TW202429079A - Method for measuring free hemoglobin and reagent for measuring free hemoglobin - Google Patents

Abstract

The present invention provides a method for measuring free hemoglobin, comprising causing an antigen-antibody reaction for measuring free hemoglobin using two or more anti-hemoglobin antibodies, wherein the antigen-antibody reaction is carried out in the presence of an anti-haptoglobin antibody. The present invention also provides a reagent for measuring free hemoglobin, a kit for measuring free hemoglobin, and a method for preventing false high values in free hemoglobin measurement. The measuring method and the measuring reagent according to the present invention make it possible to prevent false high values of the amount of free hemoglobin when free hemoglobin is specifically measured.

Description

Method for determination of free hemoglobin and reagent for determination of free hemoglobin

The present invention relates to a method and a reagent for specifically measuring free hemoglobin, and more particularly to a method and a reagent capable of suppressing false high values when measuring the amount of free hemoglobin.

Hemoglobin in the blood binds to heme-binding globulin in the body to form a heme-heme-binding globulin complex. This complex is eventually engulfed by macrophages in the liver and metabolized via the scavenger receptor CD163. However, if hemolysis occurs due to burns or artificial heart and lungs, and the heme cannot be completely processed by heme-binding globulin in the body, the blood concentration of heme that has not formed a complex with heme-binding globulin (free heme) will increase.

Since free hemoglobin has a small molecular weight, after being excreted into urine, it is taken up by the renal tubular epithelial cells through the glomerulus of the kidney and then decomposed into hemoglobin and hemoglobin. Among them, the hemoglobin iron contained in the hemoglobin acts as a catalyst to produce free radicals, causing necrosis of the proximal tubular epithelial cells and leading to renal tubular damage. Free hemoglobin can be treated with hemoglobin-binding globulin preparations, but in order to administer the appropriate amount of hemoglobin-binding globulin preparations, it is necessary to accurately measure the free hemoglobin concentration.

For example, a method for measuring the concentration of free hemoglobin has been proposed, which is to contact a sample containing free hemoglobin with a solid phase human hemoglobin binding globulin to which human hemoglobin is solid-phase bound, so that the free hemoglobin binds to the human hemoglobin binding globulin on the solid phase, and then an enzyme-labeled anti-human hemoglobin antibody is allowed to function to measure the free hemoglobin bound to the solid phase (see patent document 1). In addition, a method for measuring the concentration of free hemoglobin has been proposed, which is to add an anti-hemoglobin binding globulin antibody to a sample containing free hemoglobin, allow the hemoglobin-hemoglobin binding globulin complex present in the sample to react with the antibody, and then measure the free hemoglobin by enzyme immunoassay (see patent document 2).

[Prior technical literature] [Patent literature] [Patent literature 1] Japanese Patent Publication No. 2-231565 [Patent literature 2] Japanese Patent Publication No. 7-103978

[Problem to be solved by the invention] In the determination of free hemoglobin, there are known methods of measuring free hemoglobin by adsorbing free hemoglobin onto immobilized hemoglobin-binding globulin, washing and separating the free hemoglobin (Patent Document 1), and a method of measuring free hemoglobin by removing the hemoglobin-hemoglobin-binding globulin complex in advance (Patent Document 2). However, both methods are complicated to operate, and their rapidity and measurement processing capacity are also limited. Therefore, what is expected is a highly specific and rapid free hemoglobin immunoassay method.

In response to this, the inventors have discovered that, by using a method for measuring free hemoglobin using two or more specific anti-hemoglobin antibodies, or a method for measuring free hemoglobin in which the reactivity to a hemoglobin-hemoglobin binding globulin complex is 15% or less of the reactivity to free hemoglobin, free hemoglobin can be specifically measured even in an environment where a hemoglobin-hemoglobin binding globulin complex may exist, and thus applied for the present patent.

Furthermore, the inventors have found through research that in the specific determination method of free hemoglobin, the measured value of free hemoglobin may be higher than the actual amount of free hemoglobin (false high value).

The present invention has been made in view of the above problems, and its object is to provide a method and a reagent capable of suppressing false high values of the free hemoglobin amount when specifically measuring free hemoglobin.

[Technical means for solving the problem] The inventors of the present invention conducted research to solve the above-mentioned problem and found that when an antigen-antibody reaction for measuring free hemoglobin is performed using two or more anti-hemoglobin antibodies, if there are many hemoglobin-hemoglobin binding globulin complexes, a false high value of the amount of free hemoglobin will be generated. In addition, it was also found that if the antigen-antibody reaction for measuring free hemoglobin is performed in the presence of an anti-hemoglobin binding globulin antibody, the above-mentioned false high value can be suppressed, thereby completing the present invention. Specifically, the present invention is as follows.

[1] A method for measuring free hemoglobin comprises the following steps: Using two or more anti-hemoglobin antibodies, an antigen-antibody reaction for measuring the free hemoglobin is performed; The antigen-antibody reaction is performed in the presence of an anti-hemoglobin binding globulin antibody. [2] The method for measuring free hemoglobin as described in [1], wherein the antigen-antibody reaction is performed in an environment where a hemoglobin-hemoglobin binding globulin complex may exist. [3] The method for measuring free hemoglobin as described in [1] or [2], wherein the two or more anti-hemoglobin antibodies are a combination of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain. [4] The method for measuring free hemoglobin as described in any one of [1] to [3], wherein at least one of the two or more anti-hemoglobin antibodies is carried on an insoluble carrier. [5] The method for measuring free hemoglobin as described in [4], wherein the insoluble carrier is an insoluble particle. [6] The method for measuring free hemoglobin as described in [5], which is an immunoagglutination method. [7] A reagent for measuring free hemoglobin, which is used to measure free hemoglobin, comprising: Two or more anti-hemoglobin antibodies; and Anti-hemoglobin binding globulin antibodies; The two or more anti-hemoglobin antibodies are used in an antigen-antibody reaction for measuring the free hemoglobin; The anti-hemoglobin binding globulin antibodies constitute a reaction solution for carrying out the antigen-antibody reaction. [8] The reagent for measuring free hemoglobin as described in [7], wherein the antigen-antibody reaction is carried out in an environment where a hemoglobin-hemoglobin binding globulin complex may exist. [9] A reagent for measuring free hemoglobin as described in [7] or [8], wherein the two or more anti-hemoglobin antibodies are a combination of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain. [10] A reagent for measuring free hemoglobin as described in any one of [7] to [9], wherein at least one of the two or more anti-hemoglobin antibodies is carried on an insoluble carrier. [11] A reagent for measuring free hemoglobin as described in [10], wherein the insoluble carrier is an insoluble particle. [12] A reagent for measuring free hemoglobin as described in [11], which is a reagent for immunoagglutination method. [13] A free hemoglobin assay reagent set comprising the free hemoglobin assay reagent described in any one of [7] to [12]. [14] A method for suppressing false high values in free hemoglobin assay, comprising the following steps: Using two or more anti-hemoglobin antibodies to carry out an antigen-antibody reaction for measuring the free hemoglobin; The antigen-antibody reaction is carried out in the presence of an anti-hemoglobin binding globulin antibody.

[Effects of the invention] The measuring method and measuring reagent of the present invention can suppress false high values of the free hemoglobin amount when specifically measuring free hemoglobin.

Hereinafter, embodiments of the present invention will be described.

[Term] (Hemoglobin) Hemoglobin is a protein contained in red blood cells in the body. It has the property of binding to oxygen molecules and participates in the transport of oxygen. Hemoglobin has a tetrameric structure [α ₂ β ₂ ] with two of each of the following two subunits: the α chain (subunit) composed of 141 amino acids, and the β chain (subunit) composed of 146 amino acids. The molecular weight of the α chain is about 15,500, and the molecular weight of the β chain is about 17,000. As described later, they can be separated by electrophoresis, etc. In addition, the molecular weight of hemoglobin as a whole is about 64,500.

(Heme-binding globulin) Heme-binding globulin is a type of glycoprotein that specifically binds to heme to form a heme-heme-binding globulin complex. There are three serotypes of human heme-binding globulin, Hp1-1, Hp2-1, and Hp2-2. In the simplest structure (Hp1-1), heme-binding globulin is composed of two α chains and two β chains, which are connected by S-S bonds. In terms of molecular weight, the Hp1α chain is about 10,000, the Hp2α chain is about 18,000, and the β chain is about 39,000. The total molecular weight of heme-binding globulin, for example, is about 98,000 in Hp1-1.

(Heme-heme binding globulin complex) When heme and heme binding globulin form a complex, one molecule of tetrameric heme [α ₂ β ₂ ] dissociates into two molecules of dimer heme [αβ], and each dimer heme binds to heme binding globulin. At this time, Hp1-1 type heme binding globulin has two sites in the molecule that bind to dimer heme. Therefore, when Hp1-1 type heme binding globulin and tetrameric heme [α ₂ β ₂ ] form a complex, a complex is formed in which two 1/2 molecules of dimer heme [αβ] are bound to one molecule of Hp1-1 type heme binding globulin.

[Free hemoglobin measurement method] One embodiment of the present invention is a method for measuring free hemoglobin, which uses two or more anti-hemoglobin antibodies to carry out an antigen-antibody reaction for measuring free hemoglobin, and the antigen-antibody reaction is carried out in the presence of an anti-hemoglobin binding globulin antibody.

The inventors of the present invention believe that the mechanism by which the false high value of the free hemoglobin amount can be suppressed by carrying out the antigen-antibody reaction in the presence of an anti-hemoglobin antibody is as follows: Antibodies obtained using tetrameric free hemoglobin [α ₂ β ₂ ] as an antigen also react with dimeric hemoglobin [αβ] contained in the hemoglobin-hemoglobin complex. Therefore, it has been very difficult to obtain anti-free hemoglobin antibodies that do not bind to the hemoglobin-hemoglobin complex using conventional animal immunization methods. The free hemoglobin measurement method used in the present embodiment is to select two or more anti-hemoglobin antibodies so that the reaction with the hemoglobin-hemoglobin complex is sufficiently suppressed and a specific reaction with free hemoglobin occurs. Specifically, as shown in the reference examples described below, the reactivity to the heme-heme binding globulin complex can be made 15% or less of the reactivity to free heme. This reactivity can be said to be the result of sufficient suppression of the reaction to the heme-heme binding globulin complex, but in the antigen-antibody reaction, if there are many heme-heme binding globulin complexes, the reaction with the complex cannot be ignored. This is believed to be the reason why the free heme amount produces a falsely high value.

In contrast, in the present embodiment, when the antigen-antibody reaction for measuring free hemoglobin is carried out in the presence of an anti-hemoglobin antibody, the anti-hemoglobin antibody forms a stereogenic barrier to the anti-hemoglobin antibody in the hemoglobin-hemoglobin complex. As a result, the reaction with the hemoglobin-hemoglobin complex (especially the dimeric hemoglobin [αβ] contained in the complex) is fully inhibited. In this way, it is possible to fully suppress the false high value of the free hemoglobin amount when there are many hemoglobin-hemoglobin complexes. However, the mechanism of action that can suppress the false high value of the free hemoglobin amount is not limited to the above-mentioned mechanism of action.

(Immunological method) The method for measuring free hemoglobin in this embodiment is not particularly limited as long as it is a method using antigen-antibody reaction, that is, an immunological method, and examples thereof include immunoagglutination method (e.g., latex agglutination method, colloidal gold agglutination method, etc.), ELISA method, immunochromatography method, etc. Among these methods, the measurement method of this embodiment is preferably using immunoagglutination method, and more preferably using latex agglutination method.

(Anti-heme antibodies) In this embodiment, two or more anti-heme antibodies are used to specifically measure free heme. The above-mentioned antigen-antibody reaction for measuring free heme is preferably an antigen-antibody reaction to two or more different antigen epitopes in the same antigen (heme in this embodiment). In other words, the immunological method of this embodiment is preferably a method using the two or more antigen-antibody reactions.

The two or more anti-hemoglobin antibodies used in this embodiment are not particularly limited as long as they are a combination that can specifically measure free hemoglobin. For example, a combination of an antibody specific to the hemoglobin α chain and an antibody specific to the hemoglobin β chain; a combination of antibodies specific to the boundary between the hemoglobin α chain and the β chain, etc. can be used. Among them, a combination of an antibody specific to the hemoglobin α chain and an antibody specific to the hemoglobin β chain is particularly preferred.

Here, "antibodies specific for the heme α chain" refers to anti-heme antibodies that react much more strongly to the heme α chain than to the heme β chain. In addition, in this specification, this property of the anti-heme antibody is also referred to as the specificity of the anti-heme antibody.

For example, the specificity of the anti-hemoglobin antibody can be defined as a property that the reaction to the hemoglobin α chain is 75% or more, preferably 80% or more, relative to the sum of the reaction to the hemoglobin α chain and the reaction to the β chain. The same applies to "antibodies specific to the hemoglobin β chain". The specificity to the hemoglobin α chain is evaluated by the following steps as shown in the embodiment described below: using the anti-hemoglobin antibody to be evaluated as a primary antibody for hemoglobin, performing a Western blot method; accumulating the band brightness of the area where the α chain exists on the membrane and the band brightness of the area where the β chain exists; calculating the ratio of the α chain cumulative value to the total amount of the two cumulative values. The specificity to the hemoglobin β chain is the same.

Here, the inventors believe that the mechanism of action of free heme can be specifically determined by using a combination of an antibody specific for the heme α chain and an antibody specific for the heme β chain as follows: As for the heme portion of the heme-heme-binding globulin complex, that is, the heme in the αβ dimer structure that forms a complex with the heme-binding globulin, it is believed that it cannot bind to the α chain specific antibody and the β chain specific antibody at the same time due to factors such as stereogenic barriers. In contrast, since free hemoglobin is a tetrameric hemoglobin of α ₂ β ₂ structure and does not form a complex with heme-binding globulin, it is believed that it can bind to both α-chain specific antibodies and β-chain specific antibodies at the same time. However, the mechanism of action of specifically measuring free hemoglobin using a combination of α-chain specific antibodies and β-chain specific antibodies is not limited to the above-mentioned mechanism of action.

The type of anti-heme antibody that can be used in this embodiment is not particularly limited. For example, the animal species from which the antibody is derived is not particularly limited, and examples thereof include antibodies derived from animals such as rabbits, goats, mice, rats, horses, and sheep. Multiple antibodies obtained from the serum of an immunized animal of the object to be measured by a known method, or single antibodies obtained by cell fusion of the spleen of an immunized animal of the object to be measured and bone marrow cancer cells can be used. In addition, fragments of these antibodies [e.g., F(ab')2, Fab, Fab', or Fv] can also be used.

(Insoluble carrier) Among the two or more anti-hemoglobin antibodies used in the present embodiment, at least one may be carried on an insoluble carrier, or two or more may be carried on an insoluble carrier. The insoluble carrier is not particularly limited as long as it is a carrier that can carry antibodies, and can be appropriately selected according to the type of the above-mentioned immunological method. For example, the insoluble carrier can be exemplified by insoluble particles that can be used in immunological methods, and the insoluble particles can be exemplified by commonly used metal colloid particles such as colloidal gold particles, latex particles, silica particles, magnetic particles, fluorescent particles, red blood cells, etc. The insoluble particles are preferably latex particles, and more preferably polystyrene latex particles. The insoluble carrier is preferably in the form of particles, and its average particle size is preferably 5 to 1,000 nm, more preferably 30 to 500 nm, and further preferably 75 to 350 nm, but its use is not particularly limited to this range.

Carrying antibodies means that the antibodies are fixed on the surface of an insoluble carrier by physical adsorption or chemical binding. For example, a carrying method (immobilization method) is a known technique for immobilizing antibodies on insoluble carrier particles by mixing antibodies and insoluble carrier particles so that the antibodies are physically adsorbed on the surface of insoluble carrier particles. In addition, when insoluble carrier particles with amino or carboxyl groups introduced on the surface are used, the antibodies can be immobilized on the surface of insoluble carrier particles by chemical bonding using glutaraldehyde or carboxylic acid imide reagents. The amount of antibodies carried is not particularly limited, as long as it is 0.5~2,000 μg/mg latex, it can also be 1~1,000 μg/mg latex or 2~500 μg/mg latex. The amount of antibody carried can be calculated by subtracting the amount of antibody after immobilization on the insoluble carrier from the amount of antibody before immobilization on the insoluble carrier.

In addition, when the insoluble carrier carries two or more anti-hemoglobin antibodies, they may all be carried on the same insoluble carrier. Alternatively, a plurality of insoluble carriers carrying one anti-hemoglobin antibody may be mixed on one insoluble carrier. In this case, the insoluble carriers used to carry different types of anti-hemoglobin antibodies may be the same type of insoluble carriers or different types of insoluble carriers with different materials or particle sizes.

(Anti-HBG Antibody)

The anti-heme-binding globulin antibody used in this embodiment is not particularly limited as long as it can react with heme-binding globulin in an antigen-antibody manner. For example, it can be an antibody specific to the heme α chain or an antibody specific to the β chain. In addition, antibodies specific to the α chain or β chain of heme-binding globulin can be evaluated in the same manner as the aforementioned specificity to the heme α chain or β chain. Among them, as shown in the examples described below, in this embodiment, whether it is an antibody specific to the heme-binding globulin α chain or an antibody specific to the β chain, it can fully suppress the false high value of the free hemoglobin amount.

The types of anti-heme-binding globulin antibodies that can be used in this embodiment are not particularly limited. For example, the animal species from which the antibodies are derived are not particularly limited, and examples thereof include antibodies derived from animals such as rabbits, goats, mice, rats, horses, and sheep. Multiple antibodies obtained from the serum of an immunized animal of the object to be measured using a known method, or single antibodies obtained by cell fusion of the spleen of an immunized animal of the object to be measured with bone marrow cancer cells can be used. In addition, fragments of these antibodies [e.g., F(ab')2, Fab, Fab', or Fv] can also be used. In addition, the anti-heme-binding globulin antibodies used in this embodiment are preferably not carried on an insoluble carrier.

(Measurement object) The measurement object applied to the measurement method of this embodiment is not particularly limited as long as it can contain free hemoglobin. Examples of the measurement object include blood, serum, plasma, urine, lymph, puncture fluid, spinal fluid, sweat, saliva, gastric juice, alveolar lavage fluid, feces and other specimens from organisms; samples obtained by diluting or suspending these specimens, etc.

(Measurement) In this embodiment, after an antigen-antibody reaction for measuring free hemoglobin is carried out in the presence of an anti-hemoglobin binding globulin antibody, the measurement is then carried out by a known method. The object of measurement can be appropriately set according to the immunological method adopted. For example, if it is an immunoagglutination method, the amount of turbidity change within a specific time can be cited; if it is an ELISA method, the amount of color development/absorbance caused by the labeling of the labeled antibody can be cited. Therefore, the method for measuring these characteristics can also adopt a known method, such as an optical method, and a general optical measurement device can be used.

The measurement method of the above embodiment is to use two or more anti-heme antibodies to carry out an antigen-antibody reaction for measuring free heme, and even if it is carried out in an environment where, for example, a heme-heme binding globulin complex may exist, free heme can be specifically measured. Moreover, by carrying out the above antigen-antibody reaction in the presence of an anti-heme binding globulin antibody, even in the presence of a large amount of heme-heme binding globulin complex, false high values of the amount of free heme can be suppressed (equivalent to the method for suppressing false high values in the measurement of free heme of the present invention). According to the measurement method of this embodiment, there is no need to remove the hemoglobin-hemoglobin binding globulin complex before the antigen-antibody reaction for measuring free hemoglobin. Therefore, not only is the operation simple, but also free hemoglobin can be measured specifically and false high values of free hemoglobin can be suppressed.

[Reagent for measuring free hemoglobin] A reagent for measuring free hemoglobin in one embodiment of the present invention comprises two or more anti-hemoglobin antibodies and anti-hemoglobin binding globulin antibodies. The two or more anti-hemoglobin antibodies are used in an antigen-antibody reaction for measuring free hemoglobin. On the other hand, the anti-hemoglobin binding globulin antibody constitutes a reaction solution for carrying out the antigen-antibody reaction.

The anti-hemoglobin antibody used in the reagent of this embodiment can be the antibody described in the above-mentioned free hemoglobin measurement method. Among the above-mentioned two or more anti-hemoglobin antibodies used in the reagent of this embodiment, at least one may be carried on an insoluble carrier, and two or more may be carried on an insoluble carrier. The insoluble carrier may be an insoluble particle. The types of insoluble carriers and insoluble particles, the method of carrying antibodies, etc. are the same as those described in the above-mentioned measurement method.

In addition, the anti-heme-binding globulin antibody used in the reagent of this embodiment can be the antibody described in the above-mentioned free hemoglobin measurement method. The anti-heme-binding globulin antibody constitutes a reaction solution in the reagent of this embodiment, and the reaction solution is used for the antigen-antibody reaction for measuring free hemoglobin. Here, the point that the anti-heme-binding globulin antibody constitutes a reaction solution for antigen-antibody reaction refers to the state in which the anti-heme-binding globulin antibody exists in the reaction solution for antigen-antibody reaction. More specifically, the anti-heme-binding globulin antibody can be added to the liquid constituting the reaction solution at any stage before the start of the above-mentioned antigen-antibody reaction.

(Form of the assay reagent, etc.) The assay reagent of this embodiment is a reagent having the aforementioned structure and using an immunological method to assay free hemoglobin. As long as the reagent satisfies these conditions, the form of the assay reagent is not particularly limited, and reagents using, for example, immunoagglutination methods (e.g., latex agglutination method, colloidal gold agglutination method, etc.), ELISA method, immunochromatography method, etc., can be used. Among them, the immunoagglutination method reagent is preferred, and the latex agglutination method reagent is more preferred.

The assay reagent of this embodiment may be composed of a two-reagent system including a reagent not containing an insoluble carrier (first reagent) and a reagent containing an insoluble carrier carrying an antibody (antibody-carrying insoluble carrier) (second reagent), or may be composed of only a single reagent system including a reagent containing an antibody-carrying insoluble carrier. Here, the first reagent may be used to adjust the assay environment, for example, as a diluent to adjust the concentration of the assay object or impurities in the reaction system, or to adjust the reaction rate, etc. The second reagent contains an antibody-carrying insoluble carrier, and will produce an immunoagglutination reaction when mixed with the first reagent and the sample. The first reagent and the second reagent may contain pH buffer, salt, surfactant, coagulation promoter, preservative, etc. The pH value during the coagulation reaction is preferably 5~9.

Furthermore, the reaction solution is obtained by mixing the assay reagent with the sample, but the concentration of the insoluble carrier in the reaction solution can be appropriately selected from the range of, for example, 0.0001 mg/mL to 10 mg/mL, depending on the particle size of the insoluble carrier used and the overall design of the assay system. The concentration of the insoluble carrier carrying the anti-hemoglobin antibody in the assay reagent can be 0.01 to 5 mg/mL or 0.05 to 1 mg/mL. In addition, since the concentration of the insoluble carrier in the second reagent is diluted by mixing with the first reagent or the sample when used, the concentration of the insoluble carrier in the second reagent can be appropriately selected according to the dilution ratio. For example, when it is diluted twice, it can be appropriately adjusted to 0.0002~20 mg/mL, and when it is diluted three times, it can be appropriately adjusted to 0.0003~30 mg/mL.

When the assay reagent of this embodiment is composed of the above two reagent systems, the anti-hemoglobin binding globulin antibody can be contained in the first reagent or in the second reagent. In addition, when the assay reagent of this embodiment has a liquid for diluting or suspending the assay object, the anti-hemoglobin binding globulin antibody can be contained in the dilution/suspension liquid.

The above-mentioned free hemoglobin assay reagent has two or more anti-hemoglobin antibodies, which are used in the antigen-antibody reaction for measuring free hemoglobin. Thus, for example, even in an environment where a hemoglobin-hemoglobin binding globulin complex may exist, free hemoglobin can be specifically measured. Moreover, by forming a reaction solution with anti-hemoglobin binding globulin antibodies to perform an antigen-antibody reaction for measuring free hemoglobin, the assay reagent of this embodiment can suppress false high values of the free hemoglobin amount even in the presence of a large amount of hemoglobin-hemoglobin binding globulin complex. The assay reagent of this embodiment does not require the step of removing the hemoglobin-hemoglobin binding globulin complex before the antigen-antibody reaction for measuring free hemoglobin. Therefore, not only is the operation simple, but also free hemoglobin can be specifically measured and false high values of free hemoglobin can be suppressed.

[Other embodiments] In addition, the present invention also provides a free hemoglobin assay reagent set, which includes the free hemoglobin assay reagent described above. The assay reagent contained in the reagent set may include, for example, an anti-hemoglobin antibody carrying an insoluble carrier. In addition, the free hemoglobin assay reagent set may include, in addition to the assay reagent, a calibrator and a controller, and may also include an instrument and a container for collecting a specimen, a preservation solution for preserving the specimen, and the like.

The embodiments described above are recorded for easy understanding of the present invention and are not intended to limit the present invention. Therefore, each element disclosed in the above embodiments also includes all design changes or equivalents within the technical scope of the present invention.

[Example] The present invention is described in more detail below through the demonstration of manufacturing examples, test examples, etc., but the present invention is not limited to the following manufacturing examples, test examples, etc.

Reference Example 1 : Preparation of anti-human hemoglobin monoclonal antibodies (1) Immunization of mice Immunize mice with hemoglobin. After separate immunization, the antibody titer of the mice is measured using the RIA method of the double antibody method of hemoglobin labeled with ^125I . Finally, the mice with high antiserum titer are selected. (2) Cell fusion The spleen of the selected mouse is removed to prepare spleen cells. The prepared spleen cells and mouse bone marrow cancer cells are subjected to cell fusion by electrofusion, suspended in the fusion cell selection medium, and then inoculated into a 96-well microplate. (3) Screening of cell lines producing monoclonal antibodies Ten days after cell fusion, cells producing anti-human hemoglobin monoclonal antibodies were screened using the RIA method using double antibody method with ¹²⁵ I-labeled hemoglobin, and 10 copies were obtained.

Reference Example 2 : Confirmation of the specificity of anti-hemoglobin antibodies Human hemoglobin (manufactured by Eiken Chemical Co., Ltd.) purified from human type O blood was electrophoresed using a pre-made polyacrylamide electrophoresis gel (manufactured by ATTO) and an AE-6530 RAPIDAS mini flat-plate electrophoresis tank (manufactured by ATTO). The protein after electrophoresis was transferred to a PVDF membrane using a semi-dry transfer tank (manufactured by Bio-Rad). Western blotting was performed using the antibody obtained in Reference Example 1 (1 μg/mL) as the primary antibody and an HRP-labeled anti-mouse IgG antibody recognition antibody (rabbit-derived multi-strain antibody, manufactured by Cappel) as the secondary antibody, and the HRP was illuminated using Western Lightning ECL Pro (manufactured by PerkinElmer) to obtain band images. The band images were analyzed using CS Analyzer ver.3.0 (manufactured by ATTO Corporation), and the measurement mode was: the band brightness of the lane was calculated as a cumulative value using the designated area regional density method. Then, the band brightness cumulative values of the α chain and β chain were calculated based on the band brightness of the Western blot method. In addition, in order to correct the band brightness, the coefficient 0.783 calculated by the method described below was multiplied by the band cumulative value of the α chain. The band brightness ratio of each subunit was calculated when the α chain cumulative value + the β chain cumulative value was set to 100%. Based on this result, antibodies showing a cumulative value ratio of α chain bands of 75% or more were judged to be specific antibodies to heme α chain, and antibodies showing a cumulative value ratio of β chain bands of 75% or more were judged to be specific antibodies to heme β chain. The results are shown in Table 1.

In addition, in order to calibrate the band brightness, human hemoglobin was electrophoresed in the same manner as above using a pre-made electrophoresis gel of polyacrylamide (manufactured by ATTO) and an AE-6530 RAPIDAS mini flat-plate electrophoresis tank (manufactured by ATTO), and CBB staining was performed. Then, the cumulative values of the band brightness of the α chain and the β chain of the obtained electrophoresis image were calculated, and the ratio of the band brightness of each subunit was calculated when the cumulative value of the α chain + the cumulative value of the β chain was set to 100%. From this result, it can be seen that the band brightness of the β chain and the α chain is 43.9%:56.1%. In order to normalize the band brightness, the coefficient 0.783 was multiplied by the cumulative value of the α chain.

[Table 1] Sample Antibody Name Identify the part Cumulative value Proportion β α (after correction) β α 1 No.4 β 18732 438 97.7% 2.3% 2 No.11 β 160859 0 100.0% 0.0% 3 No.16 α 434 3180 12.0% 88.0% 4 No.8 α 465 2913 13.8% 86.2% 5 No.1 α 992 9466 9.5% 90.5% 6 No.12 α 312 1038 23.1% 76.9% 7 No.21 β 24696 362 98.6% 1.4% 8 No.23 β 9750 352 96.5% 3.5% 9 No.24 β 1118 122 90.2% 9.8% 10 No.32 β 6111 197 96.9% 3.1%

As shown in Table 1, among the antibodies obtained in Reference Example 1, the ratio of the α chain cumulative value of No. 1, 8, 12, and 16 is 75% or more relative to the total value of the α chain cumulative value and the β chain cumulative value, and therefore they are identified as α chain specific antibodies. In contrast, the ratio of the β chain cumulative value of No. 4, 11, 21, 23, 24, and 32 is 75% or more relative to the total value of the α chain cumulative value and the β chain cumulative value, and therefore they are identified as β chain specific antibodies.

Reference Example 3 : Reactivity of Monoclonal Antibodies The antibodies obtained in Reference Example 1 were used to evaluate their reactivity to free heme and to heme-heme binding globulin complexes. The following method was used to compare the degree of aggregation of each anti-human heme monoclonal antibody when it was immobilized on polystyrene latex particles and reacted with a sample containing heme or heme-heme binding globulin complexes.

(1) Immobilizing monoclonal antibodies on polystyrene latex particles Immobilizing antibodies on polystyrene latex particles is performed using known techniques. Each anti-hemoglobin monoclonal antibody is mixed with polystyrene latex particles (particle size 200 nm) and then immobilized separately. The anti-hemoglobin monoclonal antibody is carried on the surface of the polystyrene latex particles to prepare an antibody-carrying polystyrene latex particle solution.

(2) Sample preparation The heme-heme binding globulin complex sample was prepared by mixing 32.3 pmol/mL heme and 32.3 pmol/mL heme binding globulin in equal amounts in 50 mM HEPES buffer (pH 7.4). The heme-only sample was prepared by using a solution containing 16.1 pmol/mL heme in 50 mM HEPES buffer (pH 7.4).

(3) Method for measuring latex aggregation Aggregation reaction was carried out using the wells of a 96-well flat-bottom microplate. Specifically, 100 μL of 50 mM HEPES buffer (pH 7.4) was injected into each well of the microplate, and then 50 μL of a polystyrene latex particle solution immobilized with each antibody was added, followed by 30 μL of the sample prepared in (2). Using an absorbance microplate analyzer (TECAN JAPAN), the absorbance was measured at a wavelength of 660 nm 10 seconds after the sample was added and 5 minutes and 10 seconds after the sample was added, and the difference between the two was used as the aggregation index. Next, the ratio of the aggregation reactivity to heme and the aggregation reactivity to the heme-heme binding globulin complex was determined. The results are shown in Table 2.

[Table 2] Combination of monoclonal antibodies Hemoglobin (⊿OD×10,000) Heme-heme-binding globulin complex (⊿OD×10,000) ratio No.1 No.32 1790 30 1.68% No.8 No.4 1380 50 3.62% No.8 No.21 1080 0 0.00% No.8 No.23 540 0 0.00% No.8 No.24 680 0 0.00% No.8 No.32 580 0 0.00% No.16 No.4 2680 20 0.75% No.16 No.11 3120 80 2.56% No.16 No.23 1280 0 0.00% No.16 No.24 1700 0 0.00% No.16 No.32 1950 0 0.00%

As shown in Table 2, a specific reaction to free hemoglobin was confirmed in the antibody combination used. The reactivity to the hemoglobin-heme-binding globulin complex was less than 15% of the reactivity to free hemoglobin.

Reference Example 4 : Free Hemoglobin Assay Reagent Using the antibody obtained in Reference Example 1, an immunoagglutination assay reagent was prepared.

Prepare the first and second reagents as assay reagents. The first reagent uses 50mM HEPES buffer (pH7.4). The second reagent is a mixture of polystyrene latexes carrying the anti-hemoglobin monoclonal antibody (anti-Hb antibody) of Reference Example 2, and the latex concentration is adjusted to 1.0 mg/mL to prepare an immunoagglutination assay reagent. The antibody-carrying insoluble carrier is prepared by mixing each anti-hemoglobin monoclonal antibody and polystyrene latex particles (average particle size 100nm) to carry the anti-hemoglobin monoclonal antibody on the surface of the polystyrene latex particles. This reagent uses No.16 as the α-chain specific anti-heme monoclonal antibody and No.11 as the β-chain specific anti-heme monoclonal antibody.

Reference Example 5 : Comparison with other assays The assays were compared using a sample consisting of a mixture of free heme and heme-heme binding globulin complex.

(1) Preparation of purified free heme and heme-heme binding globulin complex JCCRM912-3H (Medical Standard Material Mechanism for Detection) was diluted to 2.0 mg/mL with physiological saline. In addition, 1 mg of freeze-dried heme binding globulin (SIGMA-ALDRICH) derived from human mixed plasma was dissolved in 303 μL of physiological saline to prepare a heme binding globulin solution (3.3 mg/mL).

(2) Mixing of free hemoglobin and heme-heme binding globulin complex A certain amount of free hemoglobin (free-Hb) prepared at 2.0 mg/mL was taken and mixed with a heme binding globulin (Hpt) solution prepared at 3.3 mg/mL in the volume shown in Table 3 to prepare solutions (specimens) with different mixing ratios. In addition, free-Complex in Table 3 represents the molar ratio of free hemoglobin to heme-heme binding globulin complex.

[Table 3] free-Complex 0:1 1:3 1:1 3:1 1:0 free-Hb (2.0 mg/mL) 200 μL 200 μL 200 μL 200 μL 200 μL Hpt (3.3 mg/mL) 200 μL 150 μL 100 μL 50 μL 0 μL Physiological saline 0 μL 50 μL 100 μL 150 μL 200 μL Total 400 μL 400 μL 400 μL 400 μL 400 μL Free-Hb concentration (mg/mL) 0.00 0.25 0.50 0.75 1.00

(3) Determination of free hemoglobin/hemoglobin-heme binding globulin complex mixed solution A commercially available hemoglobin colorimetric assay reagent was used for comparison with the immunoagglutination assay reagent prepared in Reference Example 4. The hemoglobin colorimetric assay reagent is a reagent that quantifies hemoglobin by dissolving the red blood cell membrane with sodium dodecyl sulfate and then measuring the absorbance of the dissolved hemoglobin. However, the hemolysis step was omitted in this test. Each mixed solution (sample) of the free hemoglobin (free-Hb)/hemoglobin-heme binding globulin (Hb-Hp) complex prepared in (2) above was mixed with the hemoglobin colorimetric assay reagent and then measured at 540nm using a spectrophotometer (manufactured by Shimadzu Corporation, UV-1900).

On the other hand, the immunoagglutination reaction assay reagent (Latex reagent) prepared in Reference Example 4 is prepared by diluting each mixed solution (specimen) prepared in (2) above by 50 times with physiological saline, and then using the biochemical automatic analyzer JCA-BM6070 for measurement under the following conditions. Sample volume: 1.0μL, first reagent: 50μL, second reagent: 50μL, measurement wavelength: 658nm The results are shown in Table 4 and Figure 1.

[Table 4] Free-Hb concentration (mg/mL) 0.000 0.250 0.500 0.750 1.000 Colorimetric method 1.036 0.990 0.984 0.977 0.971 Latex reagent (this invention) 0.065 0.387 0.544 0.706 1.010

If a commercially available hemoglobin colorimetric quantitative reagent is used, the measured value will not change even if the ratio of free hemoglobin to hemoglobin-hemoglobin complex changes, so it is impossible to distinguish the two and perform the measurement. In contrast, the immunoagglutination reaction measurement reagent (latex reagent) of Reference Example 4 is not affected by the amount of hemoglobin-hemoglobin complex, and a measurement value proportional to the free hemoglobin concentration can be obtained. The above results show that the latex reagent of Reference Example 4 can specifically measure free hemoglobin.

Example 1 : Preparation of anti-heme-binding globulin monoclonal antibody Except that the mice were immunized with human heme-binding globulin, the same operation as in Reference Example 1 was performed to obtain 5 copies of anti-human heme-binding globulin monoclonal antibody.

Example 2 : Anti-heme-binding globulin antibody assay reagent (1) Preparation of reagents One of the anti-heme-binding globulin antibodies obtained in Example 1 (AO-53 in Example 4 described below) was added to 50 mM HEPES buffer (pH 7.4) to a final concentration of 10 μg/mL to prepare a first reagent. The second reagent used was the reagent prepared in Reference Example 4.

(2) Preparation of samples and measurement conditions Total human hemoglobin common reference standard substance JCCRM912-3 (manufactured by the Institute for the Detection of Medical Standard Substances) was mixed with the Hb calibration diluent "Eiken" (manufactured by Eiken Chemical Co., Ltd.) to the target concentration. The obtained sample was measured under the following conditions using the measurement reagent prepared in (1) above (with anti-hemoglobin binding globulin antibody added) and the measurement reagent of Reference Example 4 (without anti-hemoglobin binding globulin antibody added). Sample volume: 1.0μL First reagent: 50μL Second reagent: 50μL Measurement wavelength: 658nm

The machine used for the above measurement is the automatic biochemical analyzer JCA-BM6070. In addition, the machine has a function to calculate the calibration curve based on the curve of the turbidity change (ΔOD). First, the calibration curve was prepared using the above-mentioned free hemoglobin standard substance diluted to various concentrations. The values of the turbidity change (ΔOD) at each concentration at this time are shown in Table 5 and Figure 2A. In addition, the free hemoglobin sample diluted to various concentrations was measured using the measurement reagent of Example 2, and the dilution linearity was evaluated. The results are shown in Figure 2B. In addition, the measurement values in Figure 2B are converted using the calibration curve of Example 2 obtained from Table 5, etc.

[Table 5] Hb concentration (µg/mL) Turbidity (⊿OD) Reference Example 4 (No anti-Hpt antibody) Example 2 (addition of anti-Hpt antibody) 0 -0.0012 -0.0009 10 0.0093 0.0094 25 0.0679 0.0679 50 0.4755 0.4796 75 0.7713 0.7697 100 0.8746 0.8700

As shown in Table 5 and Figure 2, for samples containing only free hemoglobin, the addition of anti-hemoglobin antibodies did not hinder the preparation of the test curve (Table 5, Figure 2A); even in the presence of anti-hemoglobin antibodies, free hemoglobin could be measured in proportion to the concentration (Figure 2B).

Embodiment 3 ：Determination of excess heme-binding globulin sample

The samples were prepared by mixing hemoglobin and heme-binding globulin to the target concentrations shown in Table 6 using the Hb calibration diluent "Eiken" (manufactured by Eiken Chemical Co., Ltd.). The total human hemoglobin common reference standard JCCRM912-3 (manufactured by the Institute for the Examination of Medical Standards) was used as hemoglobin, and heme-binding globulin derived from human mixed plasma (manufactured by SIGMA-ALDRICH) was used as heme-binding globulin.

The obtained samples were measured under the following conditions using the assay reagent of Example 2 (with anti-heme-binding globulin antibody added) and the assay reagent of Reference Example 4 (without anti-heme-binding globulin antibody added). The machine used for the above-mentioned measurement was the automatic biochemical analyzer JCA-BM6070. Sample volume: 1.0μL　First reagent: 50μL　Second reagent: 50μL　Measurement wavelength: 658nm The results are shown in Table 6 and Figure 3.

[Table 6] Test sample No. 1 2 3 4 5 6 Sample Hb (μmol/mL) 0.81 0.81 0.81 0.81 0.81 0.81 Hpt (μmol/mL) 0.00 0.24 0.49 0.93 1.05 1.30 Free-Hb target value (μg/mL) 50 35 20 0 0 0 Free-Hb measurement value (μg/mL) Reference Example 4 (without anti-Hpt antibody) 53 34 twenty two 6 5 4 Example 2 (addition of anti-Hpt antibody) 53 35 twenty two 2 1 1

As shown in Table 6 and Figure 3, when the assay reagent of Reference Example 4 (without anti-heme-binding globulin antibody), it was found that in samples 4 to 6 where a high concentration of heme-heme complex was present, the measured values tended to be higher than the target value of the amount of free heme in the assay sample, which can be considered to be a false high value. In contrast, when the assay reagent of Example 2 (with anti-heme-binding globulin antibody) was used for the assay, in samples 4 to 6, it can be considered that the measured values of the amount of free heme became close to the target value, and the false high value was obviously suppressed.

Example 4 : Assay reagents using various anti-heme-binding globulin antibodies (1) Preparation of reagents The first reagent was prepared by using other anti-heme-binding globulin antibodies obtained in Example 1 instead of the anti-heme-binding globulin antibody (AO-53) used in Example 2. That is, the anti-heme-binding globulin antibody obtained in Example 1 was added to 50 mM HEPES buffer (pH 7.4) to a final concentration of 10 μg/mL to prepare the first reagent. The second reagent was prepared using the reagent prepared in Reference Example 4.

In addition, the heme-binding globulin specificity (heme-binding globulin recognition site) of each anti-heme-binding globulin antibody was confirmed by Western blotting in the same manner as in Reference Example 2. The results are shown in Table 7.

(2) Sample preparation and measurement conditions The total human hemoglobin common reference standard substance JCCRM912-3 (manufactured by the Institute for the Detection of Medical Standard Substances) was added to normal human serum or serum residue samples as purified hemoglobin to prepare serum samples. The obtained samples were measured under the following conditions using the measurement reagent prepared in (1) above (with anti-hemoglobin binding globulin antibody added) and the measurement reagent of Reference Example 4 (without anti-hemoglobin binding globulin antibody added). Sample volume: 1.0 μL　First reagent: 50 μL　Second reagent: 50 μL　Measurement wavelength: 658 nm The results are shown in Table 7 and Figure 4.

[Table 7] antibody No Hpt antibody AO-53 AN12-3 AO-27 AO-35 AO-1 Identify the part - α chain α chain β chain β chain α chain Serum samples Sample 1 38 26 26 28 28 twenty four Sample 2 41 18 15 16 twenty two 12 Sample 3 16 2 1 1 1 0 Sample 4 36 13 9 9 14 7 Sample 5 17 4 1 1 2 1 Sample 6 16 4 2 2 2 2 Sample 7 29 8 5 4 7 3

As shown in Table 7 and Figure 4, even when anti-HBG antibodies other than AO-53 were used, an inhibitory effect on the increase (false high value) in the free hemoglobin measurement value was confirmed. In addition, it is believed that this inhibitory effect can be obtained regardless of the hemoglobin recognition site in the anti-HBG antibody.

Example 5 : Determination of free hemoglobin in feces (1) Preparation of samples Purified hemoglobin (manufactured by Eiken Chemical Co., Ltd.) was diluted with a fecal solution to obtain a target hemoglobin concentration as shown in Table 8, thereby preparing a fecal free hemoglobin determination sample. The fecal solution was prepared by adding human feces to 50 mM HEPES buffer (pH 7.4) to a concentration of 0.5% by mass. In addition, purified hemoglobin (manufactured by Eiken Chemical Co., Ltd.) and purified type 1-1 hemoglobin (manufactured by Eiken Chemical Co., Ltd.) were mixed and diluted with a fecal solution at a mixing ratio of hemoglobin:heme-binding globulin = 1:1.4 (molar ratio) so that the hemoglobin concentration reached the target concentration shown in Table 8, and a fecal hemoglobin-heme-binding globulin complex assay sample was prepared.

For each obtained test sample, the test reagent prepared in Example 2 was used to perform the test under the following conditions. Sample volume: 1.0 μL　First reagent: 50 μL　Second reagent: 50 μL　Measurement wavelength: 658 nm The results are shown in Table 8 and Figure 5.

[Table 8] Target value (μg/mL) Measured value (μg/mL) Free Hb sample Hb-Hpt test material 0 -1 0 5 5 -1 15 14 -1 20 20 -1 25 twenty four 0 30 27 0

As shown in Table 8 and FIG5 , even in a sample containing feces, a measurement value equivalent to the target value was obtained in the free hemoglobin measurement sample, and in the hemoglobin-heme-binding globulin complex measurement sample, the measurement value was close to 0. Therefore, it was confirmed that the measurement reagent of the present invention can specifically measure free hemoglobin even in a solution containing feces.

without.

FIG1 is a graph showing the results of measuring free hemoglobin (free-Hb)/hemoglobin-heme binding globulin (Hb-Hp) complexes in various mixed solutions (specimens) using the immunoagglutination assay reagent prepared in Reference Example 4 and a commercially available colorimetric assay reagent. FIG2 is a graph showing the results of measuring the amount of free hemoglobin using the assay reagent of Example 2 (with anti-hemoglobin binding globulin antibody added) and the assay reagent of Reference Example 4 (without anti-hemoglobin binding globulin antibody added). FIG2A: Shows the results of measuring the turbidity change (ΔOD) in the assay reagents of Example 2 and Reference Example 4. FIG2B: Shows the results of evaluating the dilution linearity using the assay reagent of Example 2. FIG3 is a graph showing the results of measuring the amount of free hemoglobin in a sample in which a hemoglobin-hemoglobin complex may be present using the measuring reagent of Example 2 (with anti-hemoglobin antibody added) and the measuring reagent of Reference Example 4 (without anti-hemoglobin antibody added). FIG4 is a graph showing the results of measuring the amount of free hemoglobin in a sample prepared by adding purified hemoglobin to a human negative serum or serum residual specimen using various anti-hemoglobin antibodies. FIG5 is a graph showing the results of measuring the amount of free hemoglobin in a feces measuring sample and a feces hemoglobin-hemoglobin complex measuring sample using the measuring reagent of Example 2.

without.

without.

Claims (14)

A method for measuring free hemoglobin comprises the following steps: Using two or more anti-hemoglobin antibodies to carry out an antigen-antibody reaction for measuring the free hemoglobin; The antigen-antibody reaction is carried out in the presence of an anti-hemoglobin binding globulin antibody. The method for measuring free hemoglobin as described in claim 1, wherein the antigen-antibody reaction is carried out in an environment where a hemoglobin-hemoglobin binding globulin complex may exist. The method for measuring free hemoglobin as described in claim 1, wherein the two or more anti-hemoglobin antibodies comprise a combination of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain. The method for measuring free hemoglobin as described in claim 1, wherein at least one of the two or more anti-hemoglobin antibodies is carried on an insoluble carrier. The method for measuring free hemoglobin as described in claim 4, wherein the insoluble carrier is an insoluble particle. The method for determining free hemoglobin as described in claim 5 is an immunoagglutination method. A free hemoglobin assay reagent is used to assay free hemoglobin, comprising: Two or more anti-hemoglobin antibodies; and Anti-hemoglobin binding globulin antibodies; The two or more anti-hemoglobin antibodies are used in an antigen-antibody reaction for assaying the free hemoglobin; The anti-hemoglobin binding globulin antibodies constitute a reaction solution for the antigen-antibody reaction. A reagent for measuring free hemoglobin as described in claim 7, wherein the antigen-antibody reaction is carried out in an environment where a hemoglobin-hemoglobin binding globulin complex may exist. A reagent for measuring free hemoglobin as described in claim 7, wherein the two or more anti-hemoglobin antibodies comprise a combination of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain. A reagent for measuring free hemoglobin as described in claim 7, wherein at least one of the two or more anti-hemoglobin antibodies is carried on an insoluble carrier. A reagent for measuring free hemoglobin as described in claim 10, wherein the insoluble carrier is an insoluble particle. A reagent for measuring free hemoglobin as described in claim 11, which is a reagent for immunoagglutination method. A free hemoglobin assay reagent set comprising the free hemoglobin assay reagent described in any one of claims 7 to 12. A method for suppressing false high values in free hemoglobin determination comprises the following steps: Using two or more anti-hemoglobin antibodies to carry out an antigen-antibody reaction for determining the free hemoglobin; The antigen-antibody reaction is carried out in the presence of an anti-hemoglobin binding globulin antibody.