prepared in (1-1) to a buffer solution in which 0.9 (W / V)% sodium chloride and 0.1 (W / V)% sodium azide were added to a phosphate concentration of 25 mM) A reaction inhibitor was added to a concentration of 5% by weight to obtain a buffer solution (first reagent).

(2) Lipid antigen-sensitized latex reagent (second reagent)
(2-1) Preparation of lipid antigen solution 2 ml of ethanol solution of cardiolipin (5 mg / ml, manufactured by Sigma), 10 ml of ethanol solution (10 mg / ml) of purified lecithin (manufactured by Nacalai Tesque) and cholesterol (manufactured by Nacalai Tesque) 3 ml of an ethanol solution (10 mg / ml) was mixed to obtain a lipid antigen solution.

(2-2) Preparation of lipid antigen-sensitized latex reagent (second reagent) Polystyrene latex (average particle size 0.4 μm, 10 (W / V)%, manufactured by Sekisui Chemical Co., Ltd.) 100 μl, (2-1) 250 μl of the lipid antigen solution prepared in the above was added all at once, and the mixture was gently stirred at 37 ° C. for 2 hours. Next, 3 ml of PBS (pH 6.5, phosphate concentration 100 mM) containing BSA at a concentration of 5 (W / V)% was added all at once, and further stirred at 37 ° C. for 1 hour. Centrifugation was performed at 15000 rpm and 4 ° C. for 30 minutes, the supernatant was removed, and the precipitated latex particles were resuspended in 2 ml of PBS containing BSA at a concentration of 1 (W / V)%. This operation was repeated twice to wash the latex particles. Finally, the latex particles were suspended in 10 ml of PBS containing EDTA · 4Na at a concentration of 10 mM and choline chloride at a concentration of 500 mM to obtain a lipid antigen-sensitized latex reagent (second reagent).

(Example 2)
A buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that the nonspecific reaction inhibitor was prepared by adding 12 mL of ethanol.

(Example 3)
After preparing a non-specific reaction inhibitor in the same manner as in Example 1, (1-2) except that the non-specific reaction inhibitor was added to 10% by weight in the preparation of the buffer solution (first reagent). In the same manner as in Example 1, a buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared.

Example 4
After preparing a non-specific reaction inhibitor in the same manner as in Example 2, (1-2) except that the non-specific reaction inhibitor was added to 10% by weight in the preparation of the buffer solution (first reagent). In the same manner as in Example 1, a buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared.

(Example 5)
(1-1) Preparation of Nonspecific Reaction Inhibitor 3 mL of ethanol was added to 1.0 mL of polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.05 μm and stirred at room temperature for 30 minutes. . Next, 30 mL of PBS containing 5.0% by weight of BSA that had been heat denatured at 56 ° C. for 10 minutes was added, stirred at room temperature for 120 minutes, and then washed by centrifugation at 30,000 rpm. 10 ml of PBS with a phosphate concentration of 25 mM was added to the resulting precipitate to suspend the latex particles to prepare a nonspecific reaction inhibitor.

A buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that a non-specific reaction inhibitor was prepared using the method described above.

(Comparative Example 1)
(1-2) Buffer solution (first reagent) and lipid antigen-sensitized latex in the same manner as in Example 1 except that the nonspecific reaction inhibitor was not added. A reagent (second reagent) was prepared.

(Comparative Example 2)
(1-2) In preparation of the buffer solution (first reagent), PBS (pH 6) containing pullulan (manufactured by Hayashibara) and 1.0 (W / V)% BSA at a concentration of 1.0 (W / V)% 0.5 wt% BSA in a buffer solution in which 0.9 (W / V)% sodium chloride and 0.1 (W / V)% sodium azide are added to phosphate concentration 25 mM) The buffer solution (first reagent), lipid antigen was prepared in the same manner as in Example 1 except that a buffer solution (first reagent) was prepared by adding PBS that had been heat denatured at 56 ° C. for 10 minutes. A sensitized latex reagent (second reagent) was prepared.

(Comparative Example 3)
(1-1) Preparation of nonspecific reaction inhibitor PBS containing 5.0% by weight of BSA in 1.0 mL of polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.05 μm After stirring at room temperature for 120 minutes, the mixture was washed by centrifugation at 30,000 rpm. 10 ml of PBS with a phosphate concentration of 25 mM was added to the resulting precipitate to suspend the latex particles to prepare a nonspecific reaction inhibitor.

A buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that a non-specific reaction inhibitor was prepared using the method described above.

(Comparative Example 4)
(1-1) Preparation of non-specific reaction inhibitor PBS containing 5.0% by weight of BSA in 1.0 mL of polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.05 μm 30 mL of a solution denatured by heating at 56 ° C. for 10 minutes was added, stirred at room temperature for 120 minutes, and then washed by centrifugation at 30,000 rpm. 10 ml of PBS with a phosphate concentration of 25 mM was added to the resulting precipitate to suspend the latex particles to prepare a nonspecific reaction inhibitor.

A buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that a non-specific reaction inhibitor was prepared using the method described above.

(Evaluation)
The buffer solutions (first reagent) and lipid antigen-sensitized latex reagent (second reagent) obtained in Examples 1 to 5 and Comparative Examples 1 to 4 were evaluated by the following methods.
After mixing 180 μl of buffer solution (first reagent) with 20 μl of syphilis negative serum and holding at 37 ° C. for 5 minutes, 60 μl of lipid antigen-sensitized latex reagent (second reagent) was added and stirred, and after addition at a measurement wavelength of 700 nm Changes in absorbance at 1 minute and 5 minutes were measured using a Hitachi 7170 automatic biochemical analyzer. And the measured value was calculated | required based on the analytical curve created beforehand from the obtained absorbance change.
A calibration curve was prepared by measuring a standard product with a known concentration under the same conditions and determining the relationship between the concentration and the amount of change in absorbance. Judgment criteria are negative when the measured value is less than 1.0 RU, and when the measured value is 1.0 RU or more, it is considered that the substance was erroneously judged by reacting with some substance even though it was actually negative. False positive. The results are shown in Table 1.

As shown in Table 1, Examples 1 to 5 show negative, while Comparative Examples 1 and 2 show false positive. By adding a nonspecific reaction inhibitor, It turns out that a specific reaction can be suppressed effectively. In addition, since Comparative Examples 3 and 4 show false positives, it can be seen that in the preparation of the non-specific reaction inhibitor, when the organic solvent is not added, the non-specific reaction cannot be sufficiently suppressed. From the results of Examples 1 to 5, it can be seen that the effect of suppressing non-specific reaction can be further improved by increasing the amount of organic solvent added and thermal denaturation of BSA.

(Example 6)
In the preparation of the (1-1) non-specific reaction inhibitor of Example 1, it was carried out except that 1.0 mL of polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.22 μm was used. In the same manner as in Example 1, a buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared.

(Example 7)
In the preparation of the (1-1) nonspecific reaction inhibitor of Example 1, it was carried out except that 1.0 mL of polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.15 μm was used. In the same manner as in Example 1, a buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared.

(Example 8)
In the preparation of the (1-1) nonspecific reaction inhibitor of Example 1, it was carried out except that 1.0 mL of polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.05 μm was used. In the same manner as in Example 1, a buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared.

(Comparative Example 5)
In the preparation of the (1-1) nonspecific reaction inhibitor of Example 1, except that 1.0 mL of polystyrene latex (solid content: 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.40 μm was used. In the same manner as in Example 1, a buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared.

(Evaluation)
The buffer solution (first reagent) and lipid antigen-sensitized latex reagent (second reagent) obtained in Examples 6 to 8 and Comparative Example 5 were evaluated by the following methods.
180 μl of a buffer solution (first reagent) is mixed with 20 μl of 7 types of syphilis-negative sera and held at 37 ° C. for 5 minutes, and then 60 μl of a lipid antigen-sensitized latex reagent (second reagent) is added and stirred, and the measurement wavelength is 700 nm. The change in absorbance for 1 minute and 5 minutes after the addition was measured using a Hitachi 7170 automatic biochemical analyzer. And the measured value was calculated | required based on the analytical curve created beforehand from the obtained absorbance change. The criterion was negative when the measured value was less than 1.0 RU, and false positive when the measured value was 1.0 RU or more. The results are shown in Table 2.

As shown in Table 2, when a polystyrene latex having an average particle size smaller than the latex particles of the second reagent was used as in Examples 6 to 8, negative was shown, but as in Comparative Example 5, When polystyrene latex having the same average particle diameter as the latex particles of the second reagent was used, false positives were obtained, and the nonspecific reaction suppressing effect was insufficient.

Example 9
In the preparation of (1-1) nonspecific reaction inhibitor of Example 1, 1.0 mL of polystyrene latex (solid content 10 wt%, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.05 μm was used instead of 3 mL of ethanol. A nonspecific reaction inhibitor was prepared in the same manner as in Example 1 except that 3 ml of propanol was added, and then (1-2) in the preparation of the buffer solution (first reagent), the nonspecific reaction inhibitor was changed to 10 A buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that they were added so as to be in% by weight.

(Example 10)
In the preparation of (1-1) nonspecific reaction inhibitor of Example 1, 1.0 mL of polystyrene latex (solid content 10 wt%, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.05 μm was used instead of 3 mL of ethanol. A buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that 6 ml of propanol was added.

(Example 11)
In the preparation of (1-1) nonspecific reaction inhibitor of Example 1, 1.0 mL of polystyrene latex (solid content 10 wt%, manufactured by Sekisui Chemical Co., Ltd.) having an average particle size of 0.05 μm was used instead of 3 mL of ethanol. After preparing a non-specific reaction inhibitor in the same manner as in Example 1 except that 6 ml of propanol was added, (1-2) In the preparation of the buffer solution (first reagent), 10 parts of the non-specific reaction inhibitor was prepared. A buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that they were added so as to be in% by weight.

(Example 12)
(1) Preparation of non-specific reaction inhibitor In the preparation of (1-1) non-specific reaction inhibitor of Example 1, polystyrene latex having an average particle size of 0.05 μm (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) ) A nonspecific reaction inhibitor was prepared in the same manner as in Example 1 except that 3 ml of methanol was added to 1.0 ml of ethanol instead of 3 ml of (1-2) buffer solution (first reagent). In the preparation, a buffer solution (first reagent) and a lipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that a non-specific reaction inhibitor was added to 10 wt%. .

(Evaluation)
About the buffer solution (first reagent) and the lipid antigen-sensitized latex reagent (second reagent) obtained in Examples 9 to 12, the same method as in Examples 1 to 5 and Comparative Examples 1 to 4 described above was used. evaluated. The results are shown in Table 3.

As shown in Table 3, even when an organic solvent other than ethanol was used in the preparation of the nonspecific reaction inhibitor, a remarkable nonspecific reaction inhibitory effect was observed.

According to the present invention, there are provided a nonspecific reaction inhibitor, an immunoassay reagent, and an immunoassay method capable of effectively suppressing a nonspecific reaction that occurs when performing immunoassay and realizing high detection sensitivity. can do.

Claims (4)

In the presence of a non-specific reaction inhibitor, the substance to be measured and the lipid antigen are supported on a carrier having an average particle size of 0.05 to 0.5 μm in the presence of an organic solvent, and blocking treatment is performed in the presence of the organic solvent. An immunoassay method for measuring a substance to be measured in a specimen by optically measuring or visually observing the degree of aggregation caused by antigen-antibody reaction with the applied immunoassay particles,
The non-specific reaction inhibitor is a non-specific reaction inhibitor in which an insoluble carrier having an average particle size smaller than that of the carrier is loaded with an antibody or antigen that does not immunologically react with the substance to be measured in the presence of an organic solvent. Is
An immunoassay method characterized by the above. The immunoassay method according to claim 1, wherein the lipid antigen is cardiolipin, lecithin or cholesterol. The antigen antibody comprises the substance to be measured in the sample and the immunoassay particles supported on a carrier having an average particle diameter of 0.05 to 0.5 μm in the presence of an organic solvent and subjected to blocking treatment in the presence of the organic solvent. A method for suppressing a non-specific reaction that occurs in an immunoassay method for measuring a substance to be measured in a specimen by optically measuring or visually observing the degree of aggregation caused by the reaction,
A nonspecific reaction inhibitor carrying an antibody or antigen that does not immunologically react with the substance to be measured in the presence of an organic solvent is added to an insoluble carrier having an average particle size smaller than that of the carrier.
A non-specific reaction suppression method characterized by the above. 4. The nonspecific reaction suppression method according to claim 3, wherein the lipid antigen is cardiolipin, lecithin, or cholesterol.