Hemoglobin measurement reagent, measurement kit, and measurement method

Abstract

To provide a hemoglobin measurement reagent and a measurement method, which enable accurate measurement of the hemoglobin level by suppressing the decrease in measured hemoglobin level caused by addition of haptoglobin.SOLUTION: A reagent of the present invention for measuring hemoglobin contains an insoluble carrier for carrying an anti-hemoglobin antibody and an insoluble carrier for carrying an anti-haptoglobin antibody. A method of measuring hemoglobin in a sample according to the present invention comprises: (1) preparing a sample containing a hemoglobin-haptoglobin complex by mixing a sample and haptoglobin to form the hemoglobin-haptoglobin complex; and (2) bringing the sample obtained in the step (1) into contact with the insoluble carrier for carrying an anti-hemoglobin antibody and the insoluble carrier for carrying an anti-haptoglobin antibody to cause immunoaggregation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a reagent and a method for measuring hemoglobin. [Background technology]

[0002] Detection of blood contained in specimens such as feces, urine, and saliva is useful for diagnosing many diseases. For example, fecal occult blood tests, which detect blood in feces, are used in screening for colon cancer. One known method for detecting occult blood is an immunological technique that uses antihemoglobin antibodies to detect hemoglobin contained in occult blood in specimens such as feces.

[0003] A sample to be used in an occult blood test is usually collected by a subject in a container containing a preservative solution and sent to a testing institution such as a hospital. In many cases, the preservative solution containing the sample is stored for several days before being used for testing, and during this time it is often kept at high temperatures. Hemoglobin is unstable in solution and is particularly susceptible to denaturation or decomposition under high temperature conditions. If the structure of the epitope or its surrounding region changes due to denaturation or decomposition of hemoglobin, antibodies will no longer be able to recognize the hemoglobin, thereby reducing the accuracy of hemoglobin detection by immunological methods.

[0004] Therefore, a method of adding haptoglobin to a preservation solution for a specimen is used to stabilize hemoglobin (for example, Patent Document 1). Haptoglobin is a protein present in the blood of a wide variety of animals and plays a role in recovering hemoglobin released into the blood due to hemolysis of red blood cells. It is known that haptoglobin rapidly and irreversibly binds with hemoglobin to form a stable hemoglobin-haptoglobin complex. By adding haptoglobin to a preservation solution or the like in advance, when the specimen is added to the preservation solution or the like, the hemoglobin contained in the specimen is stabilized by forming a hemoglobin-haptoglobin complex.

[0005] However, when haptoglobin is added, particularly when the amount of haptoglobin added is in excess of the free hemoglobin in the sample, a decrease in the measured hemoglobin value may be observed (Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 10-132824 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a reagent and method for measuring hemoglobin, which can suppress a decrease in the measured value of hemoglobin due to the addition of haptoglobin and thereby measure the amount of hemoglobin more accurately. [Means for solving the problem]

[0008] As a result of extensive research conducted by the present inventors to solve the above-mentioned problems of the conventional technology, they discovered that by allowing an insoluble carrier to which an antihemoglobin antibody is bound and an insoluble carrier to which an antihaptoglobin antibody is bound to coexist, the decrease in the measured hemoglobin value caused by the addition of haptoglobin can be suppressed, and the amount of hemoglobin can be measured more accurately, which led to the completion of the present invention.

[0009] That is, the present invention relates to, for example, the following inventions. [1] A reagent for measuring hemoglobin, comprising an insoluble carrier carrying an antihemoglobin antibody and an insoluble carrier carrying an antihaptoglobin antibody. [2] The reagent according to [1], which contains at least two types of antihemoglobin antibodies. [3] The reagent according to either [1] or [2], wherein the insoluble carrier is latex particles and / or gold colloid particles. [4] The reagent according to any one of [1] to [3], wherein the anti-hemoglobin antibody and the anti-haptoglobin antibody are monoclonal antibodies. [5] Any of the reagents [1] to [4], wherein the anti-haptoglobin antibody is one type. [6] A method for measuring hemoglobin in a sample, comprising: (1) mixing a specimen with haptoglobin to form a hemoglobin-haptoglobin complex and obtain a sample containing the hemoglobin-haptoglobin complex; (2) contacting the sample obtained in step (1) with an insoluble carrier carrying an anti-hemoglobin antibody and an insoluble carrier carrying an anti-haptoglobin antibody to cause immunoagglutination; A method for measuring hemoglobin, comprising: [7] The measurement method according to [6], wherein there are at least two types of antihemoglobin antibodies. [8] The measurement method according to [6] or [7], wherein the insoluble carrier is latex particles and / or gold colloid particles. [9] The measurement method according to any one of [6] to [8], wherein the antihemoglobin antibody and the antihaptoglobin antibody are monoclonal antibodies.

[10] Any of the measurement methods [6] to [9], in which the sample is feces, saliva, or urine.

[11] A measurement method according to any one of [6] to

[10] , wherein haptoglobin is contained in the sample preservation solution.

[12] The measurement method according to

[11] , wherein the haptoglobin concentration in the sample preservation solution is 0.05 units / L to 50 units / L.

[13] The method according to any one of [6] to

[12] , wherein the hemoglobin in the sample contains at least one selected from the group consisting of free hemoglobin, a hemoglobin-haptoglobin complex intermediate, and a complete hemoglobin-haptoglobin complex, and at least a portion of the free hemoglobin forms a hemoglobin-haptoglobin complex intermediate and / or a complete hemoglobin-haptoglobin complex with the haptoglobin after step (1).

[14] A method for measuring hemoglobin in a sample, comprising the steps of contacting the sample with an insoluble carrier carrying an antihemoglobin antibody and an insoluble carrier carrying an antihaptoglobin antibody, and causing immunoagglutination.

[15] A method for suppressing a decrease in the measured value of hemoglobin in a sample, comprising a step of contacting the sample with an insoluble carrier carrying an antihemoglobin antibody and an insoluble carrier carrying an antihaptoglobin antibody, and causing immunoagglutination.

[16] A kit for measuring hemoglobin in a sample, comprising an insoluble carrier carrying an antihemoglobin antibody, an insoluble carrier carrying an antihaptoglobin antibody, and haptoglobin.

[17] A kit for measuring hemoglobin in a sample, comprising any one of the reagents [1] to [5] and haptoglobin. [Effects of the Invention]

[0010] According to the present invention, a measurement reagent and a measurement method are provided that can measure the amount of hemoglobin more accurately. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Definition> [Hemoglobin-haptoglobin complex] Hemoglobin is a protein found in red blood cells and has a tetrameric structure [α2β2] composed of two types of subunits, called α subunits (or α chains) and β subunits (or β chains). Haptoglobin is a protein found in plasma that binds to free hemoglobin in the blood and has three types of structures. For example, type 1-1 haptoglobin has a tetrameric structure [α2β2] composed of two types of subunits, called α subunits (or α chains) and β subunits (or β chains).

[0012] Hemoglobin and haptoglobin form a stable complex. Typically, one molecule of hemoglobin binds to one molecule of haptoglobin. Such a complex is called a hemoglobin-haptoglobin complex, and is also referred to herein as a complete hemoglobin-haptoglobin complex.

[0013] On the other hand, when the molar ratio of hemoglobin to haptoglobin (hemoglobin:haptoglobin) is less than 1, i.e., when haptoglobin is present in excess of hemoglobin, a complex called a hemoglobin-haptoglobin complex intermediate tends to form (JVPASTEWKA et al., Biochimica et Biophysica Acta, 386 (1975) 530-537). When forming the hemoglobin-haptoglobin complex intermediate, one molecule of tetrameric hemoglobin [α2β2] dissociates into two dimers (αβ), and each αβ dimer is thought to bind to one molecule of haptoglobin. In other words, the hemoglobin-haptoglobin complex intermediate is thought to be a complex in which one molecule of haptoglobin [α2β2] binds to half a molecule of hemoglobin (αβ).

[0014] As used herein, hemoglobin-haptoglobin complexes include both complete hemoglobin-haptoglobin complexes and hemoglobin-haptoglobin complex intermediates, unless otherwise specified.

[0015] [Specimen] The term "specimen" as used herein refers to a biological sample collected from a subject and containing or potentially containing hemoglobin. The specimen may be feces, saliva, or urine, with feces being particularly preferred. The specimen may also be whole blood, serum, plasma, or the like, and measuring the hemoglobin content in these specimens may be useful, for example, as an indicator of hemolysis or treatment using a haptoglobin preparation.

[0016] [sample] The sample in this specification includes a specimen, and in particular, is obtained by mixing the specimen with haptoglobin. Free hemoglobin in the specimen mixes with haptoglobin to form a stable hemoglobin-haptoglobin complex. That is, the sample may contain free hemoglobin that is not complexed with haptoglobin, hemoglobin-haptoglobin complexes that were originally present in the specimen (specimen-derived hemoglobin-haptoglobin complexes), and / or hemoglobin-haptoglobin complexes that were formed by adding haptoglobin (prepared hemoglobin-haptoglobin complexes). In particular, from the viewpoint of stability, it is preferable that in a sample obtained by mixing a specimen and haptoglobin, all free hemoglobin forms complexes with haptoglobin to form prepared hemoglobin-haptoglobin complexes, and it is preferable that the sample consists of hemoglobin-haptoglobin complexes derived from the specimen and prepared hemoglobin-haptoglobin complexes.

[0017] The sample can be obtained, for example, by having a subject or the like collect a specimen and then quickly adding the specimen to a specimen preservation solution containing haptoglobin (hereinafter sometimes referred to as "preservation solution"). The sample may be stored at room temperature for several days until hemoglobin measurement, and is preferably stored refrigerated at 2 to 10°C. When the specimen is feces or the like containing solid matter, the preservation solution may be filtered to remove the solid matter, and the resulting sample may be used.

[0018] [Storage solution] The preservation solution refers to a sample preservation solution for preserving a sample, and preferably contains haptoglobin.

[0019] The preservation solution preferably further contains a buffer solution or the like. The buffer solution or the like may be a buffer containing a Good's buffer such as 2-morpholinoethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), or the like, or may be a phosphate buffer, Tris buffer, or glycine buffer. The pH of the preservation solution may be 5 to 10, or 6 to 8.

[0020] The concentration of haptoglobin in the preservative solution varies depending on the type and amount of the sample, but may be, for example, 0.05 units / L to 50 units / L, 0.1 units / L to 10 units / L, or 0.2 units / L to 2 units / L. Here, 1 unit refers to the amount of haptoglobin that binds to 1 mg of hemoglobin. A haptoglobin concentration within the above range is sufficient to allow all hemoglobin in the sample to form a complex with haptoglobin.

[0021] For example, when stool is suspended in 0.2 to 20 mL of preservative solution to a concentration of 0.05% to 25% (W / V%), the quantitative ratio of specimen to haptoglobin may be 0.075 g / unit to 5000 g / unit.

[0022] The haptoglobin used in the present invention is not particularly limited as long as it complexes with the hemoglobin to be measured to form a hemoglobin-haptoglobin complex. Because the binding between hemoglobin and haptoglobin is not species-specific, a wide range of haptoglobins can be used. When human hemoglobin is the target of measurement, haptoglobin from humans, horses, pigs, monkeys, dogs, rabbits, rats, and other animals can be used. It is not necessary to use highly purified haptoglobin.

[0023] The preservation solution may further contain known additives that can be used during hemoglobin storage, such as antibacterial agents such as sodium azide (NaN), pH adjusters, and salts for adjusting ionic strength. Antibacterial agents include antibiotics and lytic enzymes. Examples of additives include known components that are known to have the effect of stabilizing hemoglobin, such as amino acids such as lysine and histidine, albumin, protease inhibitors, water-soluble complexes of transition metal ions, and ethylenediaminetetraacetic acid (EDTA). Examples of albumin include serum albumins such as bovine serum albumin (BSA) and albumin derived from egg white (ovalbumin).

[0024] <Hemoglobin measurement reagent> A hemoglobin measurement reagent according to one embodiment of the present invention comprises an insoluble carrier carrying an antihemoglobin antibody and an insoluble carrier carrying an antihaptoglobin antibody. The measurement method using the reagent of the present invention is an immunological technique, which may be a known immunological technique that utilizes an antihemoglobin antibody, such as an immunoagglutination method (e.g., latex agglutination or gold colloid agglutination), immunochromatography, or ELISA. The reagent of the present invention is particularly suitable for use in an immunoagglutination method, more preferably a latex agglutination method.

[0025] A hemoglobin measurement reagent according to one embodiment of the present invention measures the total amount of hemoglobin by measuring both free hemoglobin and hemoglobin-haptoglobin complexes. In immunological assays (particularly immunoagglutination assays), the reagent of the present invention exhibits substantially equivalent reactivity as hemoglobin with each of the antigens of free hemoglobin, complete hemoglobin-haptoglobin complexes, and hemoglobin-haptoglobin complex intermediates. In particular, the presence of an anti-haptoglobin antibody bound to an insoluble carrier provides the excellent effect of exhibiting equivalent reactivity as hemoglobin with both the antigens of the complete hemoglobin-haptoglobin complex and the hemoglobin-haptoglobin complex intermediates ("equivalent reactivity to hemoglobin").

[0026] The term "equivalent reactivity as hemoglobin" means that when a sample contains the same concentration of each antigen (free hemoglobin, complete hemoglobin-haptoglobin complex, hemoglobin-haptoglobin complex intermediate, or a mixture thereof) as hemoglobin, the hemoglobin concentrations measured by immunoagglutination on an insoluble carrier are substantially equal for each antigen. Here, "substantially equal" hemoglobin concentrations measured by immunoagglutination on an insoluble carrier means that, when the measured value for free hemoglobin is taken as 100%, the lower limit of the measured hemoglobin concentration when measuring various other antigens at the same concentration as hemoglobin is 80% or more, preferably 90% or more, and more preferably 95% or more, and the upper limit of the measured value is 120% or less, preferably 110% or less, and more preferably 105% or less.

[0027] The reagent of the present invention contains an insoluble carrier carrying an anti-haptoglobin antibody in addition to a conventional insoluble carrier carrying an anti-hemoglobin antibody, thereby suppressing the decrease in the measured hemoglobin value that would otherwise be caused by the addition of haptoglobin. In particular, even when the molar ratio of the added haptoglobin to the free hemoglobin in the sample (hemoglobin / haptoglobin) is less than 1, i.e., even when a hemoglobin-haptoglobin complex intermediate is present, the decrease in the measured hemoglobin value is suppressed, allowing accurate detection and measurement of hemoglobin in the sample. Furthermore, even when no haptoglobin is added to the sample and haptoglobin (whether free haptoglobin, hemoglobin-haptoglobin complex intermediate, or hemoglobin-haptoglobin complex) is originally present in the sample, the decrease in the measured hemoglobin value is suppressed, allowing accurate detection and measurement of hemoglobin in the sample.

[0028] The present inventors believed that the decrease in hemoglobin measurement value caused by the addition of conventional haptoglobin was partly due to the presence of a hemoglobin-haptoglobin complex intermediate, and solved this problem by adding an insoluble carrier carrying an anti-haptoglobin antibody.

[0029] The insoluble carrier may be any carrier capable of carrying antihemoglobin antibodies or antihaptoglobin antibodies, and is preferably an insoluble particle that can be used in immunological techniques. Examples include, but are not limited to, commonly used metal colloid particles such as colloidal gold particles, latex particles, silica particles, magnetic particles, fluorescent particles, and red blood cells. The insoluble particle is preferably a latex particle, and more preferably a polystyrene latex particle. The insoluble carrier is preferably particulate, and its average particle diameter is preferably 5 to 1,000 nm, more preferably 30 to 500 nm, and even more preferably 75 to 350 nm, although it can be used without being particularly limited to these ranges.

[0030] "Supporting an antibody" means that the antibody is immobilized by physical adsorption or chemical bonding to the surface of an insoluble carrier. For example, a known method of immobilizing an antibody on an insoluble carrier particle is to mix the antibody with the insoluble carrier particle and physically adsorb the antibody onto the surface of the insoluble carrier particle. Furthermore, when insoluble carrier particles with amino or carboxyl groups introduced onto the surface are used, the antibody can be immobilized on the surface of the insoluble carrier particle by chemical bonding using glutaraldehyde or a carboximide reagent.

[0031] The amount of antibody supported is not particularly limited, but may be 0.5 to 2000 μg / mg latex, or may be 1 to 1000 μg / mg latex, or 2 to 500 μg / mg latex. The amount of antibody supported can be calculated by subtracting the amount of antibody after immobilization from the amount of antibody before immobilization on the insoluble carrier.

[0032] The anti-hemoglobin antibody is not particularly limited, but it is preferable that it can recognize the hemoglobin epitope in the hemoglobin-haptoglobin complex and not cross-react with haptoglobin. While a polyclonal or monoclonal antibody may be used, a monoclonal antibody is preferred in terms of specificity, and an anti-human hemoglobin monoclonal antibody is preferred. The assay reagent contains at least one type of anti-hemoglobin antibody, preferably at least two types.

[0033] On the other hand, the anti-haptoglobin antibody is not particularly limited, but preferably can recognize the haptoglobin epitope in the hemoglobin-haptoglobin complex and does not cross-react with hemoglobin. In particular, the anti-haptoglobin antibody is preferably one that does not aggregate with free haptoglobin and an insoluble carrier carrying the anti-haptoglobin antibody, at least within the range of the amount of free haptoglobin added to form the hemoglobin-haptoglobin complex. Furthermore, the anti-haptoglobin antibody is more preferably one that does not aggregate with free haptoglobin and an insoluble carrier carrying the anti-haptoglobin antibody. The anti-haptoglobin antibody may be a polyclonal or monoclonal antibody, but is preferably a monoclonal antibody, and is preferably an anti-haptoglobin monoclonal antibody. When the anti-haptoglobin antibody is a monoclonal antibody, its recognition site may be the α chain (α chain-recognizing antibody) or β chain (β chain-recognizing antibody) of haptoglobin. The measurement reagent contains at least one type of anti-haptoglobin antibody.

[0034] The antibodies that can be used in the present invention are not particularly limited by animal species, and examples include antibodies derived from animals such as rabbits, goats, mice, rats, horses, and sheep. Either polyclonal antibodies obtained from the serum of animals immunized with the target substance by known methods or monoclonal antibodies obtained by cell fusion of the spleen of animals immunized with the target substance with myeloma cells may be used. Fragments thereof [e.g., F(ab')2, Fab, Fab', or Fv] may also be used.

[0035] The insoluble carrier used to support the antihemoglobin antibody is at least one type, but may be at least two types. One type of insoluble carrier may support one or more types of antihemoglobin antibody. Furthermore, one type of antihemoglobin antibody may be supported by one or more types of insoluble carrier.

[0036] When the measurement reagent contains two or more types of antihemoglobin antibodies, the two or more types of antihemoglobin antibodies may be supported on the same insoluble carrier. Alternatively, a mixture of multiple insoluble carriers, each of which supports one type of antihemoglobin antibody, may be used. In this case, the insoluble carriers used to support different types of antihemoglobin antibodies may be the same type of insoluble carrier, or different types of insoluble carriers with different materials, particle sizes, etc.

[0037] The insoluble carrier used to support the anti-haptoglobin antibody is at least one type, but may be at least two types. One type of insoluble carrier may support one or two or more types of anti-haptoglobin antibody. Furthermore, one type of anti-haptoglobin antibody may be supported by one or two or more types of insoluble carrier. When the measurement reagent contains two or more types of anti-haptoglobin antibodies, the same procedures as those for when the measurement reagent contains two or more types of antihemoglobin antibodies are followed.

[0038] The form of the measurement reagent is not particularly limited, and may be, for example, a two-reagent system consisting of a reagent (first reagent) that does not contain an insoluble carrier and a reagent (second reagent) that contains an insoluble carrier that carries an antibody (antibody-carrying insoluble carrier), or may be a single-reagent system consisting of only a reagent that contains an antibody-carrying insoluble carrier.

[0039] The first reagent can be used to adjust the measurement environment, such as by using it as a diluent to adjust the concentration of the analyte or impurities in the reaction system, or to adjust the reaction rate. The second reagent contains an antibody-supported insoluble carrier, and is mixed with the first reagent and the sample to cause an immune agglutination reaction. The first and second reagents can contain pH buffers, salts, surfactants, agglutination promoters, preservatives, etc. as appropriate. The pH during the agglutination reaction is preferably 5 to 9.

[0040] The measurement reagent is mixed with a storage solution to obtain a reaction solution, and the concentration of the insoluble carrier in the reaction solution can be appropriately selected, for example, from the range of 0.0001 mg / mL to 10 mg / mL depending on the particle size of the insoluble carrier used and the design of the entire measurement system. The concentration of the insoluble carrier carrying the anti-hemoglobin antibody in the measurement reagent may be 0.01 to 20 mg / mL or 0.05 to 1 mg / mL, and the concentration of the insoluble carrier carrying the anti-haptoglobin antibody may be 0.01 to 20 mg / mL or 0.05 to 1 mg / mL.

[0041] The concentration of the insoluble carrier in the second reagent is diluted by mixing it with a storage solution or by mixing the first reagent with a storage solution when used, and therefore the concentration of the insoluble carrier in the second reagent can be appropriately selected depending on the dilution ratio. For example, when used after a 2-fold dilution, the concentration can be appropriately adjusted to 0.004 mg / mL to 80 mg / mL, and when used after a 3-fold dilution, the concentration can be appropriately adjusted to 0.06 mg / mL to 120 mg / mL.

[0042] <Hemoglobin measurement method> [Process (1)] In step (1), a specimen is mixed with haptoglobin to form a hemoglobin-haptoglobin complex, thereby obtaining a sample containing the hemoglobin-haptoglobin complex.

[0043] The sample and haptoglobin are mixed as described above in the "Sample" section. Here, the hemoglobin-haptoglobin complexes contained in the sample may include both complete hemoglobin-haptoglobin complexes and hemoglobin-haptoglobin complex intermediates. The sample may also include free hemoglobin derived from the sample, hemoglobin-haptoglobin complexes derived from the sample, and other components derived from the sample. It may also include free haptoglobin that is not complexed with hemoglobin. Haptoglobin to be mixed with the sample may be contained in a sample preservation solution. When haptoglobin is added in the form of a sample preservation solution, the sample may contain not only free haptoglobin that is not complexed with hemoglobin, but also other components of the preservation solution. Since it is preferable that all of the free hemoglobin derived from the sample forms complexes with haptoglobin, the amount of haptoglobin mixed with the sample may be in excess of the free hemoglobin in the sample.

[0044] [Process (2)] In step (2), the sample obtained in step (1) is contacted with an insoluble carrier carrying an anti-hemoglobin antibody and an insoluble carrier carrying an anti-haptoglobin antibody to cause an immune agglutination reaction.

[0045] The contact causes the antihemoglobin antibody to recognize the hemoglobin in the sample, which in turn causes the insoluble carrier carrying the antihemoglobin antibody to bind to hemoglobin or hemoglobin-haptoglobin complexes, and further causes aggregation of the insoluble carriers themselves. The turbidity of the solution changes due to aggregation of the insoluble carriers, and the hemoglobin concentration in the sample can be determined by measuring the change in turbidity of the solution and using a calibration curve prepared using hemoglobin or hemoglobin-haptoglobin complexes with known hemoglobin concentrations.

[0046] The change in turbidity can be measured by optically measuring the absorbance or scattered light of the immune reaction solution. For example, a general-purpose optical measurement device may be used for the optical measurement, and measurement can be performed using, for example, the automatic biochemical analyzer "JCA-BM2250" (manufactured by JEOL Ltd.) or "JCA-BM6070" (manufactured by JEOL Ltd.).

[0047] In the measurement method of the present invention, by contacting a sample with an insoluble carrier carrying an anti-haptoglobin antibody in addition to a conventional insoluble carrier carrying an anti-hemoglobin antibody, the decrease in the measured hemoglobin value caused by the addition of haptoglobin can be suppressed, and hemoglobin in the sample can be detected and measured more accurately. Furthermore, when haptoglobin is not added when measuring hemoglobin in a sample (i.e., when step (1) is not performed), even if haptoglobin (whether free haptoglobin, a hemoglobin-haptoglobin complex intermediate, or a complete hemoglobin-haptoglobin complex) is originally present in the sample, contacting the sample with an insoluble carrier carrying an anti-hemoglobin antibody and an insoluble carrier carrying an anti-haptoglobin antibody and allowing immunoagglutination (i.e., only step (2)) suppresses the decrease in the measured hemoglobin value and enables accurate detection and measurement of hemoglobin in the sample.

[0048] The present invention further provides a kit that can be used to detect hemoglobin in a sample by the above-mentioned method. The kit may include components such as a measurement reagent containing an antihemoglobin antibody-carrying insoluble carrier and an antihaptoglobin antibody-carrying insoluble carrier, haptoglobin (preferably a preservative solution containing haptoglobin), a calibrator, and a control, and may also include components such as an instrument and container for collecting the sample and a preservative solution for preserving the sample. [Example]

[0049] <Examples 1-1 to 4-2, Comparative Example 1> [Preparation of measurement reagents] As measurement reagents, a first reagent and a second reagent were prepared. As the first reagent, 50 mM HEPES buffer (pH 7.4) was used. As the second reagent, 50 mM HEPES buffer (pH 7.4), polystyrene latex carrying each of multiple types of anti-hemoglobin monoclonal antibodies (anti-Hb antibodies), and polystyrene latex carrying an anti-haptoglobin monoclonal antibody were mixed at the concentrations shown in Table 1 to prepare reagents for immunoagglutination measurement in Examples 1-1 to 1-4 and Comparative Example 1, each having the final latex concentration shown in Table 1. Similarly, as the second reagent, reagents for immunoagglutination measurement in Examples 2-1 to 2-3, Examples 3-1 to 3-3, and Examples 4-1 to 4-2 were mixed at predetermined concentrations to prepare reagents for immunoagglutination measurement in Examples 2-1 to 2-3, Examples 3-1 to 3-3, and Examples 4-1 to 4-2, each having the final latex concentration shown in Table 2.

[0050] The anti-haptoglobin monoclonal antibodies used were AO-53 (recognizing the α chain), AO-27 (recognizing the α chain), AO-35 (recognizing the α chain), and AN12-8 (recognizing the β chain).The anti-haptoglobin antibodies used were those that were confirmed not to aggregate with the insoluble carrier carrying the anti-haptoglobin antibody in the amount of free haptoglobin added in the section "Sample Preparation" below.

[0051] The antibody-supported polystyrene latex was prepared by a known method, namely, by mixing each anti-hemoglobin monoclonal antibody or anti-haptoglobin monoclonal antibody with polystyrene latex particles (average particle size 200 nm) to support the anti-hemoglobin monoclonal antibody or anti-haptoglobin monoclonal antibody on the surface of the polystyrene latex particles. [Table 1] [Table 2]

[0052] [Sample preparation] Human hemoglobin (hHb) purified from human blood and human haptoglobin (hHp) (Haptoglobin Human, Phenotype 1-1 (Sigma)) were mixed in the Hb calibrator diluent 'Eiken' (Eiken Chemical Co., Ltd.) to the concentrations shown in Table 3 to prepare samples No. 0 to 6. No. 3 contains 800 μg / L of human hemoglobin mixed with 0.8 units / L of human haptoglobin, and all of the hemoglobin and haptoglobin form complete hemoglobin-haptoglobin complexes.

[0053] [Immune aggregation] The measurement reagents from Examples 1-1 to 1-4, 2-1 to 2-3, 3-1 to 3-3, 4-1 to 4-2, and Comparative Example 1 were added to Samples No. 0 to 6, respectively, and the turbidity was measured using the BM2250 automatic analyzer. The equivalent hemoglobin concentration was calculated based on a previously prepared calibration curve. Furthermore, the relative hemoglobin value (%) relative to No. 0 (sample not containing human haptoglobin) was also calculated. The results are shown in Table 3.

[0054] The measurement conditions for JCA-BM2250 are as follows. Sample volume: 2.0 μL First reagent: 60 μL Second reagent: 30 μL Measurement wavelength: 658nm [Table 3]

[0055] Table 3 shows that when haptoglobin was added during hemoglobin measurement, the Examples in which both anti-haptoglobin antibody-carrying latex and anti-haptoglobin antibody-carrying latex were added were able to suppress the decrease in hemoglobin measurement value due to the addition of haptoglobin, i.e., obtained measurement values ​​closer to the actual values, compared to Comparative Example 1 in which only anti-haptoglobin antibody-carrying latex was added without adding anti-haptoglobin antibody-carrying latex. This result was obtained for both the α-chain-recognizing anti-haptoglobin antibody and the β-chain-recognizing anti-haptoglobin antibody used, demonstrating that the result is independent of the subunit recognized by the anti-haptoglobin antibody.

[0056] <Comparative Examples 2-1 to 2-3, 3-1 to 3-3, and 4 to 7> Instead of the anti-haptoglobin antibody-carrying latex used in Example 1-1, a free anti-haptoglobin antibody not carried on latex was mixed with anti-hemoglobin antibody-carrying latex to prepare a reagent. This was added to samples No. 0 to 6, and an agglutination reaction was similarly induced, followed by calculation of the equivalent hemoglobin concentration and the relative hemoglobin value (%). The composition of the reagents for each comparative example is shown in Table 4, and the results are shown in Table 5. The anti-haptoglobin antibodies AN11-2 and AN12-3 are β-chain-recognizing anti-haptoglobin monoclonal antibodies, and were prepared by known methods.

[0057] Table 5 shows that the addition of free anti-haptoglobin antibody that was not bound to latex could not prevent the decrease in hemoglobin measurement caused by the addition of haptoglobin. In fact, the values ​​tended to become even lower. This is probably because the excess anti-haptoglobin antibody inhibited the binding of the anti-hemoglobin antibody bound to latex with the hemoglobin-haptoglobin complex. [Table 4] [Table 5]

[0058] <Examples 5-1 to 5-5, Comparative Example 8> [Preparation of measurement reagents] As measurement reagents, a first reagent and a second reagent were prepared. As the first reagent, 50 mM HEPES buffer (pH 7.4) was used. As a second reagent, 50 mM HEPES buffer (pH 7.4), polystyrene latex carrying each of several types of anti-hemoglobin monoclonal antibodies (anti-Hb antibodies), and polystyrene latex carrying an anti-haptoglobin monoclonal antibody were mixed at the concentrations shown in Table 6 to prepare reagents for measuring immune agglutination reactions in Examples 5-1 to 5-5 and Comparative Example 8, each having the final latex concentration shown in Table 6. The anti-haptoglobin monoclonal antibodies used were AO-53 (recognizing the α chain) and AN12-8 (recognizing the β chain).The anti-haptoglobin antibodies used were those that were confirmed not to aggregate with the insoluble carrier carrying the anti-haptoglobin antibody in the amount of free haptoglobin added in the section "Sample Preparation" below.

[0059] The confirmation method was similar to the sample preparation and immunoagglutination method described below, except that the second reagent used was 1.0 mg / mL of polystyrene latex loaded with AO-53 (α-chain recognition) or AN12-8 (β-chain recognition), and the sample used was a stock solution (Hb calibrator diluent 'Eiken' (Eiken Chemical Co., Ltd.)) containing free haptoglobin at a concentration of 0 ng / mL or 24.5 pmol / mL (no hemoglobin). Note that a haptoglobin concentration of 24.5 pmol / mL corresponds to an added concentration of 1.2 units / L of free haptoglobin.

[0060] The antibody-supported polystyrene latex was prepared by a known method, namely, by mixing each anti-hemoglobin monoclonal antibody or anti-haptoglobin monoclonal antibody with polystyrene latex particles (average particle size 200 nm) to support the anti-hemoglobin monoclonal antibody or anti-haptoglobin monoclonal antibody on the surface of the polystyrene latex particles. [Table 6]

[0061] [Sample preparation] Two fecal samples (fecal samples 1 and 2) were collected from healthy individuals. Each fecal sample was mixed with human hemoglobin (hHb) and human haptoglobin (hHp) (Haptoglobin Human, Phenotype 1-1, SIGMA) purified from human blood in a storage solution (Hb Calibrator Diluent 'Eiken', Eiken Chemical Co., Ltd.) to a fecal concentration of 0.5% (w / v%). The concentrations shown in Tables 7 and 8 were also added to the sample. Sample No. 3 contained 600 μg / L of human hemoglobin and 0.6 units / L of human haptoglobin, and all of the hemoglobin and haptoglobin formed complete hemoglobin-haptoglobin complexes.

[0062] [Immune aggregation] The measurement reagents from Examples 5-1 to 5-5 and Comparative Example 8 were added to samples No. 0 to 5 of fecal specimens 1 and 2, respectively, and the turbidity was measured using an automated analyzer JCA-BM6070. The hemoglobin concentration was calculated based on a previously prepared calibration curve. Furthermore, the relative hemoglobin concentration (%) relative to No. 0 (sample not containing human haptoglobin) was also calculated. The results are shown in Tables 7 and 8.

[0063] The measurement conditions for JCA-BM6070 are as follows: Sample volume: 2.0 μL First reagent: 60 μL Second reagent: 30 μL Measurement wavelength: 658nm [Table 7] [Table 8]

Claims

1. An immunoassay method comprising the step of measuring the total amount of hemoglobin, using an immunoassay reagent comprising an insoluble carrier carrying an anti-hemoglobin antibody and an insoluble carrier carrying an anti-haptoglobin antibody, with both free hemoglobin and hemoglobin-haptoglobin complexes as measurement targets, The immunological measurement method is an immunoagglutination method, an immunochromatography method, or an ELISA method.

2. 2. The immunoassay method according to claim 1, wherein the insoluble carrier is latex particles and / or gold colloid particles.

3. 1. A method for measuring hemoglobin in a sample, comprising: (1) mixing a specimen with haptoglobin to form a hemoglobin-haptoglobin complex and obtain a sample containing the hemoglobin-haptoglobin complex; (2) measuring the total amount of hemoglobin using the sample obtained in step (1) and an immunoassay reagent containing an insoluble carrier carrying an anti-hemoglobin antibody and an insoluble carrier carrying an anti-haptoglobin antibody, with both free hemoglobin and hemoglobin-haptoglobin complexes as measurement targets; A method for immunologically measuring hemoglobin, comprising:

4. 4. The immunoassay method according to claim 3, wherein the insoluble carrier is latex particles and / or gold colloid particles.

5. 5. The immunological assay method according to claim 3, wherein the sample is feces, saliva, or urine.

6. The immunological measurement method according to any one of claims 3 to 5, wherein haptoglobin is contained in a sample preservation solution.

7. 7. The immunoassay method according to claim 6, wherein the haptoglobin concentration in the sample preservation solution is 0.05 units / L to 50 units / L.

8. The immunological measurement method according to any one of claims 3 to 7, wherein the hemoglobin in the sample contains at least one selected from the group consisting of free hemoglobin, a hemoglobin-haptoglobin complex intermediate, and a complete hemoglobin-haptoglobin complex, and at least a portion of the free hemoglobin forms a hemoglobin-haptoglobin complex intermediate and / or a complete hemoglobin-haptoglobin complex with the haptoglobin after step (1).

9. A method for measuring hemoglobin by an immunological technique, comprising a step of measuring the total amount of hemoglobin using a specimen and an immunoassay reagent including an insoluble carrier carrying an anti-hemoglobin antibody and an insoluble carrier carrying an anti-haptoglobin antibody, with both free hemoglobin and hemoglobin-haptoglobin complexes as measurement targets, The immunological method is an immunoagglutination method, an immunochromatography method, or an ELISA method.

10. The method includes measuring the total amount of hemoglobin by an immunological method using a specimen and an immunoassay reagent including an insoluble carrier carrying an anti-hemoglobin antibody and an insoluble carrier carrying an anti-haptoglobin antibody, with both free hemoglobin and hemoglobin-haptoglobin complexes as measurement targets; A method for suppressing a decrease in the measured value of hemoglobin, wherein the immunological technique is an immunoagglutination method, an immunochromatography method, or an ELISA method.

11. The method according to claim 9 or 10, wherein the insoluble carrier is latex particles and / or gold colloid particles.

12. The method according to any one of claims 9 to 11, wherein the sample is feces, saliva, or urine.

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