JP2006038823A - Nonspecific reaction inhibitor, inhibition method of nonspecific reaction, immunological measuring method, and immunological measuring reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nonspecific reaction inhibitor and an inhibition method of nonspecific reaction for immunological measurement capable of inhibiting, in immunological measurement of a measuring object in a sample, a nonspecific reaction which cannot be prevented even by contact of a blood component of a producing animal species of an antibody used for this immunological measurement with the sample, thereby preventing a wrong measurement result by a nonspecific reaction by a heterophil antibody or the like, and providing a precise measurement result, an immunological measuring method of a measuring object in a sample, and an immunological measuring reagent for a measuring object in a sample. <P>SOLUTION: A blood component of an animal species that is a bovine species and different from the producing animal species of the antibody used for the immunological measurement is brought into contact with the sample, whereby the nonspecific reaction which cannot be inhibited even by the contact of the blood component of the producing animal species of the antibody used for the immunological measurement with the sample is inhibited. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to an inhibitor of nonspecific reaction in immunological measurement, a method of suppressing nonspecific reaction in immunological measurement using the inhibitor, and an immunological analysis of a measurement target substance in a sample using the inhibitor. The present invention relates to a measurement method and an immunological measurement reagent for a substance to be measured in a sample containing the inhibitor.
The present invention is useful in the field of life science such as clinical examination, immunology and medicine, the field of chemistry such as analytical chemistry, the field of food hygiene, and the field of environmental health.

  A method for measuring a trace amount of a target substance contained in a sample using a reaction between substances having specific affinity such as antigen and antibody, sugar and lectin, nucleotide chain and complementary nucleotide chain, and ligand and receptor. Various measuring reagents are known.

  This is the presence or absence of binding between the measurement target substance contained in the sample and the specific binding substance (specific binding substance for the measurement target substance) that specifically binds to the measurement target substance. By measuring, the presence or absence of a measurement target substance contained in a sample is measured [qualitative measurement], or the content (concentration) thereof is measured [quantitative measurement].

As a measurement method and measurement reagent for a measurement target substance using a specific binding substance that specifically binds to these measurement target substances, in particular, an antigen-antibody reaction between an antigen and an antibody (immunological reaction, immune reaction) ) Is used frequently (immunological assay reagent).
As this immunological measurement method (immunological measurement reagent), latex turbidimetry using latex particles as a carrier; latex particles, organic polymer particles, inorganic particles, metal particles, magnetic particles or erythrocytes are used as carriers. Indirect agglutination measurement method used; enzyme immunoassay, fluorescence immunoassay or luminescence immunoassay using microtiter plates, beads, particles, test tubes or containers as carriers; or cellulose Alternatively, an immunochromatography method using a nonwoven fabric or the like as a carrier is frequently used.
Further, as this immunological measurement method (immunological measurement reagent), a specific method for a measurement target substance (test substance) described in JP-A-9-229936, JP-A-10-132919, etc. A method of using a carrier having a surface on which a binding substance is immobilized and coated, and particles on which a specific binding substance for the substance to be measured is immobilized, and measuring whether the particles are collected on the surface of the carrier (reagents) And the like.

However, in these immunological measurement methods (immunological measurement reagents), depending on the sample to be measured, the target substance is present even though the target substance is not present in the sample due to a non-specific reaction. Measurement results indicating that this is sometimes obtained (false positive, false positive).
On the other hand, a measurement result indicating that the measurement target substance does not exist may be obtained even though the measurement target substance exists in the sample due to the non-specific reaction (false negative, false negative).
Obtaining an erroneous measurement result due to this non-specific reaction has been a serious problem in the field of clinical laboratory tests, leading to misdiagnosis of diseases such as patients.

  This non-specific reaction is caused by the measurement target substance contained in the sample binding to a substance or carrier other than the specific binding substance for the measurement target substance at the time of measurement, or included in the sample. It is considered that a substance other than the measurement target substance is caused by binding with a specific binding substance for the measurement target substance.

As a method for suppressing this non-specific reaction, various methods have been provided so far.
For example, Japanese Patent Application Laid-Open No. 58-144748 (Patent Document 1) discloses a technique for adding bovine serum albumin, horse serum albumin or the like to a reagent in order to suppress nonspecific reaction. Japanese Patent Laid-Open No. 8-105897 (Patent Document 2) discloses a technique of adding sodium chloride in order to suppress a nonspecific reaction.

  Japanese Patent Application Laid-Open No. 8-176195 (Patent Document 3) discloses a method of using reduced albumin chemically modified to suppress nonspecific reaction as a blocking agent.

  Further, Japanese Patent Publication No. 9-510289 (Patent Document 4) discloses a technique using avidin, streptavidin or a derivative thereof for suppressing non-specific reaction.

  JP-A-59-99257 (Patent Document 5) describes host bacterial components which are the same as those used for gene recombination in order to suppress nonspecific reactions but have not been subjected to gene recombination. The technique used is shown.

  Japanese Patent Application Laid-Open No. 8-43392 (Patent Document 6) discloses a technique using a cultured cell component that does not contain a gene encoding an antigen and incorporates the same type of vector as that used for antigen production. .

Among nonspecific reactions in immunological measurements, nonspecific reactions with heterophilic antibodies are known.
This heterophilic antibody is an antibody that exists in human serum or the like and binds nonspecifically to animal immunoglobulins.
As this heterophilic antibody, a human anti-mouse antibody (HAMA) or the like is known.
For example, when an antibody against an HBs antigen is produced in a mouse and this antibody is used as a solid-phased antibody and an enzyme-labeled antibody and the HBs antigen in the sample is measured by the sandwich method of the enzyme immunoassay method, When a human anti-mouse antibody that is an antibody is present, binding of “solid phase-mouse anti-HBs antibody-human anti-mouse antibody-mouse anti-HBs antibody-labeling enzyme” occurs even if no HBs antigen is present in the sample, As a result, a signal is generated, and a measurement result indicating that the HBs antigen is present in the sample is obtained (false positive, false positive).

  As a method for suppressing the nonspecific reaction caused by this heterophilic antibody, in order to prevent the nonspecific reaction caused by the human anti-mouse antibody (HAMA) when the mouse antibody is used for measurement of the measurement target substance, the mouse is used in the measurement reagent. A technique for absorbing human anti-mouse antibody by adding serum is shown in the literature (Non-Patent Document 1).

  In addition, in order to prevent non-specific reaction due to human anti-mouse antibody (HAMA) as described above, mouse serum can be added to the measurement reagent to absorb human anti-mouse antibody and suppress non-specific reaction. Description of examples in which horse serum, sheep serum, and rabbit serum were not added to suppress non-specific reaction, but bovine serum, sheep serum, rat serum, or guinea pig serum were added instead of mouse serum In some cases, a non-specific reaction suppression effect similar to that at the time of addition of mouse serum was shown, but when cat serum, dog serum, rabbit serum or pigeon serum was added, none of the examples showed an inhibitory effect, and mice Serum from other animal species such as sheep and cattle or hybridoma-derived mouse IgG (immunog Brin) there is described that is used have been disclosed in the literature (Non-Patent Document 2).

  Furthermore, Japanese Patent Application Laid-Open No. 9-49840 (Patent Document 7) discloses a technique for suppressing non-specific reactions caused by heterophilic antibodies using α-globulin.

JP 58-144748 A

JP-A-8-105897

JP-A-8-176195

Japanese National Patent Publication No. 9-510289

JP 59-99257 A

JP-A-8-43392

Japanese Patent Laid-Open No. 9-49840

Medical Technology, Vol. 25, No. 3, pp. 174-178, published in 1997

Clinical Chemistry, Volume 23 Supplement, 175a-7 to 175a-8, 1994

As described above, in the immunological measurement method (immunological measurement reagent), depending on the sample to be measured, the measurement target substance exists despite the absence of the measurement target substance in the sample due to non-specific reaction. Measurement results may be obtained (false positive, false positive), or measurement results indicating that the measurement target substance does not exist despite the presence of the measurement target substance in the sample. (False negative, false negative).
As this non-specific reaction, a non-specific reaction by a heterophilic antibody such as a human anti-mouse antibody (HAMA) has been known.

  While the present inventors are pursuing research aiming at suppression of non-specific reaction in immunological measurement of a measurement target substance in a sample, non-specificity by a conventional human anti-mouse antibody (HAMA) has been carried out. Non-specific reaction that cannot be prevented by the method of absorbing human anti-mouse antibody by adding mouse serum to the measurement reagent in order to prevent the chemical reaction, that is, the blood component of the animal species producing the antibody used for immunological measurement It has been found that non-specific reactions that cannot be prevented by contact with the sample are present at a relatively high frequency.

  Therefore, the subject of the present invention is non-specificity that cannot be prevented by contacting the blood component of the animal species producing the antibody used in the immunological measurement with the sample in the immunological measurement of the measurement target substance in the sample. It is intended to provide a means that can suppress the target reaction, thereby preventing an erroneous measurement result from being obtained due to a non-specific reaction caused by a heterophilic antibody or the like, so that an accurate measurement result can be obtained. The purpose is to make it.

  As a result of diligent studies to solve the above problems, the present inventors have determined that antibodies that are bovine species and are used for immunological measurement as inhibitors of nonspecific reactions in immunological measurement. The present inventors have found that the above problems can be solved by using blood components of animal species different from the production animal species of the present invention, and have completed the present invention.

That is, the present invention includes the following inventions.
(1) An inhibitor of a nonspecific reaction in an immunological measurement of a measurement target substance in a sample, which is a bovine species and is different from an animal producing animal species used for the immunological measurement It consists of a blood component of a species, and by bringing the inhibitor of the nonspecific reaction into contact with the sample, the blood component of the animal species producing the antibody used for the immunological measurement is suppressed even when it is brought into contact with the sample. An inhibitor of nonspecific reaction in immunoassay, characterized by being able to suppress nonspecific reaction that cannot be performed.

(2) The inhibitor of a nonspecific reaction according to (1) above, wherein the nonspecific reaction is due to a heterophilic antibody.

(3) The inhibitor of nonspecific reaction according to (1) or (2) above, wherein the blood component is blood, plasma, serum, or immunoglobulin.

(4) The inhibitor of nonspecific reaction according to any one of (1) to (3), wherein the bovine species is goat, sheep or cow.

(5) The inhibitor of nonspecific reaction according to any one of (1) to (4), wherein the antibody used for the immunological measurement is a monoclonal antibody.

(6) An inhibitor of a nonspecific reaction in an immunological measurement of a measurement target substance in a sample, wherein the nonspecific reaction samples a blood component of an animal producing animal species used for the immunological measurement The inhibitor of the non-specific reaction is a bovine species and consists of blood components of an animal species different from the antibody-producing animal species used for the immunological measurement. An inhibitor of non-specific reaction in immunological measurement, characterized by

(7) The inhibitor of a nonspecific reaction according to (6) above, wherein the nonspecific reaction is caused by a heterophilic antibody.

(8) The inhibitor of nonspecific reaction according to (6) or (7), wherein the blood component is blood, plasma, serum, or immunoglobulin.

(9) The inhibitor of nonspecific reaction according to any one of (6) to (8), wherein the bovine species is goat, sheep or cow.

(10) The inhibitor of nonspecific reaction according to any one of (6) to (9), wherein the antibody used for the immunological measurement is a monoclonal antibody.

(11) A method for suppressing a nonspecific reaction in an immunological measurement of a substance to be measured in a sample, which is a bovine species and is different from an animal producing animal species used for the immunological measurement By contacting the blood component of the species with the sample, it suppresses a nonspecific reaction that cannot be suppressed even if the blood component of the animal species producing the antibody used for the immunological measurement is brought into contact with the sample, Method for suppressing non-specific reaction in immunological measurement.

(12) The method for suppressing a nonspecific reaction according to (11) above, wherein the nonspecific reaction is caused by a heterophilic antibody.

(13) The method for suppressing a nonspecific reaction according to (11) or (12) above, wherein the blood component is blood, plasma, serum, or immunoglobulin.

(14) The method for suppressing a nonspecific reaction according to any one of (11) to (13), wherein the bovine species is a goat, a sheep, or a cow.

(15) The method for suppressing a nonspecific reaction according to any one of (11) to (14), wherein the antibody used for the immunological measurement is a monoclonal antibody.

(16) A method for suppressing a non-specific reaction in an immunological measurement of a measurement target substance in a sample, wherein the non-specific reaction samples a blood component of an antibody-producing animal species used for the immunological measurement. Samples of blood components of animal species that are bovine species whose method of suppressing the nonspecific reaction is different from the animal-producing animal species used for the immunological measurement. A method for suppressing a non-specific reaction in an immunological measurement, which is characterized in that it is brought into contact with an immunological assay.

(17) The method for suppressing a nonspecific reaction according to (16), wherein the nonspecific reaction is caused by a heterophilic antibody.

(18) The method for suppressing a nonspecific reaction according to (16) or (17), wherein the blood component is blood, plasma, serum, or immunoglobulin.

(19) The method for suppressing a nonspecific reaction according to any one of (16) to (18), wherein the bovine species is a goat, a sheep, or a cow.

(20) The method for suppressing a nonspecific reaction according to any one of (16) to (19), wherein the antibody used for the immunological measurement is a monoclonal antibody.

(21) A method for immunological measurement of a substance to be measured in a sample, wherein a blood component of an animal species which is a bovine species and is different from an animal producing animal species used for the immunological measurement is used as a sample. By contacting, the nonspecific reaction that cannot be suppressed even if the blood component of the animal species producing the antibody used for the immunological measurement is brought into contact with the sample is suppressed. Immunological measurement method.

(22) The immunological measurement method for a substance to be measured in a sample according to (21), wherein the nonspecific reaction is caused by a heterophilic antibody.

(23) The immunological measurement method for a substance to be measured in a sample according to (21) or (22), wherein the blood component is blood, plasma, serum, or immunoglobulin.

(24) The immunological measurement method for a substance to be measured in a sample according to any one of (21) to (23), wherein the bovine species is a goat, sheep or cow.

(25) The immunological measurement method for a substance to be measured in a sample according to any one of (21) to (24), wherein the antibody used for the immunological measurement is a monoclonal antibody.

(26) An immunoassay reagent for a substance to be measured in a sample, which contains a blood component of an animal species that is a bovine species and is different from the animal species that produce the antibody used for the immunological measurement The immunology of the substance to be measured in the sample is characterized by suppressing non-specific reactions that cannot be suppressed even when the blood component of the animal species producing the antibody used for the immunological measurement is brought into contact with the sample. Measurement reagent.

(27) The reagent for immunological measurement of a substance to be measured in a sample according to the above (26), wherein the nonspecific reaction is caused by a heterophilic antibody.

(28) The reagent for immunological measurement of a substance to be measured in a sample according to (26) or (27), wherein the blood component is blood, plasma, serum, or immunoglobulin.

(29) The reagent for immunological measurement of a substance to be measured in a sample according to any one of (26) to (28), wherein the bovine species is goat, sheep or cow.

(30) The reagent for immunological measurement of a substance to be measured in a sample according to any one of (26) to (29), wherein the antibody used for the immunological measurement is a monoclonal antibody.

  Inhibitor of nonspecific reaction in immunological measurement of the present invention, method of suppressing nonspecific reaction in immunological measurement of the present invention, immunological measurement method of a substance to be measured in a sample of the present invention, and the present The reagent for immunological measurement of the substance to be measured in the sample of the invention comprises contacting the blood component of the animal species producing the antibody used for the immunological measurement with the sample in the immunological measurement of the substance to be measured in the sample. It is possible to suppress non-specific reactions that cannot be prevented even by this, thereby preventing erroneous measurement results from being obtained due to non-specific reactions with heterophilic antibodies, etc., and accurate measurement results Is obtained.

1. Nonspecific reaction Inhibitor of nonspecific reaction in the first immunological measurement of the present invention, inhibitor of nonspecific reaction in the second immunological measurement of the present invention, first of the present invention A method for suppressing a nonspecific reaction in immunological measurement of the present invention, a method for suppressing a nonspecific reaction in the second immunological measurement of the present invention, a method for immunological measurement of a measurement target substance in a sample of the present invention, and An immunological measurement reagent for a measurement target substance in a sample of the present invention (hereinafter referred to as an inhibitor of the present invention) is a bovine species in the immunological measurement of a measurement target substance in a sample and The blood component of the animal species different from the animal species producing the antibody used for the immunological measurement is brought into contact with the sample, whereby the blood component of the animal species producing the antibody used for the immunological measurement is brought into contact with the sample. To suppress nonspecific reactions that cannot be suppressed It can be done.

  For example, when immunological measurement of a substance to be measured in a sample is performed using a mouse antibody (polyclonal antibody and / or monoclonal antibody), non-specificity that cannot be suppressed even when the mouse blood component is brought into contact with the sample The target reaction can be suppressed.

  The inhibitor and the like of the present invention are suitable for nonspecific reactions caused by heterophilic antibodies.

2. Blood component The blood component of the animal species that is a bovine species and is different from the antibody-producing animal species used for the immunological measurement in the inhibitor of the present invention will be described in detail below.

(1) Bovidae species Although it is a bovine species, any species that belongs to the Bovidae Bovidae family is applicable without particular limitation.

  Examples of the animal species belonging to the bovine family include animal species belonging to cattle, animal species belonging to goats and sheep, and animal species belonging to antelopes.

  Examples of animal species belonging to the bovines include cattle.

  Examples of animal species belonging to the goats and sheep include goats and sheep.

  Examples of animal species belonging to this antelope include antelopes.

In the inhibitor and the like of the present invention, the bovine species is preferably goat, sheep or cow.
Particularly preferred is a goat.
Moreover, it is especially preferable that it is a cow.

(2) Antibody used in the immunological measurement The antibody used in the immunological measurement, but the antibody used in the immunological measurement of the substance to be measured in the sample. Thus, immunological measurement of the measurement target substance in the sample is performed.

  Examples of this antibody include antisera, polyclonal antibodies, monoclonal antibodies, and fragments thereof (antigens capable of binding to an antigen; such as F (ab) '2 or Fab').

  In the inhibitor and the like of the present invention, this antibody is particularly suitable when it is a monoclonal antibody or a fragment thereof (having an antigen-binding ability; for example, F (ab) '2 or Fab').

(3) Antibody producing animal species used for the immunological measurement Antibody producing animal species used for the immunological measurement, but used for immunological measurement of the measurement target substance in the sample described above Is the species of animal that produced
When the antibody is produced from a cell, the antibody-producing animal species is the animal species from which the antibody-producing cell is derived.
In addition, when the antibody is produced from a fused cell obtained by fusing a plurality of cells, it refers to each animal species from which each cell before fusion is derived.
Examples of animal species that produce this antibody include mice, rats, horses, rabbits, guinea pigs, goats, sheep, and cows. Furthermore, humans can be mentioned as animal species producing this antibody.

(4) Animal species different from the antibody-producing animal species used in the immunological measurement The animal species is different from the antibody-producing animal species used in the immunological measurement. An animal species different from the animal species producing the antibody used for immunological measurement of the substance.
For example, when the antibody-producing animal species is a mouse, the animal species different from the antibody-producing animal species is an animal species other than a mouse, and examples include goats, sheep or cows. .

(5) Animal species that are bovine species and are different from the antibody-producing animal species used for the immunological measurement What are bovine species and antibody-producing animal species that are used for the immunological measurement Although it is a different animal species, it is a bovine species detailed in (1) above, and is used for immunological measurement of a measurement target substance in a sample detailed in (2) to (4) above The animal species is different from the antibody-producing animal species.

In other words, it is an animal species that satisfies both of the following two conditions.
(B) It is a bovine species.
(B) The animal species is different from the antibody-producing animal species used for the immunological measurement of the measurement target substance in the sample.

For example, this animal species may be any animal species as long as it is a bovine species if the antibody producing animal species used for immunological measurement of the measurement target substance in the sample is not a bovine species.
When the antibody producing animal species used for immunological measurement of the measurement target substance in the sample is a bovine species, it may be any bovine species other than the antibody producing animal species.

(6) Blood component In the inhibitor of the present invention, the blood component is a blood component of an animal species that is a bovine species and is different from the antibody-producing animal species used for the immunological measurement. It is a blood component of an animal species that is a bovine species described in detail and that is different from the antibody-producing animal species used in the immunological measurement.

Examples of the blood component of the animal species include blood, components contained in the blood, and components derived from the blood.
More specifically, examples of the blood component include blood, plasma, and serum.

Examples of the blood component include immunoglobulins.
Examples of the immunoglobulin include immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), and immunoglobulin E (IgE).

In the inhibitor of the present invention, the blood component of an animal species that is a bovine species and is different from the antibody-producing animal species used in the immunological measurement is preferably an immunoglobulin, More preferred are immunoglobulins of the same class as the antibody used for the measurement.
As a class of antibodies used for immunological measurement, immunoglobulin G (IgG) is generally used. Therefore, immunoglobulin G (IgG) is particularly preferable as the blood component.

  The blood component of an animal species that is a bovine species and is different from the antibody-producing animal species used in the immunological measurement used in the inhibitor of the present invention may be one kind, Alternatively, a plurality of types may be used.

3. Contact with a sample In the inhibitor of the present invention, the blood component of an animal species that is a bovine species and is different from the animal species that produces the antibody used for the immunological measurement, or a non-specific comprising the blood component The contact of the reaction inhibitor with the sample will be described in detail below.

  In the inhibitor of the present invention, the blood component of an animal species that is a bovine species and is different from the animal species that produce the antibody used for the immunological measurement, or suppression of a nonspecific reaction comprising this blood component By bringing the agent into contact with the sample, the above-mentioned non-specific reaction can be suppressed.

  This contact is caused by a blood component of a bovine species that is different from the animal species that produces the antibody used in the immunological measurement, or a nonspecific reaction inhibitor comprising the blood component, a sample, Any method may be used as long as it can be contacted.

At the time of this contact, the concentration of the blood component of the bovine species that is different from the animal species that produces the antibody used for the immunological measurement, or the concentration of the nonspecific reaction inhibitor comprising this blood component. However, depending on conditions such as the mixing ratio and contact time between this blood component or inhibitor and the sample, it cannot be said in general, but when this blood component or inhibitor consists of blood, serum, or plasma, Is preferably in the range of 0.1% (w / v) to 20% (w / v), and preferably in the range of 0.5% (w / v) to 10% (w / v). More preferably, it is in a range of 1% (w / v) to 5% (w / v).
Moreover, when this blood component or inhibitor consists of immunoglobulins, it is usually preferably in the range of 0.05 μg / mL to 20 mg / mL, and in the range of 0.25 μg / mL to 10 mg / mL. Is more preferable, and is particularly preferably in the range of 0.5 μg / mL to 2.5 mg / mL.

  This contact time is different depending on the conditions such as the concentration of this blood component or inhibitor, the mixing ratio of this blood component or inhibitor and the sample, etc., but it is usually 1 minute or more. It is preferable that it is in the range of 15 minutes to 3 hours.

  Although it is the temperature at the time of contact, there is no particular limitation, but if the temperature becomes too high, the components involved in the measurement, particularly the components made of protein, will be denatured, deactivated or decomposed, and accurate measurement cannot be performed. Therefore, it is preferable to set it to 60 degrees C or less.

4). Sample In the inhibitor or the like of the present invention, a sample means a substance whose measurement target substance may be present, and the presence / absence of the measurement target substance or the content (concentration) to be measured.

  Examples thereof include body fluids such as human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, and the like.

5. Substance to be measured In the inhibitor or the like of the present invention, the substance to be measured is a substance to be measured for the presence or absence or content (concentration) in the sample.

  As the substance to be measured, any substance can be used as long as it can be measured by an immunological measurement method or an immunological measurement reagent.

  Examples of the measurement target substance include organic substances such as proteins, carbohydrates, lipids, and nucleic acids, or inorganic substances such as metals.

  More specifically, for example, virus-related antigens or antibodies such as HBs antigen, anti-HBs antibody, HBe antigen, anti-HBe antibody, anti-HBc antibody, anti-HCV antibody, anti-HIV antibody, anti-ATLV antibody; E. coli O157 antigen, Bacteria-related antigens or antibodies such as anti-treponema pallidum antibody, anti-mycoplasma antibody, anti-streptridine O antibody (ASO); immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), or immunity Immunoglobulins such as globulin E (IgE); C-reactive protein (CRP), α1-acid glycoprotein, haptoglobin, complement C3, complement C4, inflammation markers such as rheumatoid factor; α-fetoprotein, CEA, CA19-9 Tumor markers such as: human placental chorionic gonadotropin and other hormones; alleles Allergen-related antigens or antibodies such as antigens and allergen-specific 1 gE antibodies; blood coagulation-related substances such as antithrombin III (ATIII); fibrinolytic substances (FDP), fibrinolytic substances such as D-dimer; ABO blood group Blood group-related antigens or antibodies such as antibodies and irregular antibodies; viral DNA or RNA; bacterial DNA or RNA; animal or plant DNA or RNA such as humans; other diseases such as lipoprotein (a) and ferritin Substances related to the above; drugs; metals; poisonous or deleterious substances.

As this substance to be measured, an antigen is suitable.
In particular, an antigen contained in a trace amount in a sample is preferable. This is because the smaller the concentration contained, the greater the influence of non-specific reaction, and the inhibitor of the present invention is useful.

6). Immunological measurement of a measurement target substance in a sample In the inhibitor of the present invention, the immunological measurement of a measurement target substance in a sample is performed using an immunological reaction (antigen-antibody reaction). Any measurement principle can be used as long as the measurement is performed.

  As a measurement principle of immunological measurement of a measurement target substance in this sample, for example, latex turbidimetry; indirect aggregation reaction measurement method such as latex agglutination reaction measurement method; enzyme immunoassay method, fluorescence immunoassay method, radioimmunoassay Or immunoassay using a labeling substance such as luminescence immunoassay, that is, labeled immunoassay; Western blotting; ELSA method (Enzyme-Linked Ligandsorbent Assay) [Thromb. Haemost. 79, 767-772, 1998, and WO 98/23963]; or JP-A-9-229936 and JP-A-10-132919, etc. Examples thereof include a carrier having a surface on which a specific binding substance is fixed and coated, and a measurement method using particles on which a specific binding substance for a measurement target substance (test substance) is fixed.

  The labeled immunoassay is the above-described method, and the present invention can be applied to any method such as the sandwich method, the competitive method, or the homogeneous method (homogeneous method).

7). Inhibitor of nonspecific reaction in immunological measurement Inhibitor of nonspecific reaction in first immunological measurement of the present invention, and Inhibitor of nonspecific reaction in second immunological measurement of the present invention Both of them are blood species of an animal species that is a bovine species and is different from the antibody-producing animal species used for the immunological measurement, that is, it is a bovine species and the immunology concerned. Although it contains a blood component of an animal species different from the antibody-producing animal species used for the measurement, it may contain other components.

8). Inhibition of non-specific reaction in immunological measurement Both the method for suppressing non-specific reaction in the first immunological measurement of the present invention and the method of suppressing non-specific reaction in the second immunological measurement of the present invention , After the contact of the sample with the blood component of the animal species that is a bovine species and is different from the animal species that produces the antibody used for the immunological measurement, It may be performed or may be performed simultaneously with the contact between the blood component and the sample.

9. Immunological measurement method for measurement target substance in sample In the immunological measurement method for a measurement target substance in a sample of the present invention, an animal that is a bovine species and produces an antibody used for the immunological measurement. A blood component of an animal species different from the species is brought into contact with the sample. Other than that, the measurement may be performed according to a normal operation procedure in the measurement principle of the immunological measurement method.

  In the immunological measurement method for a substance to be measured in a sample of the present invention, the immunological measurement of the substance to be measured in the sample is performed by producing an antibody that is a bovine species and is used for the immunological measurement. It may be performed after the contact between the blood component of the animal species different from the animal species and the sample, or may be performed simultaneously with the contact between the blood component and the sample.

  The measurement operation in the immunological measurement method of the present invention may be performed by a method or using an apparatus such as an analyzer.

  Further, the measurement operation in the immunological measurement method of the present invention may be performed by a one-step method (one-reagent method) or by a method performed by a plurality of operation steps such as a two-step method (two-reagent method). May be.

  The following is an example of measuring an HBs antigen in a sample using an immunological measurement method (immunological measurement reagent) of a measurement target substance in a sample based on an enzyme immunoassay (ELISA method) as a measurement principle. A specific description will be given.

(1) The following are prepared as measuring reagents for HBs antigen in a sample.
“Washing solution”: phosphate buffered saline containing nonionic surfactant “nonspecific reaction inhibitor-containing solution”: phosphate buffered saline containing 1% (w / v) goat serum and casein sodium “ Antibody-immobilized microplate ": 96-well microtiter plate immobilized with mouse anti-HBs monoclonal antibody" Peroxidase-labeled antibody solution ": phosphate-buffered saline containing peroxidase-labeled mouse anti-HBs monoclonal antibody and sodium caseinate “Coloring solution”: Buffer solution containing O-phenylenediamine and hydrogen peroxide “Reaction stop solution”: Sulfuric acid aqueous solution

(2) A certain amount of the “non-specific reaction inhibitor-containing solution” is added to the well of the “antibody-immobilized microplate”.

(3) A predetermined amount of a sample such as serum is added to the well, and the sample is allowed to stand at room temperature for a certain period of time to bring the nonspecific reaction inhibitor made of goat serum into contact with the sample in the well. Is also contacted with an anti-HBs antibody immobilized on a microplate.

(4) The liquid in the well is removed by suction and washed with the “cleaning liquid”.

(5) A certain amount of the “peroxidase-labeled antibody solution” is added to the well, left at room temperature for a fixed time, and labeled with peroxidase on the HBs antigen bound to the microplate via the anti-HBs antibody. The reacted anti-HBs antibody is contacted and reacted.
Thus, when HBs antigen is present in the sample, binding of “well of microplate-anti-HBs antibody-HBs antigen-anti-HBs antibody-peroxidase” occurs.

(6) The liquid in the well is removed by suction and washed with the “cleaning liquid”.

(7) A certain amount of the “color developing solution” is added to the well and allowed to stand at room temperature for a certain period of time to react peroxidase bound to the microplate with o-phenylenediamine and hydrogen peroxide.

(8) After a certain time, a certain amount of the “reaction stop solution” is added to the well to stop the reaction.

(9) The absorbance at 490 nm of the liquid in the well is measured with a microplate reader, and the concentration of HBs antigen contained in the sample is calculated from this absorbance.

10. Reagent for immunological measurement of a substance to be measured in a sample In the reagent for immunological measurement of a substance to be measured in a sample of the present invention, an animal that is a bovine species and produces an antibody used for the immunological measurement. It is characterized by containing blood components of an animal species different from the species.

  The immunological measurement reagent of the substance to be measured in the sample of the present invention may be composed of one measurement reagent, or composed of two or more plural measurement reagents. Also good.

  When it is composed of one measurement reagent, the one measurement reagent is a bovine species and contains blood components of an animal species different from the antibody-producing animal species used in the immunological measurement cage. In addition, it contains a measurement component for performing an immunological measurement of a substance to be measured in a sample according to the measurement principle of the immunological measurement reagent.

  In the case where it is composed of two or more measurement reagents, the blood component of an animal species that is a bovine species and is different from the antibody-producing animal species used for the immunological measurement is at least Although it must be contained in one measuring reagent, it may be contained in two or more measuring reagents constituting the immunological measuring reagent as necessary.

  When it is composed of two or more measurement reagents, the measurement components for performing the immunological measurement of the measurement target substance in the sample according to the measurement principle of the immunological measurement reagent are included in one measurement reagent. Or may be included in two or more measurement reagents as appropriate.

  Furthermore, when it is composed of two or more measurement reagents, the blood component of an animal species that is a bovine species and is different from the animal species that produce the antibody used in the immunological measurement, It may coexist with the measurement component or may be contained independently.

  The concentration of blood components of a bovine species that is included in the immunological measurement reagent of the substance to be measured in this sample and that is different from the animal species that produce the antibody used for the immunological measurement. Depending on conditions such as the mixing ratio and contact time between the measurement reagent containing the blood component and the sample, it cannot be generally stated. However, when this blood component is composed of blood, serum, or plasma, it is usually 0. Preferably in the range of 1% (w / v) to 20% (w / v), more preferably in the range of 0.5% (w / v) to 10% (w / v), A range of 1% (w / v) to 5% (w / v) is particularly preferable.

  When this blood component is composed of immunoglobulin, it is usually preferably in the range of 0.05 μg / mL to 20 mg / mL, more preferably in the range of 0.25 μg / mL to 10 mg / mL. , Particularly preferably in the range of 0.5 μg / mL to 2.5 mg / mL.

  Although it is a measurement component for performing immunological measurement of a measurement target substance in a sample, when measuring by indirect aggregation reaction measurement method, for example, latex particles, organic polymer particles, inorganic particles, metal particles, magnetic Immobilized antibody or antigen capable of binding to the substance to be measured on a carrier such as particles or erythrocytes, and in some cases, a microtiter plate (microtiter plate immobilized with an antibody or antigen capable of binding to the substance to be measured) Plate) or a sample diluent.

  When measurement is performed by a labeled immunoassay method such as an enzyme immunoassay method, a fluorescence immunoassay method, or a luminescent immunoassay method, for example, measurement is performed on a carrier such as a microtiter plate (microplate), beads, particles, test tube or container. Immobilized antibody or antigen that can bind to the target substance, Microtiter plate (microplate), beads, particles, test substance or its analog immobilized on a carrier such as a test tube or container, An antibody or antigen that can be bound to a substance to be measured and an enzyme, a fluorescent substance or a labeling substance such as a substance related to a luminescence reaction, or an antibody or antigen that can bind to a human antibody, an enzyme, From immobilized substances such as fluorescent substances or substances related to luminescent reactions, from labeled substances Substances involved in the reaction to produce the Gunaru, or can be given sample diluent and the like.

  When the measurement is performed by latex agglutination reaction, for example, there can be mentioned latex particles on which an antibody or an antigen capable of binding to the substance to be measured is immobilized, or a sample diluent.

  When measurement is performed by an immunochromatography method, for example, an antibody or antigen that can be bound to a substance to be measured is immobilized on a carrier such as cellulose or non-woven fabric, or an antibody or antigen that can be bound to a substance to be measured. There may be mentioned, for example, an enzyme, a fluorescent substance or a substance that is immobilized with a labeling substance such as a substance relating to a luminescent reaction, or a sample diluent.

  Furthermore, when the measurement is performed by the immunological measurement method (immunological measurement reagent) described in JP-A-9-229936 and JP-A-10-132919, the specific binding to the substance to be measured Examples thereof include a carrier having a surface on which a substance is immobilized and coated, particles on which a specific binding substance for the substance to be measured is immobilized, or a sample diluent.

  In addition, various aqueous solvents can be used as a solvent used in the immunological measurement reagent of the measurement target substance in the sample of the present invention.

  Examples of the aqueous solvent include purified water, physiological saline, or various buffer solutions such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, or phosphate buffered saline. .

  About the pH of this buffer solution, an appropriate pH may be selected and used as appropriate. Although there is no particular limitation, it is general to select and use a pH within the range of pH 5 to pH 11.

  The reagent for immunological measurement of the measurement target substance in the sample of the present invention includes protein such as casein or a salt thereof in addition to the measurement component for performing immunological measurement of the measurement target substance in the sample. Various salts; various saccharides; skim milk powder; various animal sera such as normal rabbit serum; various preservatives such as sodium azide or antibiotics; activating substances; reaction promoting substances; sensitivity increasing substances such as polyethylene glycol; A specific reaction inhibitor; or one or more of various surfactants such as a nonionic surfactant, an amphoteric surfactant or an anionic surfactant may be appropriately included.

  And the density | concentration at the time of making these include in the immunological measurement reagent of this invention is although it does not specifically limit, 0.001-10% (W / V) is preferable, Especially 0.01-5% ( W / V) is preferred.

  Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; betaine acetate, etc. Amphoteric surfactants; or polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether acetic acid Anionic surfactants, such as and the like.

In addition, the immunological measurement reagent of the measurement target substance in the sample of the present invention can be sold alone or used for measurement of the measurement target substance in the sample.
Moreover, the immunological measurement reagent of the measurement target substance in the sample of the present invention can be sold in combination with other reagents or used for measurement of the measurement target substance in the sample.

  Examples of the other reagent include a reagent diluent, a confirmation reagent for measurement reaction, a positive sample, a negative sample, a reagent containing a substance for performing calibration (calibration), and the like.

The present invention will be further described below with reference to examples.
In addition, this invention is not limited by these.

[Example 1] Confirmation of inhibitory effect of non-specific reaction The inhibitory effect of non-specific reaction in the immunological measurement of the measurement target substance in the sample, such as the inhibitor of the present invention, was confirmed.

1. Immunological measurement reagent An immunological measurement reagent for the substance to be measured in the sample was prepared as follows.
(1) Preparation of anti-HBs antibody-immobilized microplate In each well of a 96-well microtiter plate (Sinotest), an anti-HBs monoclonal antibody solution [10 mM phosphate buffer (so that anti-HBs monoclonal antibody is 1 μg / mL) (added to pH 7.0)] (Sinotest) was dispensed in 50 μL aliquots and allowed to stand at 4 ° C. overnight.
Then, after removing the anti-HBs monoclonal antibody solution in each well by suction, 200 μL of 50 mM Tris buffer (pH 8.0) containing 0.5% (w / v) casein was added to each well at 37 ° C. It left still for 3 hours and performed the blocking process (masking process).
Thereafter, the solution in each well was removed by suction to prepare an anti-HBs antibody-immobilized microplate.

(2) Preparation of anti-HBs antibody-immobilized particle dispersion 3% (w / v) magnetic particle dispersion [trade name: Dynabeads M-450 uncoated, particle size: 4.5 μm, particle specific gravity: 1.5, particle Diameter c. v. : 5%] (Dynal) and anti-HBs monoclonal antibody solution [added to 10 mM phosphate buffer (pH 7.0) so that anti-HBs monoclonal antibody is 0.1 mg / mL] (Sinotest) ) Was mixed with 1 mL, and allowed to stand at 37 ° C. for 30 minutes.
Thereafter, 40 mL of 50 mM Tris buffer (pH 8.0) containing 0.5% (w / v) casein was added thereto, and the mixture was allowed to stand at 37 ° C. for 30 minutes to perform blocking treatment (masking treatment).
Next, the magnetic particles were washed with 50 mM Tris buffer (pH 8.0) containing 0.5% (w / v) casein.
Thereafter, the particles are re-dispersed in 50 mM Tris buffer (pH 8.0) containing 0.5% (w / v) casein so that the concentration of the magnetic particles is 0.05% (w / v). An antibody-immobilized particle dispersion was prepared.

(3) Preparation of sample diluent The following 15 types of sample diluents were prepared.
(I) Serum-free / sample diluent A 250 mM glycine buffer (pH 10.0) containing 0.5% (w / v) casein was prepared and used as a serum-free / sample diluent.

(B) 1% (w / v) mouse serum containing sample dilution solution 250 mM glycine buffer (pH 10.5) containing 1% (w / v) mouse serum (Veritas) and 0.5% (w / v) casein 0) was prepared and used as a sample diluent containing 1% (w / v) mouse serum.

(C) 5% (w / v) mouse serum-containing sample dilution 250 mM glycine buffer (pH 10.5) containing 5% (w / v) mouse serum (Veritas) and 0.5% (w / v) casein 0) was prepared and used as a sample diluent containing 5% (w / v) mouse serum.

(D) 1% (w / v) goat serum-containing sample diluent 250% glycine buffer containing 1% (w / v) goat serum (Japan Biomaterials Center) and 0.5% (w / v) casein (PH 10.0) was prepared and used as a sample diluent containing 1% (w / v) goat serum.

(E) 5% (w / v) goat serum-containing sample dilution solution 250 mM glycine buffer containing 5% (w / v) goat serum (Japan Biomaterials Center) and 0.5% (w / v) casein (PH 10.0) was prepared and used as a sample diluent containing 5% (w / v) goat serum.

(F) 1% (w / v) bovine serum containing sample dilution solution 250 mM glycine buffer containing 1% (w / v) bovine serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample diluent containing 1% (w / v) bovine serum.

(G) 5% (w / v) bovine serum containing sample dilution solution 250 mM glycine buffer containing 5% (w / v) bovine serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution solution containing 5% (w / v) bovine serum.

(H) 1% (w / v) sheep serum containing sample dilution solution 250 mM glycine buffer containing 1% (w / v) sheep serum (Nippon Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution solution containing 1% (w / v) sheep serum.

(Li) 5% (w / v) sheep serum-containing sample dilution solution 250 mM glycine buffer containing 5% (w / v) sheep serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution solution containing 5% (w / v) sheep serum.

(Nu) 1% (w / v) equine serum-containing sample dilution 250 mM glycine buffer (pH 10) containing 1% (w / v) equine serum (JRH BIOSCIENCES) and 0.5% (w / v) casein 0.0) was prepared and used as a sample dilution containing 1% (w / v) horse serum.

(L) 5% (w / v) horse serum-containing sample dilution 250 mM glycine buffer (pH 10) containing 5% (w / v) horse serum (JRH BIOSCIENCES) and 0.5% (w / v) casein 0.0) was prepared and used as a sample dilution containing 5% (w / v) horse serum.

(V) 1% (w / v) porcine serum containing sample dilution solution 250% glycine buffer containing 1% (w / v) porcine serum (JR SCIENTIFIC) D and 0.5% (w / v) casein ( pH 10.0) was prepared and used as a sample diluent containing 1% (w / v) porcine serum.

(Wa) 5% (w / v) porcine serum-containing sample dilution 250 mM glycine buffer (pH 10) containing 5% (w / v) porcine serum (JR SCIENTIFIC) and 0.5% (w / v) casein 0.0) was prepared and used as a sample diluent containing 5% (w / v) porcine serum.

(F) 1% (w / v) rabbit serum containing sample dilution solution 250 mM glycine buffer containing 1% (w / v) rabbit serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample diluent containing 1% (w / v) rabbit serum.

(Yo) 5% (w / v) rabbit serum containing sample dilution solution 250 mM glycine buffer containing 5% (w / v) rabbit serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample diluent containing 5% (w / v) rabbit serum.

2. Samples 1,161 human sera (HBs antigen-negative sera) whose HBs antigen measurement results were negative were used as samples.

3. Serum-free / immunological measurement of HBs antigen in sample using sample diluent For 1,161 HBs antigen-negative human serum samples, the serum-free sample dilution solution was used to detect HBs antigen in the sample. Immunological measurements were taken.

(1) Each of the 1,161 samples of 2 was diluted 8-fold with the serum-free sample dilution solution of (3) (i) above.

(2) Next, about each diluted sample of said (1), 25 microliters was dripped at the well of the anti- HBs antibody fixed microplate.

(3) Thereafter, 25 μL of the anti-HBs antibody-immobilized particle dispersion was dropped into the well of (2).

(4) Next, the anti-HBs antibody-immobilized microplate subjected to the treatment of (3) was stirred with a microplate mixer [model: EM-33] (Tytec Corp.) for 1 minute.

(5) Then, the anti-HBs antibody fixed microplate which performed the process of said (4) was left still at room temperature for 1 hour.

(6) One hour after standing, the aggregated image of magnetic particles in each well of the anti-HBs antibody-immobilized microplate was observed and judged.
In this determination, a case where the magnetic particles seemed to spread inside the well was determined as positive, and a case where the magnetic particles appeared to gather at one point in the center of the well was determined as negative.

(7) As a result of this measurement, among 1,161 human serum samples originally negative for HBs antigen, 1,101 samples were determined to be negative, but in 60 samples, A positive result was obtained.
That is, in the immunological measurement of the HBs antigen in the sample using this serum-free / sample dilution solution, non-specific reaction occurred in 60 samples, resulting in false positives. The incidence of non-specific reactions (false positives) at this time was 5.17% (60 / 1,161 × 100).

4). Immunological measurement of HBs antigens in samples using mouse serum containing / diluted solution About 60 human serum samples that became false positive due to non-specific reaction in samples using mouse serum containing / sample diluted solution Immunological measurements of HBs antigens were performed.

(1) A determination result of positive in the above 3 was obtained, that is, each of 60 human serum samples that became false positive due to non-specific reaction was treated with 1% (w) of (b) in (1) above. / V) Mouse serum containing / diluted 8 times with sample diluent.

(2) The subsequent operations were performed as described in (3) (2) to (6) above.

(3) In addition, except that the sample diluent is changed to the sample diluent containing 5% (w / v) mouse serum of (c) in (1) above (1), as in (1) and (2) above Measurements were made.

(4) As a result of this measurement, the sample diluent used was 1% (w / v) mouse serum-containing / sample diluent or 5% (w / v) mouse serum-containing / sample diluent. Of the 60 human serum samples that became false positives due to non-specific reactions in the measurement of 51, 51 negative samples were obtained.

  That is, in the measurement of 3 above, for 51 samples out of 60 human serum samples that became false positives, mouse serum contained in the sample diluent was brought into contact with the sample to suppress non-specific reactions. It was possible to obtain an original negative determination result.

  However, among the 60 human serum samples that became false positive due to non-specific reaction in the measurement of 3 above, 9 samples were judged to be positive, and the mouse serum contained in the sample diluent was sampled. It was not possible to suppress nonspecific reactions even when contacted with.

That is, in these nine samples, even if the blood component (serum) of a mouse which is an animal species producing the antibody (anti-HBs monoclonal antibody) used in the immunoassay reagent of 1 is contacted with the sample, The nonspecific reaction could not be suppressed.
The incidence of non-specific reactions (false positives) at this time was 0.78% (9 ÷ 1,161 × 100).

5. Immunological measurement of HBs antigen in samples using goat serum containing sample diluent etc. Human serum sample that could not suppress nonspecific reaction even when mouse serum was brought into contact with the sample and became false positive Nine were subjected to immunological measurements of HBs antigens in the samples using sample dilutions containing goat serum, bovine serum, sheep serum, horse serum, pig serum, or rabbit serum, respectively.

(1) A positive determination result was obtained in 4 above, that is, a human serum sample 9 that was not positive even when a mouse blood component (serum) was brought into contact with the sample and became a false positive. Each of the pieces was diluted 8-fold with the sample diluent containing 1% (w / v) goat serum of (1) in (1) above.

(2) The subsequent operations were performed as described in (3) (2) to (6) above.

(3) In addition, the sample diluent is a sample diluent containing (5) 5% (w / v) goat serum in (3) of (1) above, (f) a sample containing 1% (w / v) bovine serum. Diluted solution, (g) 5% (w / v) bovine serum-containing sample diluent, (x) 1% (w / v) sheep serum-containing sample diluted solution, (ii) 5% (w / v) sheep Serum-containing sample dilution, (nu) 1% (w / v) equine serum-containing sample dilution, (l) 5% (w / v) equine serum-containing sample dilution, (wo) 1% (w / V) Pig serum containing sample diluent, (W) 5% (w / v) Pig serum containing sample diluent, (f) 1% (w / v) rabbit serum containing sample diluent, or (Y ) Measurement was performed as described in (1) and (2) above, except that the measurement was performed by sequentially changing to 5% (w / v) rabbit serum-containing sample diluent.

(4) The results of this measurement are shown in Table 1.

(5) From Table 1, the following can be understood.
Nine human serum samples that could not suppress non-specific reactions even when they were brought into contact with mouse blood components (serum) were tested as false positives. Goat blood components (serum) were brought into contact with the samples. In this case, 4 samples out of these were the original negative determination results. As a result, the incidence of non-specific reactions (false positives) was 0.43% (5 ÷ 1,161 × 100). .

  Further, when bovine blood components (serum) are brought into contact with the sample, 5 out of 9 samples are the original negative determination results. As a result, the incidence of non-specific reaction (false positive) is 0. It was 34% (4 ÷ 1,161 pcs × 100).

  Further, when sheep blood component (serum) is brought into contact with the sample, 2 out of 9 samples are the original negative determination result. As a result, the incidence of non-specific reaction (false positive) is 0. 60% (7 / 1,161 × 100).

  When the blood component (serum) of the horse is brought into contact with the sample, one of nine samples is the original negative determination result. As a result, the incidence of non-specific reaction (false positive) is 0. It was 69% (8 ÷ 1,161 pcs × 100).

  In addition, when porcine blood component (serum) is brought into contact with a sample, one of nine samples is the original negative determination result. As a result, the incidence of non-specific reaction (false positive) is 0. It was 69% (8 ÷ 1,161 pcs × 100).

  Furthermore, when a rabbit blood component (serum) is brought into contact with a sample, 1 of 9 samples is the original negative determination result. As a result, the incidence of non-specific reaction (false positive) is 0. It was 69% (8 ÷ 1,161 pcs × 100).

From the above, the blood component (serum) of the mouse, which is the animal species producing the antibody (anti-HBs monoclonal antibody) used in the immunological measurement reagent of the measurement target substance (HBs antigen) in the sample, is brought into contact with the sample. Even if it is a non-specific reaction that cannot be suppressed, goats, cattle, which are bovine species and animal species different from the antibody producing animal species (mouse) used in the immunological measurement, Alternatively, it was confirmed that the non-specific reaction can be prevented by bringing the blood component (serum) of sheep into contact with the sample.
In addition, since the said nonspecific reaction can be suppressed by contacting the blood component of an animal with a sample, it is thought that it is a nonspecific reaction by a heterophilic antibody.

[Example 2] Confirmation of inhibitory effect of non-specific reaction The inhibitory effect of non-specific reaction in the immunological measurement of the measurement target substance in the sample, such as the inhibitor of the present invention, was confirmed.

1. Immunological measuring reagents The following measuring reagents were used as immunological measuring reagents for the substance to be measured in the sample.
(1) Anti-HBs antibody-immobilized microplate The anti-HBs antibody-immobilized microplate described in 1 of (1) of Example 1 was used.

(2) Anti-HBs antibody-immobilized particle dispersion The anti-HBs antibody-immobilized particle dispersion described in 1 (2) of Example 1 was used.

(3) Sample diluents The following 29 types of sample diluents were prepared.
(I) Serum-free / sample diluent A 250 mM glycine buffer (pH 10.0) containing 0.5% (w / v) casein was prepared and used as a serum-free / sample diluent.

(B) 1% (w / v) mouse serum containing sample dilution solution 250 mM glycine buffer (pH 10.5) containing 1% (w / v) mouse serum (Veritas) and 0.5% (w / v) casein 0) was prepared and used as a sample diluent containing 1% (w / v) mouse serum.

(C) 2% (w / v) mouse serum containing sample dilution solution 250 mM glycine buffer (pH 10.2) containing 2% (w / v) mouse serum (Veritas) and 0.5% (w / v) casein 0) was prepared and used as a sample diluent containing 2% (w / v) mouse serum.

(D) 5% (w / v) mouse serum containing sample dilution solution 250 mM glycine buffer (pH 10.5) containing 5% (w / v) mouse serum (Veritas) and 0.5% (w / v) casein 0) was prepared and used as a sample diluent containing 5% (w / v) mouse serum.

(E) 10% (w / v) mouse serum-containing sample dilution 250 mM glycine buffer (pH 10.) containing 10% (w / v) mouse serum (Veritas) and 0.5% (w / v) casein 0) was prepared and used as a sample dilution containing 10% (w / v) mouse serum.

(F) 1% (w / v) goat serum-containing sample diluent 250% glycine buffer containing 1% (w / v) goat serum (Nippon Biomaterials Center) and 0.5% (w / v) casein (PH 10.0) was prepared and used as a sample diluent containing 1% (w / v) goat serum.

(G) 2% (w / v) goat serum-containing sample dilution solution 250 mM glycine buffer containing 2% (w / v) goat serum (Japan Biomaterials Center) and 0.5% (w / v) casein (PH 10.0) was prepared and used as a sample diluent containing 2% (w / v) goat serum.

(H) 5% (w / v) goat serum-containing sample dilution solution 250 mM glycine buffer containing 5% (w / v) goat serum (Nippon Biomaterials Center) and 0.5% (w / v) casein (PH 10.0) was prepared and used as a sample diluent containing 5% (w / v) goat serum.

(Li) 10% (w / v) goat serum-containing sample dilution solution 250 mM glycine buffer containing 10% (w / v) goat serum (Nippon Biomaterials Center) and 0.5% (w / v) casein (PH 10.0) was prepared and used as a sample diluent containing 10% (w / v) goat serum.

(Nu) 1% (w / v) bovine serum-containing sample dilution solution 250 mM glycine buffer containing 1% (w / v) bovine serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample diluent containing 1% (w / v) bovine serum.

(L) 2% (w / v) bovine serum containing sample dilution solution 250% glycine buffer containing 2% (w / v) bovine serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution solution containing 2% (w / v) bovine serum.

(Wo) 5% (w / v) bovine serum containing sample dilution solution 250 mM glycine buffer containing 5% (w / v) bovine serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution solution containing 5% (w / v) bovine serum.

(W) Containing 10% (w / v) bovine serum Sample dilution solution 250 mM glycine buffer containing 10% (w / v) bovine serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution solution containing 10% (w / v) bovine serum.

(F) 1% (w / v) sheep serum containing sample dilution solution 250% glycine buffer containing 1% (w / v) sheep serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution solution containing 1% (w / v) sheep serum.

(Yo) 2% (w / v) sheep serum containing sample dilution solution 250% glycine buffer containing 2% (w / v) sheep serum (Nippon Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample diluent containing 2% (w / v) sheep serum.

(Ta) 5% (w / v) sheep serum-containing sample diluent 250% glycine buffer containing 5% (w / v) sheep serum (Nippon Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution solution containing 5% (w / v) sheep serum.

(L) 10% (w / v) sheep serum-containing sample diluent 250% glycine buffer containing 10% (w / v) sheep serum (Nippon Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution containing 10% (w / v) sheep serum.

(So) 1% (w / v) equine serum-containing sample dilution 250 mM glycine buffer (pH 10) containing 1% (w / v) equine serum (JRH BIOSCIENCES) and 0.5% (w / v) casein 0.0) was prepared and used as a sample dilution containing 1% (w / v) horse serum.

(Tu) 2% (w / v) equine serum-containing sample dilution 250% glycine buffer (pH 10) containing 2% (w / v) equine serum (JRH BIOSCIENCES) and 0.5% (w / v) casein 0.0) was prepared and used as a sample dilution containing 2% (w / v) horse serum.

(Ne) 5% (w / v) equine serum-containing sample dilution 250 mM glycine buffer (pH 10) containing 5% (w / v) equine serum (JRH BIOSCIENCES) and 0.5% (w / v) casein 0.0) was prepared and used as a sample dilution containing 5% (w / v) horse serum.

(Na) 10% (w / v) equine serum-containing sample diluent 250% glycine buffer (pH 10) containing 10% (w / v) equine serum (JRH BIOSCIENCES) and 0.5% (w / v) casein 0.0) was prepared and used as a sample diluent containing 10% (w / v) horse serum.

(La) 1% (w / v) porcine serum-containing sample diluent 250% glycine buffer (pH 10) containing 1% (w / v) porcine serum (JR SCIENTIFIC) and 0.5% (w / v) casein 0.0) was prepared and used as a sample diluent containing 1% (w / v) porcine serum.

(Mu) 2% (w / v) porcine serum-containing sample dilution 250% glycine buffer (pH 10) containing 2% (w / v) porcine serum (JR SCIENTIFIC) and 0.5% (w / v) casein 0.0) was prepared and used as a sample diluent containing 2% (w / v) porcine serum.

(C) 5% (w / v) porcine serum-containing sample dilution 250% glycine buffer (pH 10) containing 5% (w / v) porcine serum (JR SCIENTIFIC) and 0.5% (w / v) casein 0.0) was prepared and used as a sample diluent containing 5% (w / v) porcine serum.

(No) 10% (w / v) porcine serum-containing sample dilution 250% glycine buffer (pH 10) containing 10% (w / v) porcine serum (JR SCIENTIFIC) and 0.5% (w / v) casein 0.0) was prepared and used as a sample diluent containing 10% (w / v) porcine serum.

(E) 1% (w / v) rabbit serum-containing sample dilution solution 250 mM glycine buffer containing 1% (w / v) rabbit serum (Nippon Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample diluent containing 1% (w / v) rabbit serum.

(H) 2% (w / v) rabbit serum-containing sample diluent 250% glycine buffer containing 2% (w / v) rabbit serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample dilution solution containing 2% (w / v) rabbit serum.

(Ya) 5% (w / v) rabbit serum containing sample dilution solution 250 mM glycine buffer containing 5% (w / v) rabbit serum (Nippon Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample diluent containing 5% (w / v) rabbit serum.

(Ma) 10% (w / v) rabbit serum containing sample dilution solution 250% glycine buffer containing 10% (w / v) rabbit serum (Japan Biotest Laboratories) and 0.5% (w / v) casein A solution (pH 10.0) was prepared and used as a sample diluent containing 10% (w / v) rabbit serum.

2. Sample Human serum (HBs antigen-negative serum) that was negative as a result of measuring HBs antigen was used as a sample.

3. Immunological measurement of HBs antigen in sample using serum-free / diluted sample for HBs antigen-negative human serum sample Immunological measurement of HBs antigen in sample using serum-free / sample diluted solution Went.
This measurement operation was performed as described in 3 (1) to (6) of Example 1.
As a result of this measurement, a positive determination result was obtained for this human serum sample that was originally negative for HBs antigen.
That is, in the immunological measurement of the HBs antigen in the sample using this serum-free / sample diluent, a non-specific reaction occurred in this sample, which resulted in a false positive.

4). Immunological measurement of HBs antigens in samples using mouse serum containing / sample diluent For human serum samples that became false positive due to non-specific reaction of 3 above, samples using mouse serum containing / sample diluent The immunological measurement of the HBs antigen in was carried out.

(1) A human serum sample in which a positive determination result was obtained in 3 above, that is, a false positive due to a non-specific reaction, was obtained from 1% (w / v) mouse in (1) (3) above Serum-containing / diluted sample was diluted 8-fold.

(2) The subsequent operations were performed as described in 3 (2) to (6) of Example 1.

(3) In addition, the sample diluent contains (3) (c) 2% (w / v) mouse serum-containing sample serum and (d) 5% (w / v) mouse serum-containing sample. The measurement was carried out as described in (1) and (2) above in place of the diluted solution, (e) 10% (w / v) mouse serum-containing / sample diluted solution.

(4) As a result of this measurement, the sample diluent used is positive if it contains 1% (w / v) mouse serum or sample diluent-10% (w / v) mouse serum or sample diluent. A determination result was obtained, and even when the mouse serum contained in the sample diluent was brought into contact with the sample, the nonspecific reaction could not be suppressed.
That is, in this sample, even if the blood component (serum) of the mouse, which is the animal species producing the antibody (anti-HBs monoclonal antibody) used in the immunoassay reagent of the above 1, is contacted with the sample, it is non-specific. The reaction could not be suppressed.

5. Immunological measurement of HBs antigen in samples using goat serum containing sample diluent etc. Human serum sample that could not suppress nonspecific reaction even when mouse serum was brought into contact with the sample and became false positive Was subjected to immunological measurement of HBs antigen in the sample using sample dilutions each containing goat serum, bovine serum, sheep serum, horse serum, pig serum, or rabbit serum.

(1) A positive determination result was obtained in 4 above, that is, a human serum sample that was false positive because a non-specific reaction could not be suppressed even when a mouse blood component (serum) was brought into contact with the sample. The sample was diluted 8-fold with a sample diluent containing 1% (w / v) goat serum of (3) (f) in 1 above.

(2) The subsequent operations were performed as described in 3 (2) to (6) of Example 1.

(3) In addition, the sample diluent is a sample containing (2) (2) (w / v) goat serum in (3) of the above-mentioned sample diluent to (ma) 10% (w / v) rabbit serum. The measurement was performed as described in the above (1) and (2) except that the measurement was performed by sequentially changing to the diluted solution.

(4) The results of this measurement are shown in Table 2.

(5) From Table 2, the following can be understood.
When a human blood sample (serum) that is not positive cannot be suppressed even if it is brought into contact with the blood component (serum) of the mouse and is false positive, the blood component (serum) of the goat is brought into contact with the sample. The result was an original negative determination result.

  Similarly, when the bovine blood component (serum), sheep blood component (serum), and horse blood component (serum) were brought into contact with the sample, the original negative determination result was obtained.

  However, when a porcine blood component (serum) and a rabbit blood component (serum) were brought into contact with the sample, a positive determination result was obtained. In this case, a nonspecific reaction could not be suppressed.

  From the above, the blood component (serum) of the mouse, which is the animal species producing the antibody (anti-HBs monoclonal antibody) used in the immunological measurement reagent of the measurement target substance (HBs antigen) in the sample, is brought into contact with the sample. Even if it is a non-specific reaction that cannot be suppressed, goats, cattle, which are bovine species and animal species different from the antibody producing animal species (mouse) used in the immunological measurement, Alternatively, it was reconfirmed that the non-specific reaction can be prevented by bringing the blood component (serum) of sheep into contact with the sample.

[Example 3] Confirmation of inhibitory effect of non-specific reaction The inhibitory effect of non-specific reaction in the immunological measurement of the measurement target substance in the sample, such as the inhibitor of the present invention, was confirmed.

1. Immunological measuring reagents The following measuring reagents were used as immunological measuring reagents for the substance to be measured in the sample.
(1) Anti-HBs antibody-immobilized microplate The anti-HBs antibody-immobilized microplate described in 1 of (1) of Example 1 was used.

(2) Anti-HBs antibody-immobilized particle dispersion The anti-HBs antibody-immobilized particle dispersion described in 1 (2) of Example 1 was used.

(3) Sample diluent The following types of sample diluents were prepared.
(A) 1% (w / v) mouse serum-containing sample dilution 250 mM glycine buffer (pH 10.5) containing 1% (w / v) mouse serum (Veritas) and 0.5% (w / v) casein 0) was prepared and used as a sample diluent containing 1% (w / v) mouse serum.

(B) 5% (w / v) mouse serum-containing sample dilution 250 mM glycine buffer (pH 10.5) containing 5% (w / v) mouse serum (Veritas) and 0.5% (w / v) casein 0) was prepared and used as a sample diluent containing 5% (w / v) mouse serum.

(C) 1% (w / v) goat serum-containing sample diluent 250% glycine buffer containing 1% (w / v) goat serum (Japan Biomaterials Center) and 0.5% (w / v) casein (PH 10.0) was prepared and used as a sample diluent containing 1% (w / v) goat serum.

(D) 5% (w / v) goat serum-containing sample dilution solution 250 mM glycine buffer containing 5% (w / v) goat serum (Japan Biomaterials Center) and 0.5% (w / v) casein (PH 10.0) was prepared and used as a sample diluent containing 5% (w / v) goat serum.

(E) 1 μg / mL purified goat immunoglobulin G-containing sample diluent 250 μm glycine buffer (pH 10.0) containing 1 μg / mL purified goat immunoglobulin G (Nagase Sangyo Co., Ltd.) and 0.5% (w / v) casein 1 μg / mL purified goat immunoglobulin G-containing sample dilution.

2. Sample HBs antigen-negative human serum sample that is positive (false positive) due to non-specific reaction when immunological measurement of HBs antigen in the sample is performed using serum-free sample dilution solution 9 samples were obtained as samples.

3. Serum component-containing / immunological measurement of HBs antigen in sample using sample diluent For each of the nine human serum samples that become false-positive due to the non-specific reaction described above, a sample diluent containing serum components was prepared. Used for immunological measurement of HBs antigen in the sample.

(1) Each of the nine human serum samples that become false-positive due to the non-specific reaction of 2 is 8 in (1) (1) (1) (w / v) mouse serum-containing sample dilution solution of (3). Diluted twice.

(2) The subsequent operations were performed as described in 3 (2) to (6) of Example 1.

(3) In addition, the sample diluent is the sample (1) (3) (b) 5% (w / v) mouse serum containing sample diluent, (c) 1% (w / v) goat serum containing sample Diluted solution, (d) 5% (w / v) goat serum-containing sample dilution solution, or (e) 1 μg / mL purified goat immunoglobulin G-containing sample dilution solution, in turn (1) and (2 ).

(4) The results of this measurement are shown in Table 3.

(5) From Table 3, the following can be understood.
Nine human serum samples that could not suppress non-specific reactions even when they were brought into contact with mouse blood components (serum) were false positives. The number of samples was the original negative determination result.

  Further, purified goat immunoglobulin G was brought into contact with the sample in nine human serum samples that failed to suppress non-specific reaction even when the mouse blood component (serum) was brought into contact with the sample and became false positive. Even in this case, five of these samples were the original negative determination results.

From the above, the blood component (serum) of the mouse, which is the animal species producing the antibody (anti-HBs monoclonal antibody) used in the immunological measurement reagent of the measurement target substance (HBs antigen) in the sample, is brought into contact with the sample. Blood components of goats that are bovine species and animal species different from the antibody producing animal species (mouse) used in the immunological measurement, even if they are non-specific reactions that cannot be suppressed It was confirmed that the non-specific reaction can be prevented by contacting the sample with either serum or purified immunoglobulin G.

Claims (30)

  Blood of an animal species that is an inhibitor of a nonspecific reaction in an immunological measurement of a substance to be measured in a sample and that is a bovine species and is different from an animal producing animal species used for the immunological measurement A non-specific substance that is composed of a component and cannot be inhibited by bringing the blood component of the animal species producing the antibody used for the immunological measurement into contact with the sample by bringing the inhibitor of the non-specific reaction into contact with the sample. An inhibitor of non-specific reaction in immunological measurement, characterized in that the reaction can be suppressed.   The inhibitor of a nonspecific reaction according to claim 1, wherein the nonspecific reaction is caused by a heterophilic antibody.   The inhibitor of nonspecific reaction according to claim 1 or 2, wherein the blood component is blood, plasma, serum, or immunoglobulin.   The inhibitor of nonspecific reaction according to any one of claims 1 to 3, wherein the bovine species is goat, sheep or cow.   The inhibitor of a nonspecific reaction according to any one of claims 1 to 4, wherein the antibody used for the immunological measurement is a monoclonal antibody.   An inhibitor of a non-specific reaction in an immunological measurement of a substance to be measured in a sample, wherein the non-specific reaction contacts the blood component of the animal species producing the antibody used for the immunological measurement with the sample. However, the inhibitor of the nonspecific reaction is a bovine species and is composed of blood components of an animal species different from the antibody producing animal species used for the immunological measurement. An inhibitor of a non-specific reaction in an immunological measurement, characterized in that   The inhibitor of nonspecific reaction according to claim 6, wherein the nonspecific reaction is caused by a heterophilic antibody.   The inhibitor of a nonspecific reaction according to claim 6 or 7, wherein the blood component is blood, plasma, serum, or immunoglobulin.   The inhibitor of nonspecific reaction according to any one of claims 6 to 8, wherein the bovine species is goat, sheep or cow.   The inhibitor of a nonspecific reaction according to any one of claims 6 to 9, wherein the antibody used for the immunological measurement is a monoclonal antibody.   A method for suppressing a nonspecific reaction in an immunological measurement of a substance to be measured in a sample, wherein the blood is a bovine species that is different from the animal species that produces the antibody used for the immunological measurement. Immunologically characterized by suppressing non-specific reactions that cannot be suppressed by contacting the blood component of the animal species producing the antibody used in the immunological measurement with the sample by contacting the component with the sample. Method for suppressing nonspecific reaction in measurement.   The method for suppressing a nonspecific reaction according to claim 11, wherein the nonspecific reaction is caused by a heterophilic antibody.   The method for suppressing a nonspecific reaction according to claim 11 or 12, wherein the blood component is blood, plasma, serum, or immunoglobulin.   The method for suppressing a nonspecific reaction according to any one of claims 11 to 13, wherein the bovine species is a goat, a sheep, or a cow.   The method for suppressing a nonspecific reaction according to any one of claims 11 to 14, wherein the antibody used for the immunological measurement is a monoclonal antibody.   A method for suppressing a non-specific reaction in an immunological measurement of a measurement target substance in a sample, wherein the non-specific reaction is brought into contact with a blood component of an antibody-producing animal species used for the immunological measurement. However, the method for suppressing the non-specific reaction is a bovine species, and the blood component of an animal species different from the antibody-producing animal species used for the immunological measurement is brought into contact with the sample. A method for suppressing a non-specific reaction in an immunological measurement, characterized by   The method for suppressing a nonspecific reaction according to claim 16, wherein the nonspecific reaction is caused by a heterophilic antibody.   The method for suppressing a nonspecific reaction according to claim 16 or 17, wherein the blood component is blood, plasma, serum, or immunoglobulin.   The method for suppressing a nonspecific reaction according to any one of claims 16 to 18, wherein the bovine species is a goat, a sheep, or a cow.   The method for suppressing a nonspecific reaction according to any one of claims 16 to 19, wherein the antibody used for the immunological measurement is a monoclonal antibody.   A method for immunological measurement of a substance to be measured in a sample, wherein a blood component of an animal species that is a bovine species and is different from the animal species that produces the antibody used for the immunological measurement is brought into contact with the sample Suppresses a non-specific reaction that cannot be suppressed even when the blood component of the animal species producing the antibody used for the immunological measurement is brought into contact with the sample, Measuring method.   The method for immunological measurement of a substance to be measured in a sample according to claim 21, wherein the nonspecific reaction is caused by a heterophilic antibody.   The method for immunological measurement of a substance to be measured in a sample according to claim 21 or 22, wherein the blood component is blood, plasma, serum, or immunoglobulin.   The immunological measurement method for a substance to be measured in a sample according to any one of claims 21 to 23, wherein the bovine species is a goat, sheep or cow.   The method for immunological measurement of a substance to be measured in a sample according to any one of claims 21 to 24, wherein the antibody used for the immunological measurement is a monoclonal antibody.   A reagent for immunological measurement of a substance to be measured in a sample, comprising a blood component of an animal species that is a bovine species and is different from an animal species that produces the antibody used for the immunological measurement, A reagent for immunological measurement of a measurement target substance in a sample, characterized by suppressing a non-specific reaction that cannot be suppressed even when the blood component of the animal species producing the antibody used in the immunological measurement is brought into contact with the sample. .   The reagent for immunological measurement of a substance to be measured in a sample according to claim 26, wherein the nonspecific reaction is caused by a heterophilic antibody.   The reagent for immunological measurement of a substance to be measured in a sample according to claim 26 or 27, wherein the blood component is blood, plasma, serum, or immunoglobulin.   The immunoassay reagent for a substance to be measured in a sample according to any one of claims 26 to 28, wherein the bovine species is a goat, sheep or cow.   30. The reagent for immunological measurement of a substance to be measured in a sample according to any one of claims 26 to 29, wherein the antibody used for the immunological measurement is a monoclonal antibody.