JPH11337551A - Non-specific reaction suppression agent, immunity measuring reagent, and immunity measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain non-specific reaction suppression agent capable of restraining non-specific reaction and highly sensitively detecting by immobilizing antibody not immulogically reacting with a substance to be measured or the fragment, or antigen not immulogically reacting to insoluble support. SOLUTION: By the reason of receiving various denaturation or mixing with side-reaction product, there is a case of generating reaction to non-specific reaction inducing substance in a subject. By immobilizing antibody or the fragment not immulogically reacting with a substance to be measured to an insoluble support and using it as a non-specific reaction suppression agent, the non-specific reaction inducing substance reacts to the non-specific reaction suppression agent in this invention, hence reaction between the support having immobilized specific antibody and the non-specific reaction inducing substance is obstructed (restrained), and the non-specific reaction is considered to be able to be restrained. Consequently, high detecting sensitivity can be obtained, the measured substance in a tested sample can be correctly qantatively measured, and this is useful for discovery of a disease, grasp of disease condition, decision of a curing method, and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is used for measuring an analyte based on an immune reaction utilizing an antigen-antibody reaction using an insoluble carrier, and has a high detection sensitivity.
In addition, the present invention relates to a nonspecific reaction inhibitor having less nonspecific reaction, an immunoassay reagent, and an immunoassay method.

[0002]

2. Description of the Related Art Immunoassays have been 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 specifically measure the target substance with high sensitivity even from a sample in which various substances are latent.

However, in such a measuring method, when reacted with a sample such as serum, not only a positive sample containing an antigen which is a substance to be measured but also a negative sample not containing these antigens are reacted. May cause. The reason why such a non-specific reaction occurs is not clear, but it is thought to be at least in part due to factors such as complement contained in serum.

For example, when an antigen for a test substance is immobilized on an insoluble carrier, the rheumatoid factor in the test solution reacts with the Fc portion of the antibody denatured at the time of immobilization to cause non-specific aggregation. is there. Furthermore, in some cases, an inactive protein (such as bovine serum albumin) is added as a stabilizer to the buffer in which the insoluble carrier is suspended. However, depending on the grade of the reagent used, it may contain a large amount of impurities, In many cases, accurate measurement could not be performed, including positive / negative errors, when the protease was included.

For the purpose of reducing the non-specific reaction as described above, a sample is diluted with a buffer such as glycine,
Alternatively, a deactivation treatment for inactivating complement in serum has been conventionally performed. However, there is a problem that it is difficult to sufficiently suppress the non-specific reaction by such a treatment, and it takes time and effort. In addition, methods of adding denatured globulin from animals or adding a protease inhibitor have been reported, but in any case, the setting of denaturation conditions is delicate, and it is difficult to produce stable denatured globin. However, there are problems such as high cost and the like, and all of them are insufficient in terms of suppressing non-specific reactions.

[0006] In order to solve such a problem, a method of adding a protein such as gelatin or albumin or a polyanion to an antigen-antibody reaction has been conventionally known. Further, N, N-dialkylamide, lower alkyl sulfoxide (Japanese Patent Application Laid-Open No. 55-6085), alkylcellulose (Japanese Patent Application Laid-Open No. 2-173567), carboxyalkylcellulose (Japanese Patent Application Laid-Open No. 2-238360) and the like are added. Although a method for performing the method is disclosed, development of a better method has been desired.

[0007]

DISCLOSURE OF THE INVENTION In view of the above situation, the present invention suppresses nonspecific reactions without performing complicated processing such as serum immobilization and suppresses nonspecific reactions with high detection sensitivity. It is intended to provide an agent, an immunoassay reagent, and an immunoassay method.

[0008]

Means for Solving the Problems The present invention is used in an immunoassay method using a carrier on which an antibody or an antigen against a substance to be measured is immobilized, and the antibody or its antibody that does not immunologically react with the substance to be measured. It is a non-specific reaction inhibitor comprising a fragment or an insoluble carrier on which an immunologically unreactive antigen is immobilized. Hereinafter, the present invention will be described in detail.

The non-specific reaction inhibitor of the present invention comprises an antibody or a fragment thereof that does not immunologically react with a test substance,
Alternatively, it comprises an insoluble carrier on which an antigen that does not react immunologically is immobilized.

The antibody immobilized on the insoluble carrier is an antibody having no specific reactivity to the antigen to be measured. Examples of the above-mentioned antibodies include antibodies derived from serum of normal animals other than humans in the case where immunoassay is performed on samples of human blood or urine. The above-mentioned antibody and an antibody having specific reactivity to the antigen to be measured (hereinafter also referred to as “specific antibody”) are preferably of the same animal species and antibody class because of their higher nonspecific suppression effect. .

The above antibody is purified from serum of a normal animal by a method generally used. Examples of the purification method include a salting-out method, an electrophoresis method, a gel filtration method, a hydrophobic chromatography method, and an affinity chromatography method.

The above-mentioned purification methods may be used in combination of several types of purification methods. However, it is preferable that, in each of the purification steps, if there is a step which can be partially the same as that of the specific antibody, the specific antibody It is desirable to carry out the same method as described above.

[0013] The above antibody may be fragmented by a commonly used method. Examples of the fragmentation method include a method of decomposing an antibody molecule by various enzymes. For example, a Fab fragment is obtained by decomposition with papain or the like, and an F (ab ') ₂ fragment is obtained by decomposition with pepsin or the like. can get. The antibody and the specific antibody are preferably those that have been fragmented by the same enzyme.

As the above specific antibody, for example, a rabbit subclass IgG antibody having specific reactivity to the substance to be measured is fragmented by pepsin treatment, and purified by column chromatography (gel filtration) and affinity chromatography. When the (ab ') ₂ fragment is used, the antibody used for the non-specific reaction inhibitor of the present invention includes:
Rabbit-derived subclass IgG antibody having no specific reactivity to the test substance is fragmented by pepsin treatment,
F (ab ') ₂ fragments purified by column chromatography (gel filtration) can be used.

The antigens which do not react immunologically include:
There is no particular limitation as long as it does not show specific activity for the antigen or antibody to be measured, and examples include bovine serum albumin (BSA), casein, casamino acid and the like. Examples of the antigen that does not react immunologically include a blocking agent used for a carrier on which an antibody or an antigen to a substance to be measured (hereinafter, also referred to as “specific antibody or specific antigen”) is immobilized, or a stable reagent for immunoassay. It may be the same as or different from that used as the agent.

The antigens that do not react immunologically include:
The same substance as the substance which does not react immunologically immobilized on the carrier is preferable. The immunologically unreactive substance immobilized on the carrier usually refers to a blocking agent used in an immunoassay. The blocking is performed after immobilizing the specific antibody or the specific antigen on the carrier, and immunologically reacting with the specific antibody or the specific antigen in order to prevent the protein or the like in the sample from non-specifically adsorbing to the carrier. The purpose is to further bind unreacted proteins and the like.

[0017] Examples of the immunologically non-reactive substance immobilized on the carrier include bovine serum albumin, ovalbumin, lactalbumin, gelatin, casein and the like used in ordinary immunoassays.

Examples of the insoluble carrier include fine particle carriers generally used in immunochemical agglutination reactions and agglutination inhibition reactions, and organic fine particles that can be industrially mass-produced are preferred. However, the present invention is not limited to this.

Examples of the organic fine particles which can be industrially mass-produced include, for example, homopolymers obtained by polymerizing vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylates and methacrylates. And fine particles of copolymers (polystyrene, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, vinyl chloride-acrylate copolymer, etc.); styrene-butadiene copolymer, methyl methacrylate Butadiene copolymer fine particles such as butadiene copolymer and acrylonitrile-butadiene copolymer; organic polymers such as insoluble agarose, cellulose and insoluble dextran; carboxyl, primary amino and carbamoyl groups as functional groups ( —CONH ₂ ),
Reactive organic fine particles having a hydroxyl group, an aldehyde group, and the like and having a substrate made of the above-mentioned organic fine particles are exemplified.

Other examples of the insoluble carrier include biological particles such as animal erythrocytes and bacterial cells; and non-biological particles such as bentonite, collodion, cholesterol crystals, silica, gold, titanium, iron, nickel, kaolin, and carbon powder. Particles. As the shape of the insoluble carrier,
Examples include spherical particles, powders, and flakes.

The type, material and shape of the insoluble carrier depend on the measurement principle and the measurement method used to suppress the effect of the non-specific reaction and the reaction between the insoluble carrier on which the specific antibody or the specific antigen is immobilized and the substance to be measured. It is selected from the viewpoint of the presence or absence of interference, ease of preparation, ease of use, and the like.

The material of the insoluble carrier is preferably the same as the material of the carrier on which the specific antibody or the specific antigen is immobilized.
The shape of the insoluble carrier is preferably spherical particles from the viewpoint of easy preparation and easy use.

In the present invention, an insoluble carrier composed of latex is preferred, and polystyrene-based latex particles are more preferred because they have excellent antibody-adsorbing properties and can maintain biological activity stably for a long period of time. preferable.

The size of the insoluble carrier (average particle size in the case of spherical particles) depends on the shape and size of the carrier on which the specific antibody or specific antigen is immobilized, the detection method, the measuring instrument, and the like.
One having a thickness of 0.005 μm to 10 mm is appropriately selected.

As the method for immobilization on the insoluble carrier, a commonly used method can be employed. For example, a method of physically adsorbing on the surface of the insoluble carrier or a method of immobilizing the compound on the surface of the insoluble carrier having a functional group can be used. And a method of efficiently bonding by a chemical bonding method or a covalent bonding method. Preferably. This is a method in which the specific antibody or the specific antigen is immobilized on the carrier under the same method and conditions.

The amount of the specific antibody or the specific antigen immobilized on the insoluble carrier depends on the amount of the specific antibody or the specific antigen immobilized on the insoluble carrier, the measuring principle and the measuring method used, and the effect of suppressing the nonspecific reaction.
It is selected from the viewpoint of the presence or absence of interference with the reaction between the carrier on which the specific antibody or the specific antigen is immobilized and the analyte, the ease of preparation, and the like.

When immobilizing an antibody or a fragment thereof which does not immunologically react with the substance to be measured on the insoluble carrier,
It is desirable that the amount of the antibody immobilized (the amount of the insoluble carrier immobilized per unit surface area) is 20% to 500% of the amount of the specific antibody immobilized (the amount of immobilized carrier per unit surface area). In the above case, the insoluble carrier may be further subjected to a blocking treatment. As the blocking agent,
The above-described ones can be used, and a generally used physical adsorption or chemical bond can be adopted as a method for immobilizing the blocking agent. Preferably, the same immobilization method is performed using the same substance as the blocking agent used in the carrier on which the specific antibody is immobilized.

When immobilizing an antigen that does not react immunologically on the insoluble carrier, treatment for blocking an immunologically inactive substance on the carrier on which the specific antibody or the specific antigen is immobilized is preferably performed. It is preferably 0.05 to 50 times the amount. The treatment amount is the weight of the immunologically inactive substance with respect to the total surface area per one volume of the carrier.

The antibody or fragment thereof which does not immunologically react with the substance to be measured or the insoluble carrier on which the antigen which does not immunologically react is immobilized is usually prepared in the form of a suspension, Used as a reaction inhibitor.

As the suspension medium, various buffers depending on the type of the substance to be measured, the measurement principle used, and the measurement method are used. The buffer may be any buffer that does not deactivate the substance to be measured and has an ion concentration or pH that does not inhibit the antigen-antibody reaction. For example, phosphate buffer, Tris-HCl buffer Glycine buffer and the like. Preferably, they belong to the group of good buffers, for example, those having tris (hydroxy) aminomethane and sulfonic acid groups. More preferably, 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS
O), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), N-tris (hydroxymethyl) methyl-2-aminopropanesulfonic acid (TES) and the like.

The pH is preferably 7.0 to 8.5,
More preferably, it is 7.5 to 8.0. The buffer includes a quaternary ammonium salt such as choline chloride for enhancing specificity; a reaction accelerator; a stabilizer such as albumin and sucrose; a polyethylene glycol and dextran which are expected to have an effect of increasing sensitivity. Water-soluble polysaccharides; sodium azide as a preservative; sodium chloride for adjusting the salt concentration and the like can also be appropriately added. Any of the above additives can be used as long as they do not deactivate the substance to be measured and do not inhibit the antigen-antibody reaction.

In the present invention, a non-specific reaction in an immune reaction is suppressed by using the above-mentioned non-specific reaction inhibitor. That is, specific antibody purification, fragmentation with the enzyme, and, when immobilizing the specific antibody on an insoluble carrier, when the specific antibody has undergone some denaturation due to reagent pH, ionic strength, etc. During these treatments, substances other than the target antibody or its fragment may be mixed or produced. A reaction with a non-specific reaction inducing substance in a sample may occur due to the denaturation or the incorporation of a by-product or the like. The present invention provides a method for immobilizing a non-specific reaction-inducing substance in a sample by immobilizing an antibody or a fragment thereof which does not immunologically react with a test substance on an insoluble carrier and using the same as a non-specific reaction inhibitor. In order to cause a reaction with the specific reaction inhibitor, it is considered that the reaction between the carrier on which the specific antibody is immobilized and the non-specific reaction inducing substance is inhibited (suppressed), and the non-specific reaction can be suppressed.

The substance used as the above-mentioned blocking agent is also immunologically inactive and has no specific reactivity with the antigen or antibody to be measured, but induces nonspecific reaction in serum. Reaction with substances may occur. Also,
Due to the interaction with the insoluble carrier and the side reaction of the binding reaction,
It may have undergone some kind of denaturation, and such denaturation may cause a reaction with a non-specific reaction inducing substance in serum. The non-specific reaction inhibitor of the present invention is also characterized by using an insoluble carrier in which the same substance as the substance used as the blocking agent is immobilized, which is not immunologically reactive. Non-specific reaction is inhibited (suppressed) to cause a reaction with the reaction-inducing substance
It is thought to be done.

The present invention 2 is used in an immunoassay method using a carrier on which an antibody or an antigen against a substance to be measured is immobilized, and an insoluble antibody or antigen immobilized on the above substance to be immunologically reacted is immobilized. It is a non-specific reaction inhibitor composed of a carrier.

The antibody or antigen that reacts immunologically with the substance to be measured may be the same as the specific antibody or the specific antigen immobilized on the carrier. Is also good. As the specific antibody, 1
One or more monoclonal antibodies or polyclonal antibodies may be used.

As the insoluble carrier, those described in the present invention 1 can be used. The insoluble carrier on which the antibody or antigen that reacts immunologically with the substance to be measured is immobilized is usually prepared in the form of a suspension and used as a nonspecific reaction inhibitor. As the suspension medium, those described in the present invention 1 can be used.

The present invention 2 relates to a component (Fc) other than a component (epitope) that specifically reacts with the corresponding antibody or antigen among the components of an immunologically reactive antibody or antigen.
In order to suppress the non-specific reaction caused by, for example, before performing an immune reaction, a part or quantitative
Keep the sample in contact with the reagent in a low concentration situation,
It absorbs non-specific reactions.

Therefore, the amount of the antibody or antigen to be immobilized may be the same as the immobilization conditions of the present invention 1, but the concentration to be used should be 1/50 to 1/10 of the finally used immunoreagent. Is preferred. Alternatively, when a reagent in which two or more types of monoclonal antibodies are immobilized is used as an immunoreagent to be finally used, it is preferable to use at least one type or more.

The present invention 3 is a non-specific reaction inhibitor which is used in an immunoassay using a carrier on which an antibody or an antigen against the substance to be measured is immobilized, and which comprises an insoluble carrier.
As the insoluble carrier, those described in the present invention 1 can be used. The insoluble carrier is used without immobilization, and is usually prepared in the form of a suspension, and used as a non-specific reaction inhibitor. As the suspension medium, those described in the present invention 1 can be used.

The non-specific reaction inhibitor of the present invention is used in an immunoassay method using a carrier on which an antibody or an antigen (specific antibody or specific antigen) against a substance to be measured is immobilized.

The substance to be measured is a physiologically active substance contained in a biological sample and is not particularly limited as long as it can be measured by utilizing an antigen-antibody reaction. Examples thereof include proteins, glycoproteins and lipid proteins. , Receptors, enzymes, viral antigens / antibodies and the like.
Examples include RP, human fibrinogen, rheumatoid factor, alpha-fetoprotein (AFP), HBs antigen, ferritin, anti-streptolysin O antibody, syphilis treponemal antibody, antibody to syphilis lipid antigen, HBs antibody, HBc antibody, HBe antibody and the like.

The substance to be measured is also an important item in clinical examination, and is particularly useful for an item which has been conventionally considered to be insufficient in detection sensitivity by the latex agglutination method. Items requiring detection sensitivity up to 1 ng / mL, such as CEA and CA19-9, which are measured in the screening, are listed.

The specific antibody used in the above immunoassay may be a monoclonal antibody,
It may be a polyclonal antibody obtained by inoculating an immunogen into a normal animal such as a rabbit, a mouse, a horse, or a goat. The monoclonal antibody can be produced and obtained using the cell line by appropriately selecting a method known per se in the cell fusion technical field, and forming a monoclonal antibody-producing fusion cell line by combining them. Monoclonal antibodies, hybridomas and ELISA, written by Tatsuo Iwasaki et al., Kodansha).

The carrier used in the above-mentioned immunoassay is not particularly limited as long as it is usually used in the immunoassay, and examples thereof include an insoluble carrier used for the above-mentioned non-specific reaction inhibitor and plastic beads. , A plastic plate or the like can be used.

When an insoluble carrier composed of latex is used as the carrier used in the above immunoassay method,
The average particle size is large enough that an aggregate formed as a result of an agglutination reaction caused by an antigen-antibody reaction between a specific antibody or a specific antigen on a carrier and an analyte can be detected visually or optically. The average particle diameter is preferably 0.01 to 1.0 μm, more preferably 0.1 to 1.0 μm.
It is from 0.5 to 0.5 μm. As a method for sensitizing the surface of the carrier with a specific antibody or a specific antigen, the above-described method for immobilizing the carrier on an insoluble carrier can be employed.

[0046] The present invention 4 is an immunoassay reagent containing the non-specific reaction inhibitor of the present invention 1, 2 or 3 of the present invention. The above-mentioned nonspecific reaction inhibitor of the present invention 1, 2 or 3 is used as an immunoassay reagent by selecting its dosage form from the viewpoint of nonspecific reaction inhibitory effect, ease of use, etc., depending on the method of addition. Is done. The immunoassay reagent is, for example, a liquid reagent (suspension) containing the nonspecific reaction inhibitor and not containing a carrier on which a specific antibody or a specific antigen is immobilized.
Or a liquid reagent (suspension) containing the immobilized insoluble carrier and the carrier on which the specific antibody or specific antigen is immobilized. However, when the non-specific reaction inhibitor of the present invention 2 is used, it is prepared as a liquid reagent (suspension) containing the non-specific reaction inhibitor and not containing the carrier on which the specific antibody or the specific antigen is immobilized.

The present invention 5 relates to a method for treating a sample containing a substance to be measured in advance with the non-specific reaction inhibitor of the present invention, followed by immunoreaction, or in the presence of the non-specific reaction inhibitor of the present invention, This is an immunoassay method for immunoreacting a sample containing: The immunoassay according to the fifth aspect of the present invention comprises treating a sample containing the substance to be measured with the nonspecific reaction inhibitor of the present invention.

As a method for treating the non-specific reaction inhibitor, for example, (first method) a solution containing a non-specific reaction inhibitor is added in advance to a sample containing a substance to be measured, and the non-specific reaction-inducing substance in the sample is added. The mixture of the non-specific reaction inhibitor and the sample is brought into contact with the carrier on which the specific antibody or specific antigen is immobilized while the non-specific reaction inhibitor is added, and the specificity with the antigen or antibody as the analyte is measured. To cause a reactive reaction, (2nd
Method) A liquid containing a non-specific reaction inhibitor is added in advance to a sample containing a substance to be measured, a non-specific reaction-inducing substance in the sample is absorbed, and then the insoluble carrier, which is a non-specific reaction inhibitor, is removed. Contacting the solution with an insoluble carrier having a specific antibody or specific antigen immobilized thereon to cause a specific reaction with the antigen or antibody to be measured, and (Method 3) a non-specific reaction inhibitor and a specific antibody A sample containing a substance to be measured is added to a reaction system containing an insoluble carrier having immobilized thereon, and a reaction for absorbing a non-specific reaction inducing substance in the sample, and a specific reaction between a specific antibody and an antigen which is a substance to be measured. A method for causing the reaction to occur simultaneously is mentioned.

The method of treating the above-mentioned non-specific reaction inhibitor is based on the above-mentioned (the first method) from the viewpoint of its non-specific reaction suppressing effect, simplicity of use, etc., depending on the type of the substance to be measured, the measuring principle used, and the measuring method. ), (Second method) or (third method). When the non-specific reaction inhibitor is composed of an insoluble carrier on which an antibody or antigen that immunologically reacts with the test substance of the present invention 2 is immobilized, the method (1) or ( As in the second method), the method can be selected from methods in which a sample containing the substance to be measured is previously treated with a nonspecific reaction inhibitor.

As the above processing method, the (first method) or the (third method)
Method), the insoluble carrier used for the non-specific reaction inhibitor preferably has an average particle size of 0.01 to 20 μm. When it is less than 0.01 μm, it may take a lot of trouble when preparing as a non-specific reaction inhibitor. 2
If it exceeds 0 μm, the surface area per unit weight of the insoluble carrier becomes small, and if the number of carriers is not increased, the effect of suppressing the nonspecific reaction is not sufficient or the signal shown by the insoluble carrier is a substance to be measured. A signal indicating a specific reaction between the antigen or the antibody and the insoluble carrier on which the specific antibody or the specific antigen is immobilized becomes small, and as a result, the detection sensitivity may be deteriorated.

When the (method 2) is employed as the above treatment method, the insoluble carrier used for the non-specific reaction inhibitor comprises:
Those having an average particle size of 0.01 to 10 μm are preferred. 0.
When it is less than 01 μm, it may take a lot of trouble when preparing as a non-specific reaction inhibitor. If it exceeds 20 μm, the surface area per unit weight of the insoluble carrier becomes small, and if the number of carriers is not increased, the effect of suppressing the nonspecific reaction may not be sufficient.

In the immunoassay of the present invention 5, the concentration of the non-specific inhibitor added includes the amount of the non-specific inhibitor immobilized on the insoluble carrier, the size, type and average particle size of the insoluble carrier. The concentration, type and average particle size of the carrier on which the specific antibody or specific antigen is immobilized, the detection method, and the non-specific reaction suppressing effect, the reaction between the carrier and the carrier on which the specific antibody or specific antigen is immobilized. Is appropriately selected from the viewpoints of the presence or absence of interference with the device, ease of use, and the like.

Specifically, when the (method 1) or (method 3) is employed as a method for treating the above nonspecific reaction inhibitor,
The concentration of the insoluble carrier is preferably 0.001 to 5% by weight based on the antigen-antibody reaction solution. If the amount is less than 0.001% by weight, the effect of suppressing the nonspecific reaction may not be sufficient. If the content exceeds 5% by weight, the signal indicating the specific reaction between the antigen or antibody to be measured and the insoluble carrier having the specific antibody or the specific antigen immobilized thereon becomes smaller with respect to the signal shown by the insoluble carrier. Detection sensitivity may deteriorate.

When (Method 2) is employed as a method for treating the above nonspecific reaction inhibitor, the concentration of the insoluble carrier is preferably 0.001 to 10% by weight based on the antigen-antibody reaction solution. . If the amount is less than 0.001% by weight, the effect of suppressing the nonspecific reaction may not be sufficient. 10% by weight
When this is added to the sample, the antigen or antibody to be measured in the sample is non-specifically adsorbed to the insoluble carrier, and the specific reaction between the antigen and the insoluble carrier on which the specific antibody is immobilized is observed. The signal shown may be small, resulting in poor detection sensitivity. However, in this method, since the non-specific reaction inhibitor does not exist in the reaction system of the antigen-antibody reaction and has been removed in advance, the added amount of the non-specific reaction inhibitor is, It shall represent the concentration when not removed.

When latex particles are used as the carrier on which the specific antibody or the specific antigen is immobilized, the concentration of the insoluble carrier of the non-specific inhibitor is determined by adjusting the concentration of the latex solution to be added later when performing the antigen-antibody reaction. The concentration is not particularly limited as long as it does not exceed the measurement upper limit of the detection system. For example, when performing detection with a spectrophotometer, the absorbance after the completion of the reaction may be within a range not exceeding 3.0. There is no particular limitation on the case where the naked eye determination is performed on the plate.

The reaction between the above-mentioned carrier carrying the specific antibody or the specific antigen and the sample containing the antigen or the antibody to be measured is an antigen-antibody reaction. There is no particular limitation. The above reaction is preferably carried out at a constant temperature, particularly preferably at 25 ° C to 37 ° C. Although the reaction time is not particularly limited, it is preferably 5 seconds to 15 minutes.

In the above reaction, the buffer components other than the non-specific reaction inhibitor, that is, the buffer for diluting the specimen, which are finally contained in the reaction solution for the antigen-antibody reaction, are generally biochemical There are no particular restrictions on phosphoric acid, glycine, Tris and the like used for typical applications, and those exemplified as the medium for suspending the insoluble carrier can be mentioned.

The immunoassay method of the present invention 5 is not particularly limited as long as it is a method for quantifying an antigen or an antibody utilizing an antigen-antibody reaction. For example, immunoassay methods such as latex agglutination method and immunochromatography , EIA, RIA and the like.

In the case of the latex aggregation method, for example,
It can be performed as follows. The reagent used in the latex agglutination method includes a sample diluent, which is a liquid reagent for diluting and pretreating the sample (for the purpose of improving the specificity and sensitivity of the antigen-antibody reaction), and a specific antibody or specific antigen. It consists of a latex liquid which is a liquid reagent containing an immobilized carrier (latex particles). The composition of the reagent in the case of containing the non-specific reaction inhibitor of the present invention includes a one-reagent system containing only a latex solution containing the non-specific reaction inhibitor, a sample diluent containing the non-specific reaction inhibitor, and the non-specific reaction inhibitor. Two-reagent system with latex solution not containing, two-reagent system with sample diluent without non-specific reaction inhibitor and latex solution with non-specific reaction inhibitor, sample diluent with non-specific reaction inhibitor and non-specific A two-reagent system with a latex liquid containing a reaction inhibitor may be used.

The method of treatment with the non-specific inhibitor may be any of (Method 1) to (Method 3). As the above-mentioned processing method, for example, first, a sample and a sample diluent containing a non-specific reaction inhibitor are mixed and kept for a certain period of time. afterwards,
A latex solution containing no non-specific reaction inhibitor is added to the above mixture, and the degree of aggregation of the latex particles having the specific antibody immobilized thereon is observed or measured. As another example of the above-mentioned processing method, first, a sample and a sample diluent containing no nonspecific reaction inhibitor are mixed and left for a certain period of time. Thereafter, a latex solution containing a non-specific reaction inhibitor is added to the mixed solution,
The degree of aggregation of the latex particles having the specific antibody or specific antigen immobilized thereon is observed or measured.

When the immunoassay is a latex agglutination method, the insoluble carrier serving as the non-specific reaction inhibitor is preferably latex particles, depending on the particle size of the latex on which the specific antibody or specific antigen is immobilized. , 0.01-5
A particle having a particle size of 0 μm is appropriately selected. When using the insoluble carrier which is the non-specific reaction inhibitor at a relatively high concentration, the background becomes large when observing or measuring the degree of aggregation of the latex particles on which the specific antibody is immobilized, It is preferable to use particles having a particle size smaller than the latex particles to which the specific antibody or the specific antigen is immobilized.

The method for measuring the degree of aggregation of the latex is not particularly limited. When quantitatively measuring the aggregation, it is preferable to measure, for example, optically from the viewpoint of simplicity and accuracy, specifically, the measurement is scattered light,
The measurement is performed with an optical instrument for measuring the absorbance or transmitted light intensity. As the wavelength for the above measurement, 300 to 1200 nm can be used. The above measuring method is carried out by measuring the increase or decrease of the scattered light intensity, the absorbance or the excess light intensity by selecting the size of the insoluble carrier to be used, its concentration, and setting the reaction time according to a known method. Also, these methods can be used in combination.

As the optical measurement method for the latex agglomeration, known methods can be used. For example, a so-called turbidity method (the formation of agglomerates is regarded as an increase in turbidity), a measurement method based on particle size distribution (aggregation) The formation of agglomerates is regarded as a change in the particle size distribution or average particle size), and the integrating sphere turbidity method (the change in forward scattered light due to the formation of agglomerates is measured using an integrating sphere, and the ratio with the transmitted light intensity is compared. ) And the like.

For each of the above assays, a rate test (rate assay; taking at least two measurements at different time points) and determining the extent of aggregation based on the increment (ie the rate of increase) of the measurements between these time points. Ask) is available. Preferably, a speed test using a turbidimetric method is performed from the viewpoint of simplicity and quickness of measurement.

In the above-mentioned latex agglutination reaction, when the degree of agglutination after the start of the reaction is qualitatively or semi-quantitatively measured, the degree of agglutination is determined by comparing the degree of agglutination with the turbidity of a known sample. It is also possible to visually determine the degree of aggregation.

In the reagent for visually determining the degree of aggregation of the insoluble carrier, a solution containing the sample and the sensitized insoluble carrier is usually mixed on a determination plate and shaken for 1 to 5 minutes. Is determined. In addition to the naked eye determination, the aggregation determination may be performed by photographing with a video camera and performing image processing.

When the EIA method (two-step sandwich method) is adopted as the above-mentioned immunoassay method, for example, it can be performed as follows. In the EIA method,
Normally, a well is provided to allow the addition of a sample diluent or liquid reagent, which is a liquid reagent for diluting and pretreating a sample (for the purpose of improving the specificity and sensitivity of the antigen-antibody reaction). A reaction plate on which a specific antibody or a specific antigen is immobilized, and a specific antibody solution or a specific antigen solution which is a liquid reagent containing the specific antibody or the specific antigen modified with an enzyme or the like are used. When the non-specific reaction inhibitor of the present invention is used, examples of the composition of the reagent include a sample diluent containing the non-specific reaction inhibitor, a reaction plate, and a specific antibody solution containing no non-specific reaction inhibitor.

The method of adding and reacting the above non-specific inhibitor is as follows.
Any of (first method) to (third method) may be used. As a method of adding and reacting the non-specific inhibitor, for example, first, a sample and a sample diluent containing the non-specific reaction inhibitor are mixed and kept for a certain period of time. Thereafter, the mixture is added to the well of the reaction plate on which the specific antibody or the specific antigen is immobilized, and the mixture is left for a certain period of time. Thereafter, the mixed solution is removed from the well, the inside of the well is washed, a specific antibody solution or a specific antigen solution is added to the well, and the well is kept for a certain period of time. Again, the specific antibody solution or the specific antigen solution is removed from the wells, the inside of the wells is washed, and then the measurement is performed by a generally used method.

As the above-mentioned measurement method, a solution containing a substrate in which a reaction is caused by an enzyme modified with a specific antibody is added to a well, the substrate is allowed to react, and the resulting color development or the like is observed or measured. In the case of the EIA method, the insoluble carrier which is a non-specific reaction inhibitor is preferably a spherical or flaky polymer, and any size of 0.01 to 10 mm can be used.

[0070]

DESCRIPTION OF THE PREFERRED EMBODIMENTS (Examples) The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of Antibody for Non-Specific Reaction Inhibitor 1.8 g of sodium sulfate was added to 10 mL of normal rabbit serum while stirring at 25 ° C. After leaving for 30 minutes, 25 ° C,
Centrifuged at 7000G for 10 minutes. The precipitate was collected and combined with a 0.03 M phosphate buffer (containing 0.1 M NaCl,
(pH 6.5, hereinafter referred to as “PBS”). Then, after dialysis against PBS for 24 hours, 10
Centrifuged at 00G for 10 minutes. The supernatant was passed through a DEAE-cellulose column, which is a cation exchange resin equilibrated with PBS, to collect an IgG fraction. The obtained IgG fraction was concentrated.

(2) Preparation of Specific Antibody Subtype Ad, protein concentration 1 as HBs antigen
mg / mL, purity 95% or more (ADVANCED BI
OTECHNOLOGIES INC. 8 mg was immunized to one rabbit, and blood was collected three months after the immunization injection. Rabbit antiserum 1 immunized with HBs antigen
While stirring 0 mL at 25 ° C., 1.8 g of sodium sulfate
Was added. After leaving it for 30 minutes,
Centrifuged for 0 minutes. Collect the precipitate and add 10 mL of PBS.
Was dissolved. After dialysis against PBS for 24 hours, 10
Centrifuged at 00G for 10 minutes. The supernatant was passed through a DEAE-cellulose column equilibrated with PBS to collect an IgG fraction. Thereafter, the obtained IgG fraction was passed through a column packed with agarose gel on which HBs antigen was immobilized, and affinity purification was performed to obtain an IB having specificity for HBs antigen.
A specific antibody solution containing only the gG component was obtained. Thereafter, the obtained specific antibody solution was concentrated.

(3) Preparation of non-specific reaction inhibitor suspension Antibody for non-specific reaction inhibitor was added at a concentration of 1.0 mg / mL to P
In 3.75 mL of a solution dissolved in BS, the average particle size is 0.1 μm.
m of polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) 1.0 mL and PBS 1.25 mL were added,
Stirred at 25 ° C. for 60 minutes. Then add BSA to this solution
6 mL of PBS containing 2.0% by weight of
, And then washed by centrifugation at 18,000 rpm. The washing operation was performed three times. 10 mL of PBS containing 0.5% by weight of BSA was added to the obtained precipitate, and the latex was suspended to prepare a suspension of a nonspecific reaction inhibitor.

Example 2 (1) Preparation of HBS antibody-sensitized latex solution The specific antibody of Example 1 (2) was dissolved in PBS at a concentration of 1.0 mg / mL in 1.25 mL of a solution, and the average particle size was 0%. .3
1.0 mL of μm polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) and 3.75 mL of PBS were added,
Stirred at 25 ° C. for 60 minutes. Then add BSA to this solution
6 mL of PBS containing 2.0% by weight of
, And then washed by centrifugation at 18,000 rpm. The washing operation was performed three times. To the obtained precipitate, 1 mL of the suspension of the nonspecific reaction inhibitor of Example 1 (3) and 99 mL of PBS containing 0.5% by weight of BSA were added to suspend the latex. An HBs antibody-sensitized latex solution was prepared.

(2) Preparation of Sample Diluent Polyethylene glycol (Wako Pure Chemical Industries, average molecular weight; 500,000) was dissolved in PBS at a concentration of 0.3% by weight and BSA at a concentration of 0.5% by weight. .

(3) Reagent for measuring HBs antigen The reagent for measuring HBs antigen of this example is the same as the reagent for HBs described in the above (1).
This is a two-reagent system reagent composed of a first reagent composed of a Bs antibody-sensitized latex solution and a second reagent composed of the sample diluent of the above item (2).

(4) Standard HBs antigen solution The HBs antigen was added at 0, 50, 100, 300, 500 IU /
Human serum was used at a concentration of mL.

(5) HBs antigen-negative sample and HBs antigen-positive sample 10 samples of HBS antigen-negative sample (normal serum of a healthy person) and HBS
Five antigen-positive samples (serum of hepatitis B infected person) were used.

(6) Measuring method The above (2) was added to 20 μL of the standard HBs antigen solution of (4).
After mixing 150 μL of the sample diluent described in the above section and maintaining the mixture at 37 ° C. as appropriate, the HBs antibody-sensitized latex solution 15
0 μL was added and stirred. Thereafter, the absorbance at a wavelength of 750 mn after 1 minute and 5 minutes was measured, and the change in the absorbance during this time was taken as the change in absorbance (ΔOD). The measurement was performed using Hitachi automatic analyzer 7150 type. Measure a standard HBS antigen solution and prepare a calibration curve in advance. The HBs antigen concentration in the sample was calculated from the change in absorbance of the sample and the above calibration curve.

Example 3 HBs by Latex Coagulation Method
Measurement of Antigen Except that the procedures of (1) preparation of HBS antibody-sensitized latex solution and (2) preparation of sample diluent in Example 2 were performed as follows. HBs in the same manner as in Example 2.
An antigen measurement reagent was prepared. (1) Preparation of HBs antibody-sensitized latex solution The specific antibody of Example 1 (2) was dissolved in PBS at a concentration of 1.0 mg / mL in 1.25 mL, and the average particle size was 0.3.
1.0 mL of a μm polystyrene latex (solid content: 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) and 3.75 mL of PBS were added, and the mixture was stirred at 25 ° C. for 60 minutes. Next, B
6 mL of PBS containing 2.0% by weight of SA was added, and 2
After stirring at 5 ° C. for 60 minutes, the plate was washed by centrifugation at 18,000 rpm. The washing operation was performed three times. P containing 0.5% by weight of BSA in the obtained precipitate
100 mL of BS was added to suspend the latex, and HB was added.
An s antibody-sensitized latex solution was prepared.

(2) Preparation of Sample Diluent 1% by volume of the suspension of the nonspecific reaction inhibitor of Example 1 in PBS and 0.3% by weight of polyethylene glycol (manufactured by Wako Pure Chemical Industries, average molecular weight: 500,000) % And BSA were dissolved to a concentration of 0.5% by weight.

Example 4 HBs by Latex Coagulation Method
Measurement of Antigen Except that (1) preparation of HBs antibody-sensitized latex solution, (4) standard HBs antigen solution, and (5) sample in Example 2 were performed as follows, A reagent for measuring HBs antigen was prepared in the same manner as in Example 2. (1) Preparation of HBs antibody-sensitized latex solution The specific antibody of Example 1 (2) was dissolved in PBS at a concentration of 1.0 mg / mL in 1.25 mL, and the average particle size was 0.3.
1.0 mL of polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) and 3.75 mL of PBS were added,
Stirred at 25 ° C. for 60 minutes. Then add BSA to this solution
6 mL of PBS containing 2.0% by weight of
, And then washed by centrifugation at 18,000 rpm. The washing operation was performed three times. 100 mL of PBS containing 0.5% by weight of BSA was added to the obtained precipitate, and the latex was suspended to prepare an HBs antibody-sensitized latex solution.

(4) Standard HBs antigen solution The HBs antigen was added at 0, 50, 100, 300, 500 IU /
0.12 mL of the suspension of the non-specific reaction inhibitor of Example 1 was added to 1.6 mL of human serum containing a concentration of mL, and the mixture was used.

(5) HBS antigen-negative sample and HBS antigen-positive sample 10 samples of HBs antigen-negative sample (normal serum of a healthy person) and HBs
1.6 m of 5 antigen-positive samples (serum of hepatitis B infected person)
In L, 0.12 mL of the suspension of the nonspecific reaction inhibitor of Example 1
Was added and used.

Comparative Example 1 HBs by Latex Coagulation Method
Measurement of Antigen A reagent for measuring HBs antigen was prepared in the same manner as in Example 2 except that (1) Preparation of HBs antibody-sensitized latex solution in Example 2 was performed as follows. .

(1) Preparation of HBS antibody-sensitized latex solution The specific antibody of Example 1 (2) was dissolved in PBS at a concentration of 1.0 mg / mL in PBS (1.25 mL), and the average particle size was 0.3.
Add 1.0 mL of μm polystyrene latex (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) and 3.75 mL of PBS,
Stirred at 25 ° C. for 60 minutes. Then add BSA to this solution
6 mL of PBS containing 2.0% by weight of
, And then washed by centrifugation at 18,000 rpm. The washing operation was performed three times. 100 mL of PBS containing 0.5% by weight of BSA was added to the obtained precipitate, and the latex was suspended to prepare an HBs antibody-sensitized latex solution.

The results of Examples 2 to 4 and Comparative Example 1 are shown in Table 1. Table 1 shows measurement values (IU / mL) when HBs antigen-positive samples and HBs antigen-negative samples were measured using the reagents of Examples 2 to 4 and Comparative Example 1.
In addition, the cut-off point in Examples 2-4 and Comparative Example 1 was set to 8 IU / mL, and a sample of less than 8 IU / mL was determined as negative, and a sample of 8 IU / mL or more was determined as positive.

[0088]

[Table 1]

As is clear from Table 1, in some of the HBs antigen-negative specimens, in Examples 2 to 4, negative was determined, but in Comparative Example 1, it was determined to be positive. Suggests what is happening.

Example 5 Measurement of HBs Antigen by ELISA (1) Preparation of HBs Antibody-Immobilized Plate A specific antibody of Example 1 (2) was added to each well of a 96-well microtiter plate (manufactured by Corning). 1mg /
A solution dissolved in PBS at a concentration of mL was dispensed in 100 μL portions, and allowed to stand at room temperature for 2 hours. The HBs antibody solution in each well was aspirated, and 300 μL of PBS was placed in each well and aspirated.
This washing operation was performed seven times. Next, 200 μL of PBS containing 1% of BSA was dispensed to each well. After allowing to stand at room temperature for 2 hours, the above washing operation was performed seven times, and HBs
An antibody-immobilized plate was prepared.

(2) Standard HBs antigen solution To 0.8 mL of each of the HBs antigen negative control serum and the HBs antigen positive control serum, 0.2 mL of the suspension of the nonspecific reaction inhibitor of Example 1 was added and used. .

(3) HBs antigen-negative sample and HBs antigen-positive sample 10 samples of HBs antigen-negative sample (serum of a healthy person) and HBs antigen
0.8m of 5 antigen-positive samples (serum of hepatitis B infected person)
To L, 0.2 mL of the suspension of the nonspecific reaction inhibitor of Example 1 was added and used.

(4) Measurement Method 100 μL of the standard HBs antigen solution and the sample were dispensed into each well of the microtiter plate, and the mixture was allowed to stand at room temperature for 2 hours. After aspirating the reaction solution in each well, PBS 300μ
L was placed in each well and aspirated. This washing operation was performed seven times. Then, specific antibody was added to PB at a concentration of 0.1 mg / mL.
100 μL of the solution dissolved in S was dispensed, and allowed to stand at room temperature for 2 hours, and then the washing operation was performed seven times. 5 μg / mL peroxidase-labeled anti-rabbit IgG antibody was added to each well at 50 μg / mL.
After dispensing each μL and leaving at room temperature for 2 hours, 10 mM
50 μL of o-phenylenediamine and 25 μL of 30% aqueous hydrogen peroxide were added. After standing at room temperature for 2 hours, 1N
Sulfuric acid was added in an amount of 100 μL each, and the absorbance at a measurement wavelength of 492 nm was measured. A calibration curve was prepared based on the absorbance of the standard HBs antigen solution, and the HBs antigen concentration in the sample was calculated from the absorbance of the sample and the above calibration curve.

Comparative Example 2 Measurement of HBs Antigen by ELISA Method Example 5 was the same as Example 5 except that the sections of (2) standard HBs antigen solution and (3) specimen in Example 5 were performed as follows. Similarly, a reagent for measuring HBs antigen was prepared.

(2) Standard HBS antigen solution HBs antigen negative control serum and HBS antigen positive control serum were used.

(3) HBs antigen-negative sample and HBs antigen-positive sample 10 samples of HBs antigen-negative sample (serum of a healthy person) and HBs antigen
Five antigen-positive samples (serum of hepatitis B infected person) were used.

Table 2 shows the results of Example 5 and Comparative Example 2. Table 2 shows that the reagents of Example 5 and Comparative Example 2
The determination result when the s antigen positive sample and the HBs antigen negative sample are detected is shown. The value obtained by adding 0.07 to the absorbance of the negative control serum was defined as the cutoff point.

[0098]

[Table 2]

As is evident from Table 2, although several cases in the HBs antigen-negative samples were judged to be negative in Example 5, they were judged to be positive in Comparative Example 2 and a non-specific agglutination reaction occurred. It is suggested that

Example 6 Preparation of Non-Specific Reaction Inhibitor Suspension 18 mL of a solution containing 2% by weight of BSA dissolved in PBS
Was added with 1 mL of polystyrene latex having an average particle size of 0.1 μm (solid content: 10% by weight, manufactured by Sekisui Chemical Co., Ltd.),
After stirring at 25 ° C. for 60 minutes, the mixture was centrifuged at 18,000 rpm. The washing operation was performed three times. To the obtained precipitate, 10 mL of PBS containing 0.5% by weight of BSA was added, and the latex was suspended to prepare a suspension of a nonspecific reaction inhibitor.

Example 7 HBS by Latex Coagulation Method
Measurement of Antibody (1) Preparation of HBs Antigen-Sensitized Latex Solution To 1.5 mL of a solution of HBs antigen in PBS at a concentration of 2.0 mg / mL was added a polystyrene latex having an average particle size of 0.3 μm (solid content: 10 wt. %, Manufactured by Sekisui Chemical Co., Ltd.)
0 mL and 3.5 mL of PBS were added, and the mixture was stirred at 25 ° C. for 60 minutes (this operation is called sensitization). Next, 6 mL of PBS containing 2% by weight of BSA was added to this solution,
After stirring at 60 ° C. for 60 minutes, the plate was washed by centrifugation at 18,000 rpm. The washing operation was performed three times. To the obtained precipitate, 1 mL of the suspension of the nonspecific reaction inhibitor of Example 6 and 99 mL of PBS containing 0.5% by weight of BSA were added to suspend the latex, and the HBs antigen-sensitized latex solution was added. Prepared.

(2) Preparation of Sample Diluent Polyethylene glycol (Wako Pure Chemical Industries, average molecular weight: 500,000) was dissolved in PBS at a concentration of 0.4% by weight and BSA at a concentration of 1.0% by weight. .

(3) HBs Antibody Measurement Reagent The HBs antibody measurement reagent of the present example is the HBs antibody measurement reagent of the above item (1).
This is a two-reagent system reagent comprising a first reagent comprising a Bs antigen-sensitized latex solution and a second reagent comprising the sample diluent described in the above item (2).

(4) Standard HBs antibody solution The HBs antibody was added at 0, 150, 300, 600, 1200 m
Human serum containing IU / mL concentration was used.

(5) HBs antibody-negative sample and HBS antigen-positive sample 10 samples of HBs antibody-negative sample (serum of a healthy person) and HBS
Five antibody positive samples (serum with a history of hepatitis B) were used.

(6) Measurement method The above (2) was added to 20 μL of the standard HBs antibody solution of (4).
After mixing 120 μL of the sample diluent described in the above section and maintaining the mixture at 37 ° C. as appropriate, the HBs antigen-sensitized latex solution 12
0 μL was added and stirred. Thereafter, the absorbance at a wavelength of 750 nm after 1 minute and 5 minutes is measured, and the change in the absorbance during this time is defined as the change in absorbance (吸 光 OD). The measurement was performed using a Hitachi automatic analyzer 7150. A standard HBs antibody solution was measured and a calibration curve was prepared in advance, and the HBs antibody concentration in the sample was calculated from the change in absorbance of the sample and the above calibration curve.

Example 8 HBS by Latex Coagulation Method
Measurement of Antibodies The procedure of Example 6 was the same as Example 6, except that the steps of (1) preparation of HBs antigen-sensitized latex solution and (2) preparation of sample diluent were performed as follows. HBs
An antibody measurement reagent was prepared.

(1) Preparation of HBs antigen-sensitized latex liquid A polystyrene latex having an average particle diameter of 0.3 μm (solid content: 10 wt. %, Manufactured by Sekisui Chemical Co., Ltd.)
0 mL and 3.5 mL of PBS were added, and the mixture was stirred at 25 ° C. for 60 minutes (this operation is called sensitization). Next, 6 mL of PBS containing 2% by weight of BSA was added to this solution,
After stirring at 60 ° C. for 60 minutes, the plate was washed by centrifugation at 18,000 rpm. The washing operation was performed three times. P containing 0.5% by weight of BSA in the obtained precipitate
100 mL of BS was added to suspend the latex. HB
An s antigen-sensitized latex solution was prepared.

(2) Preparation of Sample Diluent A 1% by volume suspension of the non-specific reaction inhibitor of Example 6 in PBS and 0.4% by weight of polyethylene glycol (manufactured by Wako Pure Chemical, average molecular weight: 500,000) % And BSA were dissolved to a concentration of 1.0% by weight.

Example 9 HBs by latex coagulation method
Measurement of Antibodies In Example 7, (1) Preparation of HBs antigen-sensitized latex solution, (4) Standard HBs antibody solution and (5) Sample
A HBS antibody measurement reagent was prepared in the same manner as in Example 7, except that the following procedure was performed.

(1) Preparation of HBs antigen-sensitized latex solution In 1.5 mL of a solution of HBs antigen in PBS at a concentration of 2.0 mg / mL, a polystyrene latex having an average particle size of 0.3 μm (solid content: 10 wt. %. Sekisui Chemical Mori) 1.
0 mL and 3.5 mL of PBS were added, and the mixture was stirred at 25 ° C. for 60 minutes (this operation is called sensitization). Then. To this solution, 6 mL of PBS containing 2% by weight of BSA was added, and 25 mL of BSA was added.
After stirring at 60 ° C. for 60 minutes, the plate was washed by centrifugation at 18,000 rpm. The washing operation was performed three times. P containing 0.5% by weight of BSA in the obtained precipitate
100 mL of BS was added to suspend the latex, and HB was added.
An s antigen-sensitized latex solution was prepared.

(4) Standard HBs antibody solution The HBs antibody was added at 0, 150, 300, 600, 1200 m
A suspension of the nonspecific reaction inhibitor of Example 6 (0.12 mL) was added to 1.6 mL of human serum containing IU / mL concentration, and the mixture was used.

(5) HBs antibody negative specimen and HBs
Antibody positive sample HBs antibody negative sample (Healthy human serum) 10
0.12 mL of the suspension of the non-specific reaction inhibitor of Example 6 was added to 1.6 mL of the sample and 5 samples of the HBs antibody-positive sample (serum with a history of hepatitis B), and the mixture was used.

Comparative Example 3 HBs by Latex Coagulation Method
Measurement of Antibody A reagent for measuring HBs antibody was prepared in the same manner as in Example 7, except that the procedure of (1) Preparation of HBs antigen-sensitized latex solution in Example 7 was performed as follows. . (1) Preparation of HBs antigen-sensitized latex solution In 1.5 mL of a solution of HBs antigen dissolved in PBS at a concentration of 2.0 mg / mL, a polystyrene latex having an average particle size of 0.3 μm (solid content: 10% by weight, Chemical company) 1.
0 mL and 3.5 mL of PBS were added, and the mixture was stirred at 25 ° C. for 60 minutes (this operation is called sensitization). Next, 6 mL of PBS containing 2% by weight of BSA was added to this solution,
After stirring at 60 ° C. for 60 minutes, the plate was washed by centrifugation at 18,000 rpm. The washing operation was performed three times. P containing 0.5% by weight of BSA in the obtained precipitate
100 mL of BS was added to suspend the latex, and HB was added.
An s antigen-sensitized latex solution was prepared.

Table 3 shows the results of Examples 7 to 9 and Comparative Example 3. Table 3 shows measurement values (mIU / mL) when HBs antibody negative samples and HBs antibody positive samples were measured using the reagents of Examples 7 to 9 and Comparative Example 3. Note that the cut-off points in Examples 7 to 9 and Comparative Example 3 were 3
It was set to 0 mIU / mL, and less than 30 mIU / mL was judged as negative, and 30 mIU / mL or more was judged as positive.

[0116]

[Table 3]

As is evident from Table 3, several of the HBs antibody-negative specimens were judged to be positive in Comparative Example 1 in spite of negative judgments in Examples 7 to 9, and nonspecific agglutination was observed. Suggests what is happening.

Example 10 Preparation of a suspension of a non-specific reaction inhibitor 10 mL of a solution prepared by dissolving BSA at a concentration of 2% by weight in PBS
1.0 mL of polystyrene latex having an average particle size of 0.1 μm (solid content: 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) and PB
S9mL was added and stirred at 25 ° C for 60 minutes. Then, it was washed by centrifugation at 18,000 rpm. The washing operation was performed three times. BSA is added to the resulting precipitate.
10 mL of PBS containing 0.5% by weight was added, and the latex was suspended to prepare a suspension of a nonspecific reaction inhibitor.

Example 11 HB by Latex Coagulation Method
Measurement of s antigen Example 2 (1) In the preparation of the HBs antibody-sensitized latex solution, the suspension of the non-specific reaction inhibitor of Example 1 (3) was replaced with the non-specific reaction inhibitor of Example 10. The suspension was used to prepare BSA in Example 2 (2).
HBs antigen was measured in the same manner as in Example 2 except that the concentration of HBs antigen was changed to 1.0% by weight.

Example 12 HB by Latex Coagulation Method
Measurement of s Antigen Example 3 (2) In the preparation of the sample diluent, the suspension of the nonspecific reaction inhibitor of Example 10 was used instead of the suspension of the nonspecific reaction inhibitor of Example 1. Except for this, HBs antigen was measured in the same manner as in Example 3.

Example 13 HB by Latex Coagulation Method
Example 4 (4) In preparing a standard HBs antigen solution, Example 1 was replaced with the suspension of the non-specific reaction inhibitor of Example 1.
Except that a suspension of 0 non-specific reaction inhibitors was used.
HBs antigen was measured in the same manner as in Example 4.

Comparative Example 4 HBs by Latex Coagulation Method
Measurement of Antigen The HBs antigen was measured in the same manner as in Comparative Example 1 except that the concentration of BSA was changed to 1.0% by weight in preparing the sample diluent of Comparative Example 1.

Table 4 shows the results of Examples 11 to 13 and Comparative Example 4. Table 4 shows measurement values (IU / mL) when HBs antigen-negative samples and HBs antigen-positive samples were measured using the reagents of Examples 11 to 13 and Comparative Example 4. The cut-off points in Examples 11 to 13 and Comparative Example 4 were set to 8 IU / mL, and it was determined that less than 8 IU / mL was negative and more than 8 IU / mL was positive.

[0124]

[Table 4]

As is evident from Table 4, several cases in the HBs antigen-negative samples were judged to be negative in Examples 11 to 13 but were judged to be positive in Comparative Example 4, and nonspecific agglutination was not observed. Suggests what is happening.

Example 14 Measurement of HBs Antibody by ELISA (1) Preparation of HBs Antigen Immobilized Plate HBs antigen was added at a concentration of 0.2 mg / mL to each well of a 96-well microtiter plate (manufactured by Corning).
100 μL of a solution dissolved in 0 mM Tris-HCl buffer (pH 7.4) was dispensed and left at room temperature for 2 hours. The HBs antigen solution in each well was aspirated, and 300 μL of PBS was placed in each well and aspirated. This washing operation was performed seven times. Next, 200 μL of PBS containing 1% BSA was added to each well.
Was dispensed. After allowing to stand at room temperature for 2 hours, the above-mentioned washing operation was performed seven times to prepare an HBs antigen-immobilized plate.

(2) Standard HBs Antibody Solution 0.2 mL of the suspension of the nonspecific reaction inhibitor of Example 6 was added to 0.8 mL of each of the HBs antibody negative control serum and the HBs antibody positive control serum, and the mixture was used.

(3) HBs Antibody Negative Sample and HBs Antibody Positive Sample HBs antibody negative sample (normal human serum)
5 antibody-positive samples (serum with a history of hepatitis B)
0.2 mL of the suspension of the nonspecific reaction inhibitor of Example 6 in 8 mL
L was added and used.

(4) Measurement method 100 μL of a standard HBs antibody solution and a sample were dispensed into each well of a microtiter plate and allowed to stand at room temperature for 2 hours. After aspirating the reaction solution in each well, PBS 300μ
1 was placed in each well and aspirated. This washing operation was performed seven times. Next, 50 μL of 5 μg / mL peroxidase-labeled anti-human IgG antibody was dispensed to each well, and 2 μl at room temperature.
After standing for 10 hours, 10 mMo-phenylenediamine 50
μL and 25 μL of 30% hydrogen peroxide solution were added. After allowing to stand at room temperature for 2 hours, 1N sulfuric acid was added in an amount of 100 μL each, and the absorbance at a measurement wavelength of 492 mn was measured.

Comparative Example 5 Measurement of HBs Antibody by ELISA Method (2) Standard HBs antibody solution and (3) in Example 14
Except that the sample section was performed as follows. A reagent for measuring HBs antibody was prepared in the same manner as in Example 14.

(2) Standard HBs antibody solution An HBs antibody negative control serum and an HBs antibody positive control serum were used. (3) HBs antibody-negative sample and HBs antibody-positive sample 10 samples of HBs antibody-negative sample (healthy human serum) and HBs
Five antibody positive samples (serum with a history of hepatitis B) were used.

Table 5 shows the results of Example 14 and Comparative Example 5. Table 5 shows that the reagents of Example 14 and Comparative Example 5
4 shows the results of determination when a HBs antibody negative sample and an HBS antibody positive sample were detected. The value obtained by adding 0.07 to the absorbance of the negative control serum was defined as the cutoff point.

[0133]

[Table 5]

As is evident from Table 5, several cases in the HBs antibody-negative samples were determined to be negative in Comparative Example 5 in spite of the negative determination in Example 14, and a non-specific agglutination reaction occurred. It is suggested that

Example 15 Measurement of HBs antigen by ELISA Example 5 Suspension of the nonspecific reaction inhibitor of Example 1 in (2) standard HBs antigen solution and (3) HBs antigen negative sample and HBs antigen positive sample The measurement of HBs antigen was carried out in the same manner as in Example 5, except that the suspension of the nonspecific reaction inhibitor of Example 10 was used instead of the liquid.

Example 15 and Comparative Example 2 for comparison
Table 6 shows the results. Table 6 shows the determination results when HBs antigen-negative samples and HBs antigen-positive samples were detected by the reagents of Example 15 and Comparative Example 2. The value obtained by adding 0.07 to the absorbance of the negative control serum was defined as the cutoff point.

[0137]

[Table 6]

As is evident from Table 6, several cases in the HBs antigen-negative samples were judged to be negative in Comparative Example 2 in spite of the negative judgment in Example 15, and the non-specific agglutination reaction occurred. It is suggested that

Example 16 The reagents and materials used in this example are as follows. Anti-human CEA monoclonal antibody: two types of anti-human CE
A monoclonal antibody (Clone No. CEA 4)
-G67, IgG-1, and Clone No. CE
A4-G11, IgG-1; manufactured by DAKO) was used. Latex: Polystyrene latex of 0.40 μm and 0.30 μm (both solids content 10% (W / V), manufactured by Sekisui Chemical Co., Ltd.) was used. Latex suspension buffer: 1 mM Hepes (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution adjusted to pH 7.50 by adding 0.1 N NaOH to bovine serum albumin (F).
fractionV, Reagent Grade, Mi
les Corp.) and NaN ₃ (reagent grade, Nacalai Tesque) at 0.1% (W / V), and latex. Used as a suspension buffer. Blocking buffer: 100 mM Na ₂ HPO ₄ and 100 mM NaH ₂ PO ₄ are mixed to a pH of 7.40, BSA is adjusted to 1% (W / V), and NaN ₃ is adjusted to 0.1% ( (W / V) was used as a buffer for blocking. CEA standard products: CEA standard products (Dynabot, CEA
・ 0, 5, 20, 100,
500 ng / mL was used as it was. CEA-positive specimen: human serum 2 whose CEA concentration was measured using the accessory of CEA / Rear beads kit, Dynabot
0 samples were used.

(1) Preparation of Reagent for CEA Measurement A polystyrene latex (average particle size: 0.3 μm, solid content: 10% (W / V)) was mixed with a latex suspension buffer
The solution was added and diluted to obtain a 1.0% latex solution. Anti-CE
Antibody A (Clone No. CEA 4-G67)
The antibody was diluted with an antibody dilution buffer to a protein concentration of 12.5 μg / mL to obtain a sensitized antibody solution. 1.0% (W
/ V) While stirring 600 μL of the latex solution in a 25 ° C. incubator with a magnetic stirrer, 1200 μL of the antibody sensitizing solution was quickly added, followed by stirring at 25 ° C. for 1 hour. Thereafter, 3.0 mL of the blocking buffer was added.
The mixture was stirred at 25 ° C. for 2 hours. Then 1
The mixture was centrifuged at 15,000 rpm at 5 ° C. for 15 minutes.
4.0 mL of a latex suspension buffer was added to the obtained precipitate, and the precipitate was washed by centrifugation in the same manner. The washing operation was performed three times. 1.8 mL of a blocking buffer was added to the precipitate, and the mixture was stirred well, and then subjected to a dispersion treatment with an ultrasonic crusher. To this, 1.8 mL of a blocking buffer was added to obtain a CEA measuring reagent [1] having a solid content of 0.17% (W / V). The CEA measuring reagent [1] thus prepared was stored at 4 ° C. Clo
ne No. CEA 4-G11 was immobilized on latex in the same manner, and the solid content was 0.17% (W / V).
Was used as a CEA measurement reagent [2].

(2) Preparation of Dilution Solution for Sample Dilution (R1 Solution) The same procedure as in (1) above except that 9 volumes of 1% BSA was added instead of immobilizing the anti-CEA antibody. Latex suspension (solid content 0.17%)
(W / V)) to obtain a non-specific reaction inhibitor to which BSA was immobilized. To the blocking buffer, polyethylene glycol 6000 (average molecular weight 7,500, manufactured by Wako Pure Chemical Industries, Ltd.) was added to 3% (W / V), and further,
The non-specific reaction inhibitor was suspended at 0.0001%, and used as a diluent for diluting a sample (R1 solution).

(3) Measurement of CEA Amount The CEA amount was measured using an automatic analyzer for biochemistry Model 7150 (manufactured by Hitachi, Ltd.). An R2 solution (0.17% (W / V) solid content) obtained by mixing equal amounts of the CEA measurement reagents [1] and [2] with a solid content of 0.17% (W / V) obtained in (1) above was mixed. V)). The measurement conditions are as follows. Sample volume 20 μL Sample diluent (R1 solution) 180 μL Reagent (R2 solution) 90 μL Measurement wavelength 700 nm Measurement temperature 37 ° C. Difference between absorbance approximately 80 seconds and 320 seconds after addition of reagent (R2 solution) ( ΔOD570), and the difference in absorbance multiplied by 10,000 was defined as the change in absorbance. The amount of change in absorbance was measured using a known concentration of the CEA standard, and a calibration curve was prepared. Table 7 shows the measurement results of the CEA standard product.

(4) Measurement of CEA Amount in Patient Serum Using CEA-positive serum as a sample, the amount of change in absorbance was measured in accordance with the above (3) Measurement of CEA amount, and CEA was determined using the calibration curve obtained in (3) above. The concentration was determined. Table 8 shows the results of these measurements. Table 8 also shows the results of measuring the amount of CEA in CEA-positive serum by the RIA method using a CEA-Rear beads kit (Dynabot). R
FIG. 1 is a graph showing the correlation between the measured value of the amount of CEA in CEA-positive serum measured by the IA method and the result of measuring the amount of CEA obtained in Example 16.

Example 17 In the preparation of (2) diluent for sample dilution (R1 solution) in Example 16, 9 volume of 1% casein was added instead of 9 volume of 1% BSA to immobilize casein. The same operation as in Example 16 was performed, except that the nonspecific reaction inhibitor was prepared, and the CEA amount was measured. The results are shown in Tables 7 and 8 and FIG.

Example 18 In Example 16, (2) Preparation of a diluent for diluting a sample (solution R1), 9% of 1% casamino acid was added instead of 9% of 1% BSA to fix casamino acid. The same operation as in Example 16 was performed, except that the non-specific reaction inhibitor was prepared, and the CEA amount was measured. Table 7 shows the results.
The results are shown in Table 8 and FIG.

Example 19 The procedure of Example 16 was repeated except that (2) the preparation of the diluent for diluting the sample (R1 solution) and (3) the preparation of the R2 solution in the measurement of the CEA amount were carried out as follows. The same operation as in Example 16 was performed to measure the CEA amount. The results are shown in Tables 7 and 8 and FIG.

(2) Preparation of diluent for sample dilution (R1 solution) Polyethylene glycol 60 was added to the blocking buffer.
00 (average molecular weight 7,500, manufactured by Wako Pure Chemical Industries, Ltd.)
% (W / V) was used as a diluent for sample dilution (R1 solution).

(3) Measurement of CEA Amount (1) The procedure of Example 16 (1) was repeated except that 1% BSA was added instead of immobilizing the anti-CEA antibody.
Latex suspension (solid content 0.17)
% (W / V)) to obtain a non-specific reaction inhibitor having BSA immobilized thereon. The solid content obtained in Example 16 (1) was 0.1%.
17% (W / V) CEA measuring reagents [1] and [2]
Are mixed in equal amounts, and the above-mentioned non-specific reaction inhibitor is further added to 0.00
What was suspended so that it might become 01% was used as R2 liquid.

Example 20 Example 19 (3) In the preparation of R2 solution for measuring the CEA amount, 1% casein was added instead of 1% BSA, and the non-specific reaction inhibitor having immobilized casein was added. Was prepared in the same manner as in Example 19, except that
The amount of A was measured. The results are shown in Tables 7, 8 and FIG.

Example 21 Example 19 (3) Nonspecific reaction in which casamino acid was immobilized by adding 1% casamino acid instead of 1% BSA in preparation of R2 solution for measuring CEA amount Except for preparing the inhibitor, the same operation as in Example 19 was performed,
The amount of CEA was measured. The results are shown in Tables 7, 8 and FIG.

Comparative Example 6 Polyethylene glycol 6000 (average molecular weight 7.5, 5) was used in a blocking buffer without using a nonspecific reaction inhibitor.
00, manufactured by Wako Pure Chemical Industries, Ltd.) was used in the same manner as in Example 16, except that a diluent for diluting the sample (R1 solution) was used in an amount of 3% (W / V). , CE
The amount of A was measured. The results are shown in Tables 7 and 8 and FIG.

[0152]

[Table 7]

[0153]

[Table 8]

Example 22 Example 16 (2) The CEA amount was measured in the same manner as in Example 16, except that a diluent for sample dilution (R1 solution) was prepared as described below. Was. Table 9 and Table 1 show the results.
0 and FIG.

(2) Preparation of Diluent for Sample Dilution (R1 Solution) Polyethylene glycol 60 was added to the blocking buffer.
00 (average molecular weight 7,500, manufactured by Wako Pure Chemical Industries, Ltd.)
% (W / V).
4 μm of polystyrene latex was suspended to 0.0001% and used as a sample diluent (R1 solution).

Example 23 Example 16 (1) C was prepared in the same manner as in Example 22 except that polystyrene latex having an average particle diameter of 0.4 μm was added.
The amount of CEA was measured in the same manner as in Example 22 except that the reagent [1] for CEA measurement obtained in the preparation of the reagent for EA measurement was added. The results are shown in Table 9, Table 10, and FIG.

Example 24 The procedure of Example 22 was repeated, except that polystyrene latex having an average particle size of 0.4 μm was added.
The amount of CEA was measured in the same manner as in Example 22, except that the reagent for CEA measurement [2] obtained in the preparation of the reagent for EA measurement was added. The results are shown in Table 9, Table 10, and FIG.

[0158]

[Table 9]

[0159]

[Table 10]

As is clear from Tables 7 to 10 and FIGS. 1 to 10, the calibration curves measured using the high-purity standard products did not show a large difference between the examples and comparative examples.
In the actual sample, in the comparative example, many positive errors due to non-specific reactions considered to be caused by the coexisting substance in the sample were observed, whereas in the present invention, an accurate result which was in good agreement with the result of the RIA method was obtained. Was. This is due to the fact that the latex solution, which is a non-specific reaction inhibitor added in advance, absorbs non-specific agglutination due to the materials used and coexisting substances in the sample, and accurately measures the original antigen-antibody reaction. Inferred.
From the above results, it was confirmed that the CEA quantification method according to the present invention is a more sensitive, stable and superior quantification method than the conventional latex agglutination immunoreagent.

[0161]

The non-specific reaction inhibitor of the present invention, and the immunoassay reagent and immunoassay method using the same have the above-mentioned constitutions. Specific reaction can be suppressed, and high detection sensitivity can be obtained. This makes it possible to accurately quantify the substance to be measured in the test sample, which is useful for finding a disease, grasping a disease state, and determining a treatment method.

[Brief description of the drawings]

FIG. 1 is a correlation diagram showing the results of measurement of the CEA concentration in 20 human serum samples using the RIA method and the reagent in Example 16. The vertical axis indicates C by the reagent in Example 16.
The EA concentration measurement result (ng / mL), and the horizontal axis is RI
It is a CEA concentration measurement result (ng / mL) by Method A.
In the figure, a linear regression equation and a correlation coefficient of the result of Example 16 with respect to the result of the RIA method are shown.

FIG. 2 is a correlation diagram of the results of measuring the CEA concentration in 20 human serum samples using the RIA method and the reagent in Example 17. The vertical axis indicates C by the reagent in Example 17.
The EA concentration measurement result (ng / mL), and the horizontal axis is RI
It is a CEA concentration measurement result (ng / mL) by Method A.
In the figure, a linear regression equation and a correlation coefficient of the result of Example 17 with respect to the result of the RIA method are shown.

FIG. 3 is a correlation diagram of the results of measuring the CEA concentration in 20 human serum samples using the RIA method and the reagent in Example 18. The vertical axis indicates C by the reagent in Example 18.
The EA concentration measurement result (ng / mL), and the horizontal axis is RI
It is a CEA concentration measurement result (ng / mL) by Method A.
In the figure, a linear regression equation and a correlation coefficient of the result of Example 18 with respect to the result of the RIA method are shown.

FIG. 4 is a correlation diagram showing the results of measurement of the CEA concentration in 20 human serum samples using the RIA method and the reagent in Example 19. The vertical axis indicates C by the reagent in Example 19.
The EA concentration measurement result (ng / mL), and the horizontal axis is RI
It is a CEA concentration measurement result (ng / mL) by Method A.
In the figure, a linear regression equation and a correlation coefficient of the result of Example 19 with respect to the result of the RIA method are shown.

FIG. 5 is a correlation diagram showing the results of measurement of the CEA concentration in 20 human serum samples using the RIA method and the reagent in Example 20. The vertical axis indicates C by the reagent in Example 20.
The EA concentration measurement result (ng / mL), and the horizontal axis is RI
It is a CEA concentration measurement result (ng / mL) by Method A.
In the figure, a linear regression equation and a correlation coefficient of the result of Example 20 with respect to the result of the RIA method are shown.

FIG. 6 is a correlation diagram of the results of measuring the CEA concentration in 20 human serum samples using the RIA method and the reagent in Example 21. The vertical axis indicates C by the reagent in Example 21.
The EA concentration measurement result (ng / mL), and the horizontal axis is RI
It is a CEA concentration measurement result (ng / mL) by Method A.
In the figure, a linear regression equation and a correlation coefficient of the result of Example 21 with respect to the result of the RIA method are shown.

FIG. 7 is a correlation diagram of the results of measuring the CEA concentration in 20 human serum samples by the RIA method and the reagent in Comparative Example 6. The vertical axis represents CEA with the reagent in Comparative Example 6.
The concentration measurement result (ng / mL) is shown, and the horizontal axis is the CEA concentration measurement result (ng / mL) by the RIA method. The figure shows the linear regression equation and the correlation coefficient of the result of Comparative Example 6 with respect to the result of the RIA method.

FIG. 8 is a correlation diagram of the results of measuring the CEA concentration in 20 human serum samples using the RIA method and the reagent in Example 22. The vertical axis indicates C by the reagent in Example 22.
The EA concentration measurement result (ng / mL), and the horizontal axis is RI
It is a CEA concentration measurement result (ng / mL) by Method A.
The figure shows a linear regression equation and a correlation coefficient of the result of Example 22 with respect to the result of the RIA method.

FIG. 9 is a correlation diagram of the results of measuring the CEA concentration in 20 human serum samples using the RIA method and the reagent in Example 23. The vertical axis indicates C by the reagent in Example 23.
The EA concentration measurement result (ng / mL), and the horizontal axis is RI
It is a CEA concentration measurement result (ng / mL) by Method A.
In the figure, a linear regression equation and a correlation coefficient of the result of Example 23 with respect to the result of the RIA method are shown.

FIG. 10. RIA was used to measure the CEA concentration in 20 human serum samples.
FIG. 19 is a correlation diagram of the results measured by the method and the reagent in Example 24. The vertical axis is the CEA concentration measurement result (ng / mL) using the reagent in Example 24, and the horizontal axis is R
It is a CEA concentration measurement result (ng / mL) by the IA method. In the figure, a linear regression equation and a correlation coefficient of the result of Example 24 with respect to the result of the RIA method are shown.

Claims (7)

[Claims] 1. An antibody or a fragment thereof which is used in an immunoassay using a carrier on which an antibody or an antigen against a substance to be measured has been immobilized and does not react immunologically with the substance to be measured, or A non-specific reaction inhibitor comprising an insoluble carrier to which an antigen that does not react is immobilized. 2. The non-specific reaction inhibitor according to claim 1, wherein the immunologically non-reactive antigen is the same substance as the immunologically non-reactive substance immobilized on a carrier. 3. An immunoassay method using a carrier on which an antibody or an antigen against a substance to be measured is immobilized, comprising an insoluble carrier on which an antibody or antigen immunologically reactive with the substance to be measured is immobilized. Non-specific reaction inhibitor characterized by the above-mentioned. 4. A non-specific reaction inhibitor which is used in an immunoassay using a carrier on which an antibody or an antigen against a substance to be measured is immobilized, and which comprises an insoluble carrier. 5. The non-specific reaction inhibitor according to claim 1, wherein the insoluble carrier is a latex. 6. An immunoassay reagent comprising the non-specific reaction inhibitor according to claim 1, 2, 3, 4, or 5. 7. The method according to claim 1, wherein a sample containing the substance to be measured is provided.
The immunoreaction after pre-treatment with the non-specific reaction inhibitor according to 3, 4, or 5, or an immunoreaction, or
An immunoassay method comprising immunoreacting a sample containing the substance to be measured in the presence of the nonspecific reaction inhibitor according to 3, 4, or 5.