TW202417843A - Free hemoglobin measurement reagent, free hemoglobin measurement method, and anti-hemoglobin antibody - Google Patents

Abstract

A method for measuring free hemoglobin according to the present invention involves performing an antigen-antibody reaction for measuring free hemoglobin: using at least two anti-hemoglobin antibodies that include a combination of an antibody specific to the α chain of hemoglobin and an antibody specific to the [beta] chain of hemoglobin; or under conditions under which reactivity to the hemoglobin-haptoglobin complex is no more than 15% of reactivity to free hemoglobin. The present invention also provides a free hemoglobin measurement reagent, an anti-hemoglobin antibody combination that combines an antibody specific to the [alpha] chain of hemoglobin and an antibody specific to the [beta] chain of hemoglobin, and a free hemoglobin measurement kit. The measurement method and measurement reagent of the present invention are easy to use and make it possible to specifically measure free hemoglobin.

Description

Free hemoglobin assay reagent, free hemoglobin assay method, and anti-hemoglobin antibody

The present invention relates to a reagent and a method for specifically measuring free hemoglobin, and an anti-hemoglobin antibody used in the above reagent and method.

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.

The present invention is made in view of the above problems, and its purpose is to provide a reagent and method that are simple to operate and can specifically measure free hemoglobin, as well as an antibody that can be used in the aforementioned reagent and method.

[Technical means for solving the problem] The inventors of the present invention have conducted research to solve the above-mentioned problem, and as a result, have found that a free hemoglobin assay reagent and assay method using two or more specific anti-hemoglobin antibodies, or a free hemoglobin assay reagent and assay method whose reactivity to a hemoglobin-hemoglobin binding globulin complex is 15% or less of the reactivity to free hemoglobin, and two or more anti-hemoglobin antibodies used in the aforementioned reagent and method can solve the above-mentioned problem, thereby completing the present invention. Specifically, the present invention is as follows.

[1] A method for measuring free hemoglobin, comprising using two or more anti-hemoglobin antibodies, including a combination of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain, to carry out an antigen-antibody reaction for measuring the free hemoglobin. [2] The method for measuring free hemoglobin as described in [1], wherein the antigen-antibody reaction for measuring the free hemoglobin is carried out under conditions where the reactivity to a hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin. [3] The method for measuring free hemoglobin as described in [1] or [2], wherein the antigen-antibody reaction for measuring the free hemoglobin is carried out in an environment where a hemoglobin-hemoglobin binding globulin complex may be present. [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 method for measuring free hemoglobin, comprising using two or more anti-hemoglobin antibodies to carry out an antigen-antibody reaction for measuring the free hemoglobin under conditions where the reactivity to a hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin. [8] A reagent for measuring free hemoglobin, for measuring free hemoglobin, comprising: Two or more anti-hemoglobin antibodies, wherein the anti-hemoglobin antibodies comprise a combination of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain. [9] The reagent for measuring free hemoglobin as described in [8], wherein the reactivity to the hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin. [10] A reagent for measuring free hemoglobin as described in [8] or [9], wherein the antigen-antibody reaction for measuring free hemoglobin is carried out in an environment where a hemoglobin-hemoglobin binding globulin complex may be present. [11] A reagent for measuring free hemoglobin as described in any one of [8] to [10], wherein at least one of the two or more anti-hemoglobin antibodies is carried on an insoluble carrier. [12] A reagent for measuring free hemoglobin as described in [11], wherein the insoluble carrier is an insoluble particle. [13] A reagent for measuring free hemoglobin as described in [12], which is a reagent for immunoagglutination method. [14] A free hemoglobin assay reagent for the determination of free hemoglobin, comprising: Two or more anti-hemoglobin antibodies; The reactivity of the reagent to the hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin. [15] A combination of anti-hemoglobin antibodies, which is composed of an antibody specific to the hemoglobin α chain and an antibody specific to the hemoglobin β chain. [16] A free hemoglobin assay reagent set, which comprises the free hemoglobin assay reagent described in any one of [8] to [14].

[Effects of the invention] The assay method and assay reagent of the present invention can be used to specifically assay free hemoglobin while being easy to operate.

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 two of each of the following two subunits to form a tetramer structure [α ₂ β ₂ ]: 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.

Antibodies obtained using tetrameric free heme [α ₂ β ₂ ] as antigens also react with dimeric heme [αβ] contained in the heme-heme binding globulin complex. Therefore, it has been very difficult to obtain anti-free heme antibodies that do not bind to the heme-heme binding globulin complex using conventional animal immunization methods. In contrast, in the present embodiment, it was found that by selectively using two or more anti-heme antibodies, it is possible to react with free heme instead of the heme-heme binding globulin complex.

Here, in evaluating the "reactivity to the heme-heme binding globulin complex" described later, the heme-heme binding globulin complex used as an antigen refers to a complex of heme and Hp1-1 type heme binding globulin. However, when the reactivity to this complex is much lower than the reactivity to free heme, the reactivity to the complex of other heme binding globulins (Hp2-1 type, Hp2-2 type) and heme will also be much lower than the reactivity to free heme.

[Method for measuring free hemoglobin: first embodiment] The method for measuring free hemoglobin in one embodiment of the present invention uses two or more anti-hemoglobin antibodies, including a combination of antibodies specific to the hemoglobin α chain and antibodies specific to the hemoglobin β chain, to perform an antigen-antibody reaction to measure free hemoglobin. In addition, the embodiment described below may be referred to as the first embodiment of the method for measuring free hemoglobin.

(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. The measurement method in this embodiment is preferably to use the immunoagglutination method among these methods, and more preferably to use the latex agglutination method.

(Antibody) The anti-heme antibody used in this embodiment is a combination of two or more anti-heme antibodies including an antibody specific to the heme α chain and an antibody specific to the heme β chain. Here, "antibody specific to the heme α chain" refers to an anti-heme antibody that reacts much more to the heme α chain than to the 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 set as the property that the reaction to the hemoglobin α chain relative to the sum of the reaction to the hemoglobin α chain and the reaction to the β chain is 75% or more, preferably 80% or more. 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 Western blot; 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.

The inventors of the present invention believe that the mechanism of action of specifically measuring free heme by using a combination of an antibody specific for the heme α chain and an antibody specific for the heme β chain is as follows: As for the heme portion of the heme-heme binding globulin complex, that is, the heme of 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 steric barriers. In contrast, since free heme is a heme of the α ₂ β ₂ tetramer structure and does not form a complex with the heme binding globulin, it is believed that it can bind to the α chain specific antibody and the β chain specific antibody 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 as long as it satisfies the above-mentioned specificity. 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 0.5~2,000μg/mg latex, it can also be 1~1,000μg/mg latex or 2~500μg/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.

(Reactivity) In this embodiment, it is preferred to measure free hemoglobin by performing an antigen-antibody reaction under conditions where the reactivity to the hemoglobin-heme binding globulin complex is less than 15% of the reactivity to free hemoglobin. "Reactivity to free hemoglobin" refers to the reactivity when free hemoglobin is added alone as an antigen. "Reactivity" is evaluated using the indicators used to quantify antigens in the above-mentioned immunological methods. For example, in the case of the immunoagglutination method, the amount of turbidity change within a specific time period can be cited; in the case of the ELISA method, the amount of color development/absorbance caused by the labeling of the labeled antibody can be cited. The reactivity to free hemoglobin can be evaluated, for example, under conditions where the free hemoglobin in the reaction solution of the antigen-antibody reaction is 2.7 pmol/mL (1 μg/mL), 27 pmol/mL (10 μg/mL), or 270 pmol/mL (100 μg/ml).

Furthermore, "reactivity to a heme-heme binding globulin complex" refers to reactivity when a heme-heme binding globulin complex is added as an antigen. Here, the addition of a heme-heme binding globulin complex includes, in addition to the addition of a pre-formed heme-heme binding globulin complex, the addition of free heme and an equimolar amount of heme binding globulin to form a heme-heme binding globulin complex in the mixed solution.

The reactivity to the heme-heme binding globulin complex was evaluated under the same reaction conditions as the above-mentioned reactivity to free heme, except that the antigen was changed to an equimolar heme-heme binding globulin complex. For example, when the evaluation of the reactivity to free heme was performed under the condition that the free heme was 2.7 pmol/mL, the evaluation of the reactivity to the heme-heme binding globulin complex was performed under the condition that the complex was 2.7 pmol/mL. Here, the so-called same reaction conditions refer to the anti-heme antibody used for the antigen-antibody reaction, the concentration of the antibody, the components of the reaction solution for the antigen-antibody reaction (pH buffer, salt, type/concentration of additives) and pH value, and the reaction time and temperature of the antigen-antibody reaction are all the same. In addition, when the anti-heme antibody is carried on an insoluble carrier as described later, the same type of antibody-carrying carrier will be used.

The "antigen-antibody reaction for measuring free hemoglobin" is an antigen-antibody reaction performed by adding a measurement object (e.g., a specimen, etc.) for measuring free hemoglobin, and is different from the antigen-antibody reaction for evaluating the reactivity described above. In the above measurement object, in addition to free hemoglobin, a hemoglobin-hemoglobin binding globulin complex is often present. In other words, the antigen-antibody reaction for measuring free hemoglobin is often performed in an environment where a hemoglobin-hemoglobin binding globulin complex may be present.

In this embodiment, the antigen-antibody reaction for measuring free hemoglobin is preferably carried out under conditions where the reactivity to the hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin. The reactivity ratio may be less than 10%, and further may be less than 5%. The so-called "antigen-antibody reaction for measuring free hemoglobin is carried out under the conditions" means that it is carried out under the same reaction conditions as when evaluating the aforementioned "reactivity to free hemoglobin" and "reactivity to the hemoglobin-hemoglobin binding globulin complex". The definition of the same reaction conditions is as described above.

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

[Method for measuring free hemoglobin: Second embodiment] The second embodiment of the method for measuring free hemoglobin of the present invention can be as follows: using two or more anti-hemoglobin antibodies; and measuring free hemoglobin by performing an antigen-antibody reaction under the condition that the reactivity to the hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin.

The definitions of "reactivity to free heme" and "reactivity to heme-heme-binding globulin complex" are as described in the first embodiment.

The two or more anti-hemoglobin antibodies used in this embodiment are not particularly limited as long as they satisfy the above-mentioned reactivity. 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. Among them, the combination of an antibody specific to the hemoglobin α chain and an antibody specific to the hemoglobin β chain is particularly preferred.

In the present embodiment, since two or more anti-heme antibodies are used, the above-mentioned antigen-antibody reaction is preferably an antigen-antibody reaction to two or more different antigenic epitopes in the same antigen (heme in the present embodiment). In other words, the immunological method of the present embodiment is preferably a method utilizing the two or more antigen-antibody reactions.

The other structures (non-soluble carrier, etc.) of this embodiment are the same as those of the first embodiment.

The measurement method of the above embodiment is to perform an antigen-antibody reaction for measuring free hemoglobin in the following manner: Using two or more anti-hemoglobin antibodies including a combination of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain; or Performing the measurement under conditions where the reactivity to the hemoglobin-hemoglobin binding globulin complex is 15% or less of the reactivity to free hemoglobin. Thus, for example, even if the measurement is performed in an environment where the hemoglobin-hemoglobin binding globulin complex may be present, the amount of free hemoglobin can be measured without being affected by the amount of the hemoglobin-hemoglobin binding globulin complex. According to the measurement method of this embodiment, there is no need to remove the hemoglobin-hemoglobin binding globulin complex in advance before the antigen-antibody reaction for measuring free hemoglobin. Therefore, not only is the operation simple, but free hemoglobin can also be measured specifically.

[Free hemoglobin assay reagent: first embodiment] A free hemoglobin assay reagent according to one embodiment of the present invention comprises two or more anti-hemoglobin antibodies comprising a combination of an antibody specific to the hemoglobin α chain and an antibody specific to the hemoglobin β chain. In addition, the embodiment described below may be referred to as the first embodiment of the free hemoglobin assay reagent.

The anti-hemoglobin antibody used in the reagent of this embodiment can be the antibody described in the first embodiment of the above-mentioned method for measuring free hemoglobin.

Among the two or more anti-hemoglobin antibodies used in the reagent of this 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 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.

The reagent of this embodiment preferably has a reactivity to the heme-heme binding globulin complex that is 15% or less of the reactivity to free heme. "Reactivity to free heme" and "reactivity to the heme-heme binding globulin complex" are the same as those described in the above-mentioned measurement method.

The assay reagent of this embodiment, in addition to the composition of the reagent (components of the reaction solution, etc.) described below, usually also has attachments such as instructions for use. The reaction conditions for measuring free hemoglobin are specified by the composition of the reagent and the attachments. More specifically, the attachments of the assay reagent specify the addition amount of two or more anti-hemoglobin antibodies (including the state in which such anti-hemoglobin antibodies are carried in an insoluble carrier), the total liquid volume of the reaction solution, the order of addition, the reaction time and the reaction temperature, etc. As long as the assay reagent of this embodiment is used in accordance with this attachment, the reaction conditions for measuring free hemoglobin can be specified. A reagent whose reactivity to a heme-heme binding globulin complex is 15% or less of its reactivity to free heme means that, for the reagent, when the "reactivity to free heme" and the "reactivity to a heme-heme binding globulin complex" are evaluated and compared under specific reaction conditions, the reactivity is 15% or less. The reactivity described here may be 10% or less, or may be 5% or less. In addition, "reactivity to free hemoglobin" can be evaluated by reactivity to free hemoglobin at a specific concentration (e.g., 2.7 pmol/mL, 27 pmol/mL, 270 pmol/mL). Similarly, "reactivity to hemoglobin-hemoglobin binding globulin complex" can be evaluated by reactivity to hemoglobin-hemoglobin binding globulin complex at a molar equivalent to a specific concentration of free hemoglobin.

(Form of the assay reagent, etc.) The assay reagent of this embodiment has the aforementioned structure and is a reagent for measuring free hemoglobin using an immunological method. As long as it is a reagent that meets these conditions, there is no particular limitation on the form of the assay reagent. For example, a reagent using an immunoagglutination method (such as latex agglutination method, colloidal gold agglutination method, etc.), ELISA method, immunochromatography method, etc. can be used. Among them, a reagent using an immunoagglutination method is preferred, and a reagent using a latex agglutination method is more preferred.

The assay reagent of this embodiment, for example, may be composed of two reagent types, namely, a reagent not containing an insoluble carrier (first reagent) and a reagent containing an insoluble carrier for carrying an antibody (antibody-carrying insoluble carrier) (second reagent), or may be composed of only one reagent type, namely, a reagent containing an antibody-carrying insoluble carrier. Here, the first reagent may be used to adjust the assay environment, such as being used as a diluent from the perspective of adjusting the concentration of the assay object or impurities in the reaction system or adjusting the reaction rate. The second reagent contains an antibody-carrying insoluble carrier and can be mixed with the first reagent and the sample to produce an immunoagglutination reaction. 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.

In addition, the concentration of the insoluble carrier in the reaction solution obtained by mixing the assay reagent with the sample can be appropriately selected from the range of, for example, 0.0001 mg/mL to 10 mg/mL in accordance with the particle size of the insoluble carrier used and the overall design of the assay system. In the assay reagent, the concentration of the insoluble carrier carrying the anti-hemoglobin antibody can be 0.01 to 5 mg/mL or 0.05 to 1 mg/mL. In addition, the concentration of the insoluble carrier in the second reagent is diluted when it is mixed with the first reagent or sample, etc. during use, so 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 mg/mL~20 mg/mL, and when it is diluted three times, it can be appropriately adjusted to 0.0003 mg/mL~30 mg/mL.

[Free hemoglobin assay reagent: second embodiment] The second embodiment of the free hemoglobin assay reagent of the present invention may include: two or more anti-hemoglobin antibodies; and the reactivity to the hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin.

The antibodies used in the assay reagent of this embodiment are the same as those described in the second embodiment of the above-mentioned free hemoglobin assay method. In addition, "reactivity to free hemoglobin" and "reactivity to hemoglobin-hemoglobin binding globulin complex" are the same as those described in the first embodiment of the free hemoglobin assay reagent. Other components of the assay reagent of this embodiment (insoluble carrier, form of the reagent, etc.) are also the same as those described in the first embodiment related to the free hemoglobin assay reagent.

The free hemoglobin assay reagent described above is: containing two or more anti-hemoglobin antibodies comprising a combination of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain; or having a reactivity to the hemoglobin-hemoglobin binding globulin complex of less than 15% of the reactivity to free hemoglobin. Thus, the amount of free hemoglobin can be measured without being affected by the amount of the hemoglobin-hemoglobin binding globulin complex. Therefore, even if the antigen-antibody reaction for measuring free hemoglobin is carried out in an environment where the hemoglobin-hemoglobin binding globulin complex may be present, free hemoglobin can be specifically measured. This eliminates the need to remove the heme-heme binding globulin complex before the antigen-antibody reaction for measuring free heme, making the process simple to operate and enabling specific measurement of free heme.

[Other embodiments] The present invention also provides a combination of an antibody specific for heme α chain and an antibody specific for heme β chain. The definitions of "specific reaction for heme α chain" and "specific reaction for heme β chain" are as described above. The combination of an antibody specific for heme α chain and an antibody specific for heme β chain is particularly suitable for measuring free heme.

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, for example, include an anti-hemoglobin antibody carrying an insoluble carrier. In addition, the free hemoglobin assay reagent set may include structures such as a calibrator and a controller in addition to the assay reagent, and may also include an instrument and 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, but 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.

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 a 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 double antibody RIA method using ¹²⁵ I-labeled hemoglobin, and 10 copies were obtained.

Example 2 : Confirmation of antibody specificity 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 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 Western Lightning ECL Pro (manufactured by PerkinElmer) was used to make HRP illuminate and obtain band images. The band images were analyzed using CS Analyzer ver.3.0 (manufactured by ATTO Corporation). The measurement mode was: the band brightness of the lane was calculated as a cumulative value using the designated area density method (Figure 1). Then, the cumulative values of the band brightness of the α chain and the 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 cumulative value of the α chain. 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%. 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 the results, 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 (Figure 2).

[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 Example 1, the ratio of the α chain cumulative value of No. 1, 8, 12, and 16 was 75% or more relative to the total value of the α chain cumulative value and the β chain cumulative value, and thus they were 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 was 75% or more relative to the total value of the α chain cumulative value and the β chain cumulative value, and thus they were identified as β chain specific antibodies.

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

(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. By allowing the anti-hemoglobin monoclonal antibody to be carried on the surface of the polystyrene latex particles, an antibody-carrying polystyrene latex particle solution is prepared.

(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, and the difference between the two was used as an 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, the antibody combination used showed a specific reaction to free heme. The reactivity to the heme-heme-binding globulin complex was less than 15% of the reactivity to free heme.

Example 4 : Reactivity of Monoclonal Antibodies -2 The reactivity to free heme and to heme-heme binding globulin complex was evaluated for all combinations of the 10 antibodies obtained in Example 1. The confirmation was performed by the same method as that used in Example 3.

Reactivity was evaluated according to the following criteria. A: Aggregation degree (ΔOD×10,000, the same below) when reacting with the heme-heme binding globulin complex is less than 15% of the aggregation degree when reacting with free heme B: Aggregation degree when reacting with the heme-heme binding globulin complex is more than 50% of the aggregation degree when reacting with free heme (except for the following "-") -: Aggregation degree of both free heme and heme-heme binding globulin complex is significantly low (ΔOD×10,000 is less than 500)

The above B includes the case where the aggregation degree of both free hemoglobin and hemoglobin-hemoglobin binding globulin complex exceeds the measurement limit of the device (ΔOD×10,000 exceeds 3000), resulting in the above ratio. The results are shown in Table 3. In addition, in this experiment, no combination was obtained in which the aggregation degree when reacting with the hemoglobin-hemoglobin binding globulin complex was more than 15% of the aggregation degree when reacting with free hemoglobin and less than 50% of the aggregation degree when reacting with free hemoglobin.

[table 3] antibody α chain specificity β-chain specificity 1 8 12 16 4 11 twenty one twenty three twenty four 32 α chain specificity No.1 - - - A A A A A A No.8 - - - A A A A A A No.12 - - - A A A A A A No.16 - - - A A A A A A β-chain specificity No.4 A A A A - B B B - No.11 A A A A ― - - B - No.21 A A A A B - - - - No.23 A A A A B - - - - No.24 A A A A B B - - - No.32 A A A A - - - - -

As shown in Table 3, by combining the α-chain specific antibody and the β-chain specific antibody, a specific reaction to free hemoglobin was confirmed.

Example 5 : Free hemoglobin assay reagent The antibody obtained in Example 1 was used to prepare an immunoagglutination assay reagent.

(1) Preparation of reagents The first reagent and the second reagent were prepared as assay reagents. The first reagent used a 50 mM HEPES buffer (pH 7.4). The second reagent was prepared by mixing each polystyrene latex carrying the anti-hemoglobin monoclonal antibody (anti-Hb antibody) of Example 2, adjusting the latex concentration to 1.0 mg/mL, and preparing an immunoagglutination reaction assay reagent. The antibody-carrying insoluble carrier was prepared by mixing each anti-hemoglobin monoclonal antibody and polystyrene latex particles (average particle size 100 nm) so that the anti-hemoglobin monoclonal antibody was carried 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.

(2) Preparation of samples and measurement conditions The total human hemoglobin common reference standard substance JCCRM912-3 (manufactured by the Institute of Detection of Medical Standard Substances) was mixed with the Hb calibration diluent "Eiken" (manufactured by Eiken Chemical Co., Ltd.) to the target concentrations shown in Tables 4 and 5, and then measured using the measurement reagent prepared in (1) above and the biochemical automatic analyzer JCA-BM6070. A linear test was performed for the low concentration range (0~10μg/mL, FHB_L) and the high concentration range (0~100μg/mL, FHB_H).

=Measurement conditions for low concentration region (FHB_L)= Sample volume: 5.0μL　First reagent: 50μL　Second reagent: 25μL　Measurement wavelength: 658nm =Measurement conditions for high concentration region (FHB_H)= Sample volume: 1.0μL　First reagent: 50μL　Second reagent: 50μL　Measurement wavelength: 658nm The results are shown in Tables 4 to 5 and Figures 3 to 4.

(3) Reactivity to free heme and heme-heme binding globulin complex The common reference standard substance for total human heme, JCCRM912-3 (manufactured by the Institute of Detection of Medical Standard Substances), and heme binding globulin from human mixed plasma (manufactured by SIGMA-ALDRICH) were mixed with the Hb calibration diluent "Eiken" (manufactured by Eiken Chemical Co., Ltd.) to prepare the samples for the measurement of free heme and heme-heme binding globulin complex. The samples for the measurement of free heme were prepared at 1μg/mL (2.7pmol/mL), 10μg/mL (27pmol/mL), and 100μg/mL (270pmol/ml), respectively. In addition, the heme-heme-binding globulin complex assay sample is formed by adding an equimolar amount of heme-binding globulin to the assay sample.

The prepared samples were measured using the test reagent prepared in (1) above and the biochemical automatic analyzer JCA-BM6070. The results are shown in Table 6.

[Table 4] Low concentration area (FHb_L) Theoretical value (µg/mL) Turbidity (⊿OD) Measured value (µg/mL) 0.0 0.0002 0.0 1.0 0.0073 1.0 2.5 0.0498 2.5 5.0 0.2786 5.0 7.5 0.5082 7.5 10.0 0.5903 10.0

[Table 5] High concentration area (FHb_H) Theoretical value (µg/mL) Turbidity (⊿OD) Measured value (µg/mL) 0 0.0001 0 10 0.0100 10 25 0.0596 25 50 0.4350 50 75 0.7736 75 100 0.8972 100

[Table 6] Low concentration area (FHb_L) High concentration area (FHb_H) Hb concentration in sample (µg/mL) Measured value (µg/mL) Hb concentration in sample (µg/mL) Measured value (µg/mL) Free Hb Hb-Hp complex Free Hb Hb-Hp complex 0 0 0 0 0 0 1 1 0 10 10 0 10 10 0 100 102 5

As shown in Tables 4 to 5 and Figures 3 to 4, the immunoagglutination assay reagent of this embodiment can measure free hemoglobin in proportion to the concentration. In addition, as shown in Table 6, the immunoagglutination assay reagent of this embodiment has a reactivity to the hemoglobin-hemoglobin binding globulin complex of less than 15% of the reactivity to free hemoglobin at 2.7 pmol/mL, 27 pmol/mL and 270 pmol/mL (= 1 μg/mL, 10 μg/mL and 100 μg/ml in free hemoglobin, respectively).

Example 6 : Comparison with other assays The assays were compared using samples containing 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 7 to prepare solutions (specimens) with different mixing ratios. In addition, free-Complex in Table 7 represents the molar ratio of free hemoglobin to heme-heme binding globulin complex.

[Table 7] 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 Example 5. 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 Example 5 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 8 and Figure 5.

[Table 8] 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 binding globulin complex changes, so it is impossible to distinguish the two and perform the measurement. In contrast, the immunoagglutination reaction measurement reagent (latex reagent) of the present invention is not affected by the amount of hemoglobin-hemoglobin binding globulin complex, and a measurement value proportional to the free hemoglobin concentration can be obtained. The above results show that the latex reagent of the present invention can specifically measure free hemoglobin.

without.

FIG1 is an electrophoresis image showing the confirmation result of the specificity of the antibody obtained in Example 1. Using the antibody obtained in Example 1 as the primary antibody, the Western blot method was performed to obtain a band image. The obtained band image was analyzed, and the band brightness of the lane was calculated as a cumulative value. FIG2 is an electrophoresis image obtained by electrophoresis of human hemoglobin and CBB staining. Based on the obtained electrophoresis image, the cumulative values of the band brightness of the α chain and the β chain were calculated respectively, and the coefficient for normalizing the band brightness was calculated (the coefficient 0.783 multiplied by the cumulative band value of the α chain). FIG3 is a graph showing the results of measuring hemoglobin in the low concentration area (0~10μg/mL) using the immunoagglutination reaction assay reagent prepared in Example 5. FIG. 4 is a graph showing the results of measuring hemoglobin in the high concentration region (0-100 μg/mL) using the immunoagglutination assay reagent prepared in Example 5. FIG. 5 is a graph showing the results of measuring each mixed solution (specimen) of free hemoglobin (free-Hb)/hemoglobin-heme binding globulin (Hb-Hp) complex using the immunoagglutination assay reagent prepared in Example 5 and a commercially available colorimetric assay reagent.

without.

without.

Claims (16)

A method for measuring free hemoglobin uses two or more anti-hemoglobin antibodies, including a combination of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain, to perform an antigen-antibody reaction for measuring the free hemoglobin. The method for measuring free hemoglobin as described in claim 1 is to carry out the antigen-antibody reaction for measuring the free hemoglobin under the condition that the reactivity to the hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin. The method for measuring free hemoglobin as described in claim 1, wherein the antigen-antibody reaction for measuring the free hemoglobin 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 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 method for measuring free hemoglobin comprises using two or more anti-hemoglobin antibodies to carry out an antigen-antibody reaction for measuring the free hemoglobin under the condition that the reactivity to a hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin. A free hemoglobin assay reagent is used to assay free hemoglobin, comprising: Two or more anti-hemoglobin antibodies, wherein the anti-hemoglobin antibodies contain a combination of antibodies specific to the hemoglobin α chain and antibodies specific to the hemoglobin β chain. The free hemoglobin measuring reagent as described in claim 8 has a reactivity to the hemoglobin-hemoglobin binding globulin complex that is less than 15% of the reactivity to free hemoglobin. A free hemoglobin assay reagent as described in claim 8, wherein the antigen-antibody reaction for assaying free hemoglobin is carried out in an environment where a hemoglobin-hemoglobin binding globulin complex may be present. A free hemoglobin assay reagent as described in claim 8, wherein at least one of the two or more anti-hemoglobin antibodies is carried on an insoluble carrier. The free hemoglobin assay reagent as described in claim 11, wherein the insoluble carrier is insoluble particles. A free hemoglobin assay reagent as described in claim 12, which is a reagent for immunoagglutination method. A free hemoglobin assay reagent is used to assay free hemoglobin, comprising: Two or more anti-hemoglobin antibodies; The reactivity of the reagent to the hemoglobin-hemoglobin binding globulin complex is less than 15% of the reactivity to free hemoglobin. An anti-hemoglobin antibody combination is composed of an antibody specific for the hemoglobin α chain and an antibody specific for the hemoglobin β chain. A free hemoglobin assay reagent kit comprises the free hemoglobin assay reagent described in any one of claims 8 to 14.