WO2024053586A1 - Free hemoglobin measurement reagent, free hemoglobin measurement method, and anti-hemoglobin antibody - Google Patents

Free hemoglobin measurement reagent, free hemoglobin measurement method, and anti-hemoglobin antibody Download PDF

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WO2024053586A1
WO2024053586A1 PCT/JP2023/032151 JP2023032151W WO2024053586A1 WO 2024053586 A1 WO2024053586 A1 WO 2024053586A1 JP 2023032151 W JP2023032151 W JP 2023032151W WO 2024053586 A1 WO2024053586 A1 WO 2024053586A1
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hemoglobin
free
antibody
free hemoglobin
reagent
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French (fr)
Japanese (ja)
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恵 油井
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栄研化学株式会社
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood

Definitions

  • the present invention relates to reagents and methods for specifically measuring free hemoglobin, and anti-hemoglobin antibodies used therefor.
  • hemoglobin In the blood, hemoglobin combines with haptoglobin in the body to form a hemoglobin-haptoglobin complex. Such complexes are eventually taken up by macrophages in the liver via the scavenger receptor CD163, where they are degraded and metabolized. However, when hemoglobin cannot be completely processed by haptoglobin in the body due to hemolysis caused by burns or cardiopulmonary bypass, the blood concentration of hemoglobin that does not form a complex with haptoglobin (free hemoglobin) increases.
  • Free hemoglobin has a small molecular weight, so when it is excreted in the urine, it passes through the glomerulus of the kidney, is taken up by renal tubular epithelial cells, and is broken down into heme and globin.
  • heme iron contained in heme acts as a catalyst to produce free radicals, which cause necrosis of proximal renal tubular epithelial cells and cause renal tubular damage.
  • Free hemoglobin can be treated with haptoglobin preparations, but in order to administer an appropriate amount of haptoglobin preparations, it is necessary to accurately measure the free hemoglobin concentration.
  • a specimen containing free hemoglobin is brought into contact with immobilized human haptoglobin in which human haptoglobin is bound to a solid phase, and the free hemoglobin is bound to human haptoglobin on the solid phase.
  • a method has been proposed in which free hemoglobin bound on a solid phase is measured by applying an enzyme-labeled anti-human hemoglobin antibody (see Patent Document 1).
  • a method has been proposed in which an anti-haptoglobin antibody is added to a sample containing free hemoglobin, the antibody is reacted with the hemoglobin-haptoglobin complex present in the sample, and then free hemoglobin is measured by enzyme immunoassay (See Patent Document 2).
  • Free hemoglobin can be measured by adsorbing it to immobilized haptoglobin and then washing and separating it to formulate free hemoglobin (Patent Document 1), or by removing the hemoglobin-haptoglobin complex in advance and measuring free hemoglobin.
  • Patent Document 2 and the like are known, but all of them require complicated operations and have limitations in speed and measurement processing capacity, so a rapid and highly specific free hemoglobin immunoassay method has been desired.
  • the present invention has been made in view of the above problems, and aims to provide a reagent and method that are easy to operate and can specifically measure free hemoglobin, as well as antibodies that can be used therefor. purpose.
  • the present inventors conducted research to solve the above problems, and found that a free hemoglobin measurement reagent and measurement method using two or more specific anti-hemoglobin antibodies, or a reactivity to a hemoglobin-haptoglobin complex that differs from that of free hemoglobin.
  • the inventors have discovered that a reagent and method for measuring free hemoglobin with a free hemoglobin concentration of 15% or less, and two or more anti-hemoglobin antibodies used therein can solve the above problems, and have completed the present invention.
  • the present invention is as follows.
  • a method for measuring free hemoglobin comprising: Performing an antigen-antibody reaction for measuring the free hemoglobin using two or more types of anti-hemoglobin antibodies including a combination of an antibody specific for hemoglobin ⁇ chain and an antibody specific for hemoglobin ⁇ chain, Free hemoglobin measurement method.
  • an antigen-antibody reaction for measuring the free hemoglobin is performed under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
  • [3] The method for measuring free hemoglobin according to [1] or [2], wherein the antigen-antibody reaction for measuring the free hemoglobin is performed in an environment where a hemoglobin-haptoglobin complex can exist.
  • [4] The method for measuring free hemoglobin according to any one of [1] to [3], wherein at least one of the two or more anti-hemoglobin antibodies is supported on an insoluble carrier.
  • [5] The method for measuring free hemoglobin according to [4], wherein the insoluble carrier is an insoluble particle.
  • [6] The method for measuring free hemoglobin according to [5], which is an immunoagglutination method.
  • a method for measuring free hemoglobin comprising: Using two or more types of anti-hemoglobin antibodies, Performing the antigen-antibody reaction for measuring the free hemoglobin under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin. Free hemoglobin measurement method.
  • a reagent for measuring free hemoglobin comprising: Two or more types of anti-hemoglobin antibodies, including a combination of an antibody specific for hemoglobin ⁇ chain and an antibody specific for hemoglobin ⁇ chain, Free hemoglobin measurement reagent.
  • a reagent for measuring free hemoglobin comprising: Contains two or more types of anti-hemoglobin antibodies, The reactivity to hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin, Free hemoglobin measurement reagent.
  • a combination of anti-hemoglobin antibodies which is a combination of an antibody specific to hemoglobin ⁇ chain and an antibody specific to hemoglobin ⁇ chain.
  • a free hemoglobin measurement kit comprising the free hemoglobin measurement reagent according to any one of [8] to [14].
  • free hemoglobin can be specifically measured while the operation is simple.
  • Example 1 is an electrophoresis image showing the results of confirming the specificity of the antibody obtained in Example 1.
  • Western blotting was performed using the antibody obtained in Example 1 as a primary antibody, and band images were obtained.
  • the obtained band image was analyzed, and the band brightness of the migration lane was calculated as an integrated value.
  • This is an electrophoresis image obtained by electrophoresing human hemoglobin and performing CBB staining.
  • the integrated value of the band brightness of the ⁇ chain and ⁇ chain was calculated from the obtained electrophoretic image, and a coefficient for normalizing the band brightness (multiplying the integrated band value of the ⁇ chain by a coefficient of 0.783) was calculated.
  • 2 is a graph showing the results of measuring hemoglobin in a low concentration range (0 to 10 ⁇ g/mL) using the immunoagglutination reaction measurement reagent prepared in Example 5.
  • 2 is a graph showing the results of measuring hemoglobin in a high concentration range (0 to 100 ⁇ g/mL) using the immunoagglutination reaction measurement reagent prepared in Example 5.
  • Each mixture (sample) of free hemoglobin (free-Hb)/hemoglobin-haptoglobin (Hb-Hp) complex was tested using the immunoagglutination reaction measurement reagent prepared in Example 5 and a commercially available colorimetric measurement reagent. It is a graph showing the measured results.
  • Hemoglobin is a protein contained in red blood cells in living bodies, has the property of binding to oxygen molecules, and is involved in oxygen transport. Hemoglobin has a tetrameric structure [ ⁇ 2 ⁇ 2 ]. The molecular weight of the ⁇ chain is about 15,500, and the molecular weight of the ⁇ chain is about 17,000, and as described below, these can be separated by electrophoresis or the like. The molecular weight of hemoglobin as a whole is approximately 64,500.
  • Haptoglobin is a type of glycoprotein and specifically binds to hemoglobin to form a hemoglobin-haptoglobin complex.
  • Hp1-1 type haptoglobin is composed of two ⁇ chains and two ⁇ chains, which are linked by SS bonds.
  • the molecular weights are approximately 10,000 for Hp1 ⁇ chain, approximately 18,000 for Hp2 ⁇ chain, and approximately 39,000 for ⁇ chain.
  • the overall molecular weight of haptoglobin is, for example, Hp1-1 type, approximately 98,000.
  • Hp1-1 type haptoglobin has two binding sites with dimeric hemoglobin in its molecule. Therefore, when a complex is formed between Hp1-1 type haptoglobin and tetrameric hemoglobin [ ⁇ 2 ⁇ 2 ], one molecule of Hp1-1 type haptoglobin contains two molecules of dimeric hemoglobin [ ⁇ ]. It becomes a complex with two bonds.
  • Antibodies obtained using tetrameric free hemoglobin [ ⁇ 2 ⁇ 2 ] as an antigen also react with dimeric hemoglobin [ ⁇ ] contained in the hemoglobin-haptoglobin complex. Therefore, it has been extremely difficult to obtain anti-free hemoglobin antibodies that do not bind to hemoglobin-haptoglobin complexes through conventional animal immunization. In contrast, in the present embodiment, it has been found that by selecting and using two or more types of anti-hemoglobin antibodies, the anti-hemoglobin antibodies do not react with the hemoglobin-haptoglobin complex, but react with free hemoglobin.
  • the hemoglobin-haptoglobin complex used as an antigen means a complex of hemoglobin and Hp1-1 type haptoglobin.
  • the reactivity to such complexes is sufficiently lower than the reactivity to free hemoglobin, the reactivity to other complexes of haptoglobin (Hp2-1 type, Hp2-2 type) and hemoglobin will also be This is sufficiently lower than the reactivity to free hemoglobin.
  • a method for measuring free hemoglobin according to an embodiment of the present invention uses two or more types of anti-hemoglobin antibodies, including a combination of an antibody specific to hemoglobin ⁇ chain and an antibody specific to hemoglobin ⁇ chain, to measure free hemoglobin.
  • the antigen-antibody reaction is carried out to measure the Note that the embodiment described here below may be referred to as the first embodiment of the free hemoglobin measurement method.
  • the free hemoglobin measuring method of the present embodiment is not particularly limited as long as it is a method that utilizes an antigen-antibody reaction, that is, an immunological method, and includes, for example, an immunoagglutination method (for example, a latex agglutination method, a colloidal gold agglutination method, etc.), Examples include ELISA method and immunochromatography method. Among these, the measurement method of the present embodiment is suitably used for immunoagglutination methods, more preferably for latex agglutination methods.
  • the anti-hemoglobin antibodies used in this embodiment are two or more types of anti-hemoglobin antibodies including a combination of an antibody specific to hemoglobin ⁇ chain and an antibody specific to hemoglobin ⁇ chain.
  • antibody specific for hemoglobin ⁇ chain means an anti-hemoglobin antibody whose reaction against hemoglobin ⁇ chain is sufficiently higher than that against ⁇ chain.
  • specificity of the anti-hemoglobin antibody is sometimes referred to as the specificity of the anti-hemoglobin antibody.
  • the specificity of the anti-hemoglobin antibody can be such that, for example, the reaction against hemoglobin ⁇ chain is 75% or more, or even 80% or more, of the total reaction against hemoglobin ⁇ chain and ⁇ chain.
  • the specificity for the hemoglobin ⁇ chain is determined as shown in the example below: Western blotting is performed using the anti-hemoglobin antibody to be evaluated as the primary antibody for hemoglobin; the region on the membrane where the ⁇ chain is present. The evaluation can be made by integrating the band brightness of the region where the ⁇ chain is present and the band brightness of the region where the ⁇ chain is present; and calculating the ratio of the ⁇ chain integrated value to the total amount of both integrated values.
  • the specificity for hemoglobin ⁇ chain is also similar.
  • the present inventors believe that the mechanism of action by which free hemoglobin can be specifically measured by a combination of an antibody specific to the hemoglobin ⁇ chain and an antibody specific to the hemoglobin ⁇ chain is as follows.
  • ⁇ chain-specific antibodies and ⁇ chain-specific antibodies cannot be used due to steric hindrance. It is considered that they cannot be combined at the same time.
  • free hemoglobin is hemoglobin with a tetrameric structure of ⁇ 2 ⁇ 2 and does not form a complex with haptoglobin, so that ⁇ chain-specific antibodies and ⁇ chain-specific antibodies can bind at the same time. considered to be a thing.
  • the mechanism of action by which free hemoglobin can be specifically measured by the combination of an ⁇ -chain-specific antibody and a ⁇ -chain-specific antibody is not necessarily limited to the above-mentioned mechanism of action.
  • the type of anti-hemoglobin antibody that can be used in this embodiment is not particularly limited as long as it satisfies the specificity described above.
  • 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, and animals that have been immunized with the measurement target by a known method.
  • Either a polyclonal antibody obtained from the serum of an animal to be measured or a monoclonal antibody obtained by cell fusion of the spleen of an animal immunized with the object to be measured with myeloma cells may be used.
  • fragments thereof eg, F(ab')2, Fab, Fab', or Fv] may be used.
  • At least one of the two or more anti-hemoglobin antibodies used in this embodiment may be supported on an insoluble carrier, or two or more of them may be supported on an insoluble carrier.
  • an insoluble carrier is not particularly limited as long as it can support antibodies, and can be appropriately selected depending on the type of immunological technique described above.
  • examples of insoluble carriers include insoluble particles that can be used in immunological techniques, and examples of insoluble particles include commonly used metal colloid particles such as colloidal gold particles, latex particles, silica particles, and magnetic particles. , fluorescent particles, red blood cells, etc.
  • latex particles are preferred, and polystyrene latex particles are more preferred.
  • the insoluble carrier is preferably in the form of particles, and its average particle diameter is preferably 5 to 1,000 nm, more preferably 30 to 500 nm, and even more preferably 75 to 350 nm, but it can be used without being particularly limited to this range. It is possible to do so.
  • Carrying an antibody means that the antibody is immobilized on the surface of an insoluble carrier by physical adsorption or chemical bonding.
  • the supporting method includes, for example, a known technique in which the antibody and insoluble carrier particles are mixed and the antibody is physically adsorbed onto the surface of the insoluble carrier particles. can be immobilized.
  • insoluble carrier particles having amino groups or carboxyl groups introduced onto their surfaces are used, antibodies can be immobilized on the surface of the insoluble carrier particles by chemical bonding using glutaraldehyde or carboximide reagents.
  • the amount of antibody supported is not particularly limited, but may be 0.5 to 2,000 ⁇ g/mg latex, 1 to 1,000 ⁇ g/mg latex, or 2 to 500 ⁇ g/mg latex.
  • the amount of antibody supported can be calculated by subtracting the amount of antibody after immobilization from the amount of antibody before immobilization on the insoluble carrier.
  • both may be supported on the same insoluble carrier.
  • a mixture of a plurality of insoluble carriers in which one type of anti-hemoglobin antibody is supported on one type of insoluble carrier may be used.
  • the insoluble carriers used to support different types of anti-hemoglobin antibodies may be the same type of insoluble carriers, or may be different types of insoluble carriers with different materials, particle sizes, etc.
  • reactivity In this embodiment, it is preferable to perform an antigen-antibody reaction to measure free hemoglobin under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
  • Reactivity to free hemoglobin refers to reactivity when free hemoglobin is added alone as an antigen.
  • Reactivity is evaluated by an index for quantifying the antigen in the above-mentioned immunological method. For example, in the case of an immunoagglutination method, the amount of change in turbidity over a predetermined time, and in the case of an ELISA method, the amount of color development/light absorption caused by the labeling of a labeled antibody, etc. can be mentioned.
  • Reactivity to free hemoglobin is determined, for example, by conditions under which free hemoglobin is 2.7 pmol/mL (1 ⁇ g/mL), 27 pmol/mL (10 ⁇ g/mL), or 270 pmol/mL (100 ⁇ g/mL) in the antigen-antibody reaction reaction solution. It can be evaluated by
  • reactivity to hemoglobin-haptoglobin complex refers to reactivity when a hemoglobin-haptoglobin complex is added as an antigen.
  • adding a hemoglobin-haptoglobin complex means not only adding a hemoglobin-haptoglobin complex formed in advance, but also adding free hemoglobin and haptoglobin in an equimolar amount to each other in a mixed solution. Includes embodiments in which a hemoglobin-haptoglobin complex is formed.
  • Reactivity to hemoglobin-haptoglobin complexes is evaluated under the same reaction conditions as the reactivity to free hemoglobin described above, except that the antigen is changed to equimolar hemoglobin-haptoglobin complexes.
  • the same reaction conditions refer to the anti-hemoglobin antibody used for the antigen-antibody reaction, the concentration of the antibody, the composition of the reaction solution for the antigen-antibody reaction (types and concentrations of pH buffer, salt, and additives), and the pH. , meaning that the reaction time and temperature of the antigen-antibody reaction are the same.
  • the anti-hemoglobin antibody is supported on an insoluble carrier as described below, the same type of antibody-supported carrier shall be used.
  • Antigen-antibody reaction for measuring free hemoglobin is an antigen-antibody reaction performed by adding a measurement target (e.g., a specimen) to which free hemoglobin is to be measured. Distinguished from antibody reactions. In addition to free hemoglobin, a hemoglobin-haptoglobin complex is often present in the above measurement target. In other words, antigen-antibody reactions for measuring free hemoglobin are often performed in an environment where hemoglobin-haptoglobin complexes can exist.
  • the antigen-antibody reaction in order to measure free hemoglobin, is preferably performed under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin. Such a reactivity ratio may be 10% or less, or even 5% or less.
  • the antigen-antibody reaction is carried out under the conditions for measuring free hemoglobin, which means that it is carried out under the same reaction conditions as those used to evaluate the above-mentioned "reactivity to free hemoglobin" and "reactivity to hemoglobin-haptoglobin complex.” It means that.
  • the definition of the same reaction conditions is as described above.
  • free hemoglobin is measured by a known method after performing an antigen-antibody reaction.
  • the target to be measured can be set as appropriate depending on the immunological method employed. For example, in the case of immunoagglutination, the amount of turbidity change over a given time, and in the case of ELISA, the amount of change in turbidity due to the labeling of the labeled antibody. Examples include the color development and amount of light absorption that occur. Therefore, as a method for measuring these, a known method such as an optical method can be adopted, and a general-purpose optical measuring device can be used.
  • the method for measuring free hemoglobin of the present invention uses two or more types of anti-hemoglobin antibodies; Antigen-antibody reactions can be performed to measure hemoglobin;
  • the two or more types of anti-hemoglobin antibodies used in this embodiment are not particularly limited as long as the combination satisfies the above reactivity.
  • a combination of an antibody specific to hemoglobin ⁇ chain and an antibody specific to hemoglobin ⁇ chain; a combination of antibodies specific to the vicinity of the boundary between hemoglobin ⁇ chain and ⁇ chain; and the like can be used.
  • a combination of an antibody specific for hemoglobin ⁇ chain and an antibody specific for hemoglobin ⁇ chain is particularly preferred.
  • the antigen-antibody reaction described above is an antigen-antibody reaction against two or more different epitopes in the same antigen (hemoglobin in this embodiment).
  • the immunological method of this embodiment is preferably a method that utilizes the two or more antigen-antibody reactions.
  • the measurement method uses an antigen-antibody reaction to measure free hemoglobin: carried out using two or more anti-hemoglobin antibodies, including a combination of an antibody specific for hemoglobin ⁇ chain and an antibody specific for hemoglobin ⁇ chain; or The test is carried out under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
  • a reagent for measuring free hemoglobin according to an embodiment of the present invention contains two or more types of anti-hemoglobin antibodies, including a combination of an antibody specific to hemoglobin ⁇ chain and an antibody specific to hemoglobin ⁇ chain. Note that the embodiment described here below may be referred to as the first embodiment of the free hemoglobin measurement reagent.
  • the anti-hemoglobin antibody used in the reagent of this embodiment the antibody described in the first embodiment of the method for measuring free hemoglobin mentioned above can be used.
  • At least one of the two or more anti-hemoglobin antibodies used in the reagent of this embodiment may be supported on an insoluble carrier, or two or more of them may be supported on an insoluble carrier.
  • insoluble carriers may be insoluble particles. The types of insoluble carriers and insoluble particles, the method of supporting antibodies, etc. are as explained in the measurement method described above.
  • the reactivity of the reagent of this embodiment with respect to the hemoglobin-haptoglobin complex is 15% or less of the reactivity with respect to free hemoglobin.
  • "Reactivity to free hemoglobin” and “reactivity to hemoglobin-haptoglobin complex” are as explained in the measurement method described above.
  • the measurement reagent in addition to the reagent composition (composition of the reaction solution, etc.) described later, the measurement reagent according to this embodiment usually includes an attached document such as an instruction manual, and the reaction conditions for measuring free hemoglobin are specified in the reagent. identified by its structure and attached documents. More specifically, the package insert of the measurement reagent includes the amount of two or more anti-hemoglobin antibodies (including embodiments in which these are supported on an insoluble carrier), the total volume of the reaction solution, the addition procedure, and the reaction. The time, reaction temperature, etc. are specified, and by using the measurement reagent of this embodiment in accordance with these, the reaction conditions for measuring free hemoglobin will be specified.
  • a reagent whose reactivity to hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin is defined as "reactivity to free hemoglobin” and "reactivity to hemoglobin-haptoglobin complex” under the reaction conditions specified for the reagent. It is sufficient if it is 15% or less when evaluating and comparing the "reactivity" respectively.
  • the reactivity mentioned here may be 10% or less, or even 5% or less.
  • reactivity to free hemoglobin can be evaluated by reactivity to a predetermined concentration of free hemoglobin (for example, 2.7 pmol/mL, 27 pmol/mL, 270 pmol/mL), and similarly "reactivity to hemoglobin-haptoglobin complex"
  • the "reactivity to the body” can be evaluated by the reactivity to a hemoglobin-haptoglobin complex that is equimolar to a predetermined concentration of free hemoglobin.
  • the measurement reagent of this embodiment has the above-described configuration and is a reagent for measuring free hemoglobin using an immunological technique.
  • the form of the measurement reagent is not particularly limited as long as it satisfies these requirements; for example, reagents using immunoagglutination methods (e.g., latex agglutination method, colloidal gold agglutination method, etc.), ELISA method, immunochromatography method, etc. It can be done. Among these, reagents for immunoagglutination are preferred, and reagents for latex agglutination are more preferred.
  • the measurement reagent of this embodiment is composed of a two-reagent system, for example, a reagent that does not contain an insoluble carrier (first reagent) and a reagent that contains an insoluble carrier that supports an antibody (antibody-supported insoluble carrier) (second reagent).
  • the reagent system may consist of only one reagent containing an antibody-supported insoluble carrier.
  • the first reagent can be used to adjust the measurement environment, such as by being used as a diluent for adjusting the concentration of the object to be measured or impurities in the reaction system, adjusting the reaction rate, etc.
  • the second reagent contains an antibody-supported insoluble carrier and is mixed with the first reagent and sample to produce an immunoagglutination reaction.
  • the first reagent and the second reagent can appropriately contain a pH buffer, a salt, a surfactant, an aggregation promoter, a preservative, and the like.
  • the pH during the aggregation reaction is preferably
  • the measurement reagent is mixed with the sample to obtain a reaction solution, and the concentration of the insoluble carrier in the reaction solution is determined depending on the particle size of the insoluble carrier used and the overall design of the measurement system, for example, 0.0001 mg/mL to 10 mg/mL. An appropriate selection can be made from the range of /mL.
  • the concentration of the insoluble carrier carrying the anti-hemoglobin antibody in the measurement reagent may be 0.01 to 5 mg/mL, or 0.05 to 1 mg/mL. Note that the concentration of the insoluble carrier in the second reagent is diluted by mixing with the first reagent, sample, etc. during use, so the concentration of the insoluble carrier in the second reagent should be selected appropriately depending on the dilution ratio.
  • diluting 2 times and using it when diluting 2 times and using it, it is 0.0002 mg/mL to 20 mg/mL, and when diluting 3 times and using it, it can be adjusted appropriately so that it is 0.0003 mg/mL to 30 mg/mL. can do.
  • the free hemoglobin measurement reagent of the present invention contains two or more types of anti-hemoglobin antibodies; has reactivity with hemoglobin-haptoglobin complexes that is 15% or less of the reactivity with free hemoglobin; good.
  • the antibody used in the measurement reagent of this embodiment is as described in the second embodiment of the method for measuring free hemoglobin described above. Furthermore, "reactivity to free hemoglobin” and “reactivity to hemoglobin-haptoglobin complex” are as described in the first embodiment regarding the reagent for measuring free hemoglobin. Other configurations of the measurement reagent of this embodiment (insoluble carrier, form of reagent, etc.) are also as explained in the first embodiment related to the free hemoglobin measurement reagent.
  • the free hemoglobin measuring reagents mentioned above are: comprising two or more anti-hemoglobin antibodies, including a combination of an antibody specific for hemoglobin alpha chain and an antibody specific for hemoglobin beta chain; or
  • the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin. This allows the amount of free hemoglobin to be measured regardless of the amount of hemoglobin-haptoglobin complex. Therefore, even when the antigen-antibody reaction for measuring free hemoglobin is performed in an environment where a hemoglobin-haptoglobin complex may exist, free hemoglobin can be specifically measured. This eliminates the need for steps such as removing the hemoglobin-haptoglobin complex before the antigen-antibody reaction to measure free hemoglobin, making it possible to specifically measure free hemoglobin while simplifying the operation. can.
  • the invention further provides a combination of an antibody specific for hemoglobin alpha chain and an antibody specific for hemoglobin beta chain.
  • the definitions of "reacts specifically with hemoglobin ⁇ chain” and “reacts specifically with hemoglobin ⁇ chain” are as described above.
  • a combination of an antibody specific for hemoglobin ⁇ chain and an antibody specific for hemoglobin ⁇ chain is particularly suitable for specifically measuring free hemoglobin.
  • the present invention further provides a free hemoglobin measurement kit containing the above-mentioned free hemoglobin measurement reagent.
  • the measurement reagent included in the kit may include, for example, an anti-hemoglobin antibody-supported insoluble carrier.
  • the free hemoglobin measurement kit may also include components such as a calibrator and a control, and also includes components such as an instrument and container for collecting a specimen, and a storage solution for preserving the specimen. May contain.
  • Example 1 Preparation of anti-human hemoglobin monoclonal antibody (1) Immunization of mice Mice were immunized with hemoglobin. After each immunization, the antibody titer of the mice was measured by the two-antibody RIA method using 125 I-labeled hemoglobin. As a result, mice with high antiserum titers were selected. (2) Cell fusion The spleen was removed from the selected mouse, and splenocytes were prepared. The prepared splenocytes and mouse myeloma cells were fused by electrofusion method, suspended in a fused cell selection medium, and seeded in a 96-well microplate.
  • Example 2 Confirmation of antibody specificity Human hemoglobin purified from human type O blood (manufactured by Eiken Kagaku Co., Ltd.) was transferred to a polyacrylamide precast gel for electrophoresis (manufactured by ATTO) and an AE-6530 Rapidus mini-slab electrophoresis tank (manufactured by ATTO). (manufactured by ATTO). The migrated proteins were transferred to a PVDF membrane using a Transblot SD cell (manufactured by Bio-Rad).
  • Example 1 Western blotting was performed using the antibody obtained in Example 1 (1 ⁇ g/mL) as the primary antibody and an HRP-labeled anti-mouse IgG antibody recognition antibody (rabbit-derived polyclonal antibody, manufactured by Cappel) as the secondary antibody. HRP was caused to emit light using Western Lightning ECL Pro (manufactured by PerkinElmer), and a band image was obtained. The band image was analyzed using CS Analyzer ver. 3.0 (manufactured by ATTO), and the band brightness of the electrophoresis lane was calculated as an integrated value in measurement mode: designated area zone densitometry (FIG. 1). Next, the integrated value of the band brightness of the ⁇ chain and ⁇ chain was determined from the band brightness of the Western blot.
  • HRP-labeled anti-mouse IgG antibody recognition antibody goat-derived polyclonal antibody, manufactured by Cappel
  • HRP was caused to emit light using Western Lightning ECL Pro (manufactured by PerkinElmer), and
  • the band integrated value of the ⁇ chain was multiplied by a coefficient of 0.783 calculated by the method described later.
  • the band brightness ratio of each subunit was determined when ⁇ chain integrated value + ⁇ chain integrated value was set as 100%. From this result, antibodies that showed an integrated value ratio of ⁇ chain band of 75% or more were considered hemoglobin ⁇ chain-specific antibodies, and antibodies that showed an integrated value ratio of ⁇ chain bands of 75% or more were considered hemoglobin ⁇ chain-specific antibodies. It was judged. The results are shown in Table 1.
  • Example 1 As shown in Table 1, among the antibodies obtained in Example 1, Nos. 1, 8, 12, and 16 have a ratio of ⁇ chain integrated value to the total ⁇ chain integrated value and ⁇ chain integrated value. was 75% or more, so it was recognized as an ⁇ chain-specific antibody. On the other hand, in Nos. 4, 11, 21, 23, 24, and 32, the ratio of the ⁇ -chain integrated value to the total of the ⁇ -chain integrated value and the ⁇ -chain integrated value was 75% or more, so It was recognized that it is a ⁇ chain-specific antibody.
  • Example 3 Monoclonal antibody reactivity-1 Using the antibody obtained in Example 1, reactivity to free hemoglobin and reactivity to hemoglobin-haptoglobin complex was evaluated. By the method described below, each anti-human hemoglobin monoclonal antibody was immobilized on polystyrene latex particles, and the degree of aggregation when reacted with a sample containing hemoglobin or hemoglobin-haptoglobin complex was compared.
  • Example 4 Monoclonal antibody reactivity-2 All combinations of the 10 types of antibodies obtained in Example 1 were evaluated for reactivity to free hemoglobin and reactivity to hemoglobin-haptoglobin complex. The method was confirmed using the same method as used in Example 3.
  • Reactivity was evaluated based on the following criteria.
  • - The degree of aggregation of both free hemoglobin and hemoglobin-haptoglobin complex was extremely low ( ⁇ OD ⁇ 10,000 was 500 or less for both)
  • Example 5 Free hemoglobin measurement reagent The antibody obtained in Example 1 was used to prepare an immunoagglutination reaction measurement reagent.
  • a first reagent and a second reagent were prepared as measurement reagents.
  • As the first reagent 50 mM HEPES buffer (pH 7.4) was used.
  • As a second reagent each polystyrene latex carrying each of the anti-hemoglobin monoclonal antibodies (anti-Hb antibody) of Example 2 was mixed and the latex concentration was adjusted to 1.0 mg/mL to prepare an immunoagglutination reaction measurement reagent.
  • the antibody-supported insoluble carrier was prepared by mixing each anti-hemoglobin monoclonal antibody with polystyrene latex particles (average particle size: 100 nm) and supporting the anti-hemoglobin monoclonal antibody on the surface of the polystyrene latex particles.
  • No. 16 was used as the ⁇ -chain-specific anti-hemoglobin monoclonal antibody
  • No. 11 was used as the ⁇ -chain-specific anti-hemoglobin monoclonal antibody.
  • Samples for measuring free hemoglobin were prepared to have concentrations of 1 ⁇ g/mL (2.7 pmol/mL), 10 ⁇ g/mL (27 pmol/mL), and 100 ⁇ g/mL (270 pmol/mL), respectively. Further, in the measurement sample of the hemoglobin-haptoglobin complex, an equimolar amount of haptoglobin was added to form a complex in the measurement sample.
  • the prepared sample was measured using the measurement reagent prepared in (1) above and an automatic biochemical analyzer JCA-BM6070. The results are shown in Table 6.
  • the immunoagglutination reaction measurement reagent of this example enabled measurement of free hemoglobin in proportion to concentration.
  • the reactivity to the hemoglobin-haptoglobin complex was 15% or less of the reactivity to free hemoglobin.
  • Example 6 Comparison with other measurement methods A comparison of measurement methods was carried out using a sample containing a mixture of free hemoglobin and hemoglobin-haptoglobin complex.
  • a commercially available hemoglobin colorimetric measurement reagent was used and compared with the immunoagglutination reaction measurement reagent prepared in Example 5.
  • a hemoglobin colorimetric measurement reagent is a reagent that dissolves red blood cell membranes with sodium lauryl sulfate and quantifies the eluted hemoglobin by measuring its absorbance. However, in this study, the hemolysis step was omitted.
  • Each mixture (sample) of free hemoglobin (free-Hb)/hemoglobin-haptoglobin (Hb-Hp) complex prepared in (2) above was mixed with a hemoglobin colorimetric measurement reagent, and a spectrophotometer (manufactured by Shimadzu Corporation) was used. , UV-1900) at 540 nm.
  • the immunoagglutination reaction measurement reagent (Latex reagent) prepared in Example 5 was prepared by diluting each mixed solution (sample) prepared in (2) above 50 times with physiological saline and using the following conditions. , was measured using an automatic biochemical analyzer JCA-BM6070. Sample amount: 1.0 ⁇ L, 1st reagent: 50 ⁇ L, 2nd reagent: 50 ⁇ L, measurement wavelength: 658 nm The results are shown in Table 8 and FIG.
  • the measured value does not change even if the ratio of free hemoglobin and hemoglobin-haptoglobin complex changes, and it was not possible to distinguish between the two.
  • the immunoagglutination reaction measurement reagent (latex reagent) of the present invention was not affected by the amount of hemoglobin-haptoglobin complex, and a measurement value proportional to the free hemoglobin concentration was obtained. The above results showed that the latex reagent of the present invention specifically measures free hemoglobin.

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 β 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 α chain of hemoglobin and an antibody specific to the β 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

遊離ヘモグロビン測定試薬、遊離ヘモグロビン測定方法および抗ヘモグロビン抗体Free hemoglobin measurement reagent, free hemoglobin measurement method, and anti-hemoglobin antibody
　本発明は、遊離ヘモグロビンを特異的に測定するための試薬および方法、ならびにこれらに用いる抗ヘモグロビン抗体に関するものである。 The present invention relates to reagents and methods for specifically measuring free hemoglobin, and anti-hemoglobin antibodies used therefor.
　血中においてヘモグロビンは、生体内のハプトグロビンと結合し、ヘモグロビン－ハプトグロビン複合体を形成する。かかる複合体は、スカベンジャー受容体のＣＤ１６３を介して、最終的に肝臓のマクロファージに取り込まれることにより、分解代謝される。しかし、熱傷や人工心肺などによる溶血により、ヘモグロビンを生体内のハプトグロビンで処理しきれなくなると、ハプトグロビンとの複合体を形成していないヘモグロビン（遊離ヘモグロビン）の血中濃度が上昇する。 In the blood, hemoglobin combines with haptoglobin in the body to form a hemoglobin-haptoglobin complex. Such complexes are eventually taken up by macrophages in the liver via the scavenger receptor CD163, where they are degraded and metabolized. However, when hemoglobin cannot be completely processed by haptoglobin in the body due to hemolysis caused by burns or cardiopulmonary bypass, the blood concentration of hemoglobin that does not form a complex with haptoglobin (free hemoglobin) increases.
　遊離ヘモグロビンは、分子量が小さいため、尿中に排出されると腎臓の糸球体を通過し、尿細管上皮細胞に取り込まれてヘムとグロビンに分解される。このうちヘムに含まれるヘム鉄が触媒となって、フリーラジカルを産生させ、近位の尿細管上皮細胞を壊死させ、尿細管障害を引き起こす。遊離ヘモグロビンは、ハプトグロビン製剤により治療が可能であるが、適切な量のハプトグロビン製剤を投与するためには、遊離ヘモグロビン濃度を正確に測定する必要がある。 Free hemoglobin has a small molecular weight, so when it is excreted in the urine, it passes through the glomerulus of the kidney, is taken up by renal tubular epithelial cells, and is broken down into heme and globin. Among these, heme iron contained in heme acts as a catalyst to produce free radicals, which cause necrosis of proximal renal tubular epithelial cells and cause renal tubular damage. Free hemoglobin can be treated with haptoglobin preparations, but in order to administer an appropriate amount of haptoglobin preparations, it is necessary to accurately measure the free hemoglobin concentration.
　遊離ヘモグロビン濃度を測定する方法として、例えば、ヒトハプトグロビンを固相に結合させた固相化ヒトハプトグロビンに、遊離ヘモグロビン含有検体を接触させて遊離ヘモグロビンを固相上のヒトハプトグロビンと結合させたのち、さらに酵素標識した抗ヒトヘモグロビン抗体を作用させ、固相上に結合した遊離ヘモグロビンを測定する方法が提案されている（特許文献１参照）。
　また、抗ハプトグロビン抗体を遊離ヘモグロビン含有検体に添加し、検体中に存在するヘモグロビン－ハプトグロビン複合体と該抗体を反応せしめた後、遊離ヘモグロビンを酵素免疫測定法により測定する方法が提案されている（特許文献２参照）。 As a method for measuring the free hemoglobin concentration, for example, a specimen containing free hemoglobin is brought into contact with immobilized human haptoglobin in which human haptoglobin is bound to a solid phase, and the free hemoglobin is bound to human haptoglobin on the solid phase. Furthermore, a method has been proposed in which free hemoglobin bound on a solid phase is measured by applying an enzyme-labeled anti-human hemoglobin antibody (see Patent Document 1).
Furthermore, a method has been proposed in which an anti-haptoglobin antibody is added to a sample containing free hemoglobin, the antibody is reacted with the hemoglobin-haptoglobin complex present in the sample, and then free hemoglobin is measured by enzyme immunoassay ( (See Patent Document 2).
特開平２－２３１５６５号公報Japanese Patent Application Publication No. 2-231565 特開平７－１０３９７８号公報Japanese Unexamined Patent Publication No. 7-103978
　遊離ヘモグロビンの測定には、固定化ハプトグロビンに吸着させた後に、洗浄・分離を行い、遊離ヘモグロビンを策定する方法（特許文献１）、ヘモグロビン－ハプトグロビン複合体をあらかじめ除去して遊離ヘモグロビンを測定する方法（特許文献２）等が知られているが、いずれも操作が煩雑であり、迅速性や測定処理能力に限界があるため、特異性の高い迅速な遊離ヘモグロビン免疫測定法が求められていた。 Free hemoglobin can be measured by adsorbing it to immobilized haptoglobin and then washing and separating it to formulate free hemoglobin (Patent Document 1), or by removing the hemoglobin-haptoglobin complex in advance and measuring free hemoglobin. (Patent Document 2) and the like are known, but all of them require complicated operations and have limitations in speed and measurement processing capacity, so a rapid and highly specific free hemoglobin immunoassay method has been desired.
　本発明は、上記問題に鑑みてなされたものであって、操作が簡便であり、遊離ヘモグロビンを特異的に測定することができる試薬および方法、ならびにこれに用いることのできる抗体を提供することを目的とする。 The present invention has been made in view of the above problems, and aims to provide a reagent and method that are easy to operate and can specifically measure free hemoglobin, as well as antibodies that can be used therefor. purpose.
　本発明者らは上記問題を解決すべく研究を行った結果、特定の抗ヘモグロビン抗体を２種以上用いた遊離ヘモグロビン測定試薬および測定方法、あるいはヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下である遊離ヘモグロビン測定試薬および測定方法、ならびにこれらに用いる２種以上の抗ヘモグロビン抗体が、上記課題を解決し得ることを見出し、本発明を完成させた。具体的には、本発明は以下のとおりである。 The present inventors conducted research to solve the above problems, and found that a free hemoglobin measurement reagent and measurement method using two or more specific anti-hemoglobin antibodies, or a reactivity to a hemoglobin-haptoglobin complex that differs from that of free hemoglobin. The inventors have discovered that a reagent and method for measuring free hemoglobin with a free hemoglobin concentration of 15% or less, and two or more anti-hemoglobin antibodies used therein can solve the above problems, and have completed the present invention. Specifically, the present invention is as follows.
〔１〕　遊離ヘモグロビンを測定する方法であって、
　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせを含む２種以上の抗ヘモグロビン抗体を用い、前記遊離ヘモグロビンを測定するための抗原抗体反応を行う、
遊離ヘモグロビン測定方法。
〔２〕　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下となる条件で、前記遊離ヘモグロビンを測定するための前記抗原抗体反応を行う、〔１〕に記載の遊離ヘモグロビン測定方法。
〔３〕　前記遊離ヘモグロビンを測定するための前記抗原抗体反応を、ヘモグロビン－ハプトグロビン複合体の存在し得る環境下で行う、〔１〕または〔２〕に記載の遊離ヘモグロビン測定方法。
〔４〕　前記２種以上の抗ヘモグロビン抗体の少なくとも１種が不溶性担体に担持されている、〔１〕～〔３〕のいずれか一項に記載の遊離ヘモグロビン測定方法。
〔５〕　前記不溶性担体が不溶性粒子である、〔４〕に記載の遊離ヘモグロビン測定方法。
〔６〕　免疫凝集法である、〔５〕に記載の遊離ヘモグロビン測定方法。
〔７〕　遊離ヘモグロビンを測定する方法であって、
　２種以上の抗ヘモグロビン抗体を用い、
　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下となる条件で、前記遊離ヘモグロビンを測定するための抗原抗体反応を行う、
遊離ヘモグロビン測定方法。
〔８〕　遊離ヘモグロビンを測定するための試薬であって、
　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせを含む２種以上の抗ヘモグロビン抗体を含む、
遊離ヘモグロビン測定試薬。
〔９〕　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下である、〔８〕に記載の遊離ヘモグロビン測定試薬。
〔１０〕　遊離ヘモグロビンを測定するための抗原抗体反応が、ヘモグロビン－ハプトグロビン複合体の存在し得る環境下で行われる、〔８〕または〔９〕に記載の遊離ヘモグロビン測定試薬。
〔１１〕　前記２種以上の抗ヘモグロビン抗体の少なくとも１種が不溶性担体に担持されている、〔８〕～〔１０〕のいずれか一項に記載の遊離ヘモグロビン測定試薬。
〔１２〕　前記不溶性担体が不溶性粒子である、〔１１〕に記載の遊離ヘモグロビン測定試薬。
〔１３〕　免疫凝集法の試薬である、〔１２〕に記載の遊離ヘモグロビン測定試薬。
〔１４〕　遊離ヘモグロビンを測定するための試薬であって、
　２種以上の抗ヘモグロビン抗体を含み、
　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下である、
遊離ヘモグロビン測定試薬。
〔１５〕　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体とを組み合わせてなる、抗ヘモグロビン抗体の組み合わせ。
〔１６〕　〔８〕～〔１４〕のいずれか一項に記載の遊離ヘモグロビン測定試薬を含む、遊離ヘモグロビン測定キット。 [1] A method for measuring free hemoglobin, comprising:
Performing an antigen-antibody reaction for measuring the free hemoglobin using two or more types of anti-hemoglobin antibodies including a combination of an antibody specific for hemoglobin α chain and an antibody specific for hemoglobin β chain,
Free hemoglobin measurement method.
[2] The free hemoglobin measurement according to [1], wherein the antigen-antibody reaction for measuring the free hemoglobin is performed under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin. Method.
[3] The method for measuring free hemoglobin according to [1] or [2], wherein the antigen-antibody reaction for measuring the free hemoglobin is performed in an environment where a hemoglobin-haptoglobin complex can exist.
[4] The method for measuring free hemoglobin according to any one of [1] to [3], wherein at least one of the two or more anti-hemoglobin antibodies is supported on an insoluble carrier.
[5] The method for measuring free hemoglobin according to [4], wherein the insoluble carrier is an insoluble particle.
[6] The method for measuring free hemoglobin according to [5], which is an immunoagglutination method.
[7] A method for measuring free hemoglobin, comprising:
Using two or more types of anti-hemoglobin antibodies,
Performing the antigen-antibody reaction for measuring the free hemoglobin under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
Free hemoglobin measurement method.
[8] A reagent for measuring free hemoglobin, comprising:
Two or more types of anti-hemoglobin antibodies, including a combination of an antibody specific for hemoglobin α chain and an antibody specific for hemoglobin β chain,
Free hemoglobin measurement reagent.
[9] The reagent for measuring free hemoglobin according to [8], wherein the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
[10] The free hemoglobin measurement reagent according to [8] or [9], wherein the antigen-antibody reaction for measuring free hemoglobin is performed in an environment where a hemoglobin-haptoglobin complex can exist.
[11] The reagent for measuring free hemoglobin according to any one of [8] to [10], wherein at least one of the two or more anti-hemoglobin antibodies is supported on an insoluble carrier.
[12] The reagent for measuring free hemoglobin according to [11], wherein the insoluble carrier is an insoluble particle.
[13] The free hemoglobin measuring reagent according to [12], which is a reagent for immunoagglutination.
[14] A reagent for measuring free hemoglobin, comprising:
Contains two or more types of anti-hemoglobin antibodies,
The reactivity to hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin,
Free hemoglobin measurement reagent.
[15] A combination of anti-hemoglobin antibodies, which is a combination of an antibody specific to hemoglobin α chain and an antibody specific to hemoglobin β chain.
[16] A free hemoglobin measurement kit comprising the free hemoglobin measurement reagent according to any one of [8] to [14].
　本発明の測定方法および測定試薬によれば、操作が簡便でありながら、遊離ヘモグロビンを特異的に測定することができる。 According to the measurement method and measurement reagent of the present invention, free hemoglobin can be specifically measured while the operation is simple.
実施例１で得られた抗体の特異性を確認した結果を表す泳動画像である。実施例１で得られた抗体を一次抗体として用いたウエスタンブロットを実施し、バンド画像を取得した。得られたバンド画像を解析し、泳動レーンのバンド輝度を積算値として算出した。1 is an electrophoresis image showing the results of confirming the specificity of the antibody obtained in Example 1. Western blotting was performed using the antibody obtained in Example 1 as a primary antibody, and band images were obtained. The obtained band image was analyzed, and the band brightness of the migration lane was calculated as an integrated value. ヒトヘモグロビンを電気泳動しＣＢＢ染色を行い得られた泳動画像である。得られた泳動画像からα鎖、β鎖のバンド輝度の積算値をそれぞれ算出し、バンド輝度のノーマライズのための係数（α鎖のバンド積算値に係数０．７８３を乗じる）を算出した。This is an electrophoresis image obtained by electrophoresing human hemoglobin and performing CBB staining. The integrated value of the band brightness of the α chain and β chain was calculated from the obtained electrophoretic image, and a coefficient for normalizing the band brightness (multiplying the integrated band value of the α chain by a coefficient of 0.783) was calculated. 実施例５で作製した免疫凝集反応測定試薬を用い、低濃度域（０～１０μg/mL）のヘモグロビンを測定した結果を表すグラフである。2 is a graph showing the results of measuring hemoglobin in a low concentration range (0 to 10 μg/mL) using the immunoagglutination reaction measurement reagent prepared in Example 5. 実施例５で作製した免疫凝集反応測定試薬を用い、高濃度域（０～１００μg/mL）のヘモグロビンを測定した結果を表すグラフである。2 is a graph showing the results of measuring hemoglobin in a high concentration range (0 to 100 μg/mL) using the immunoagglutination reaction measurement reagent prepared in Example 5. 遊離ヘモグロビン（free-Ｈｂ）／ヘモグロビン－ハプトグロビン（Ｈｂ－Ｈｐ）複合体の各混合液（検体）を、実施例５で作製した免疫凝集反応測定試薬と、市販の比色測定試薬とを用いて測定した結果を表すグラフである。Each mixture (sample) of free hemoglobin (free-Hb)/hemoglobin-haptoglobin (Hb-Hp) complex was tested using the immunoagglutination reaction measurement reagent prepared in Example 5 and a commercially available colorimetric measurement reagent. It is a graph showing the measured results.
　以下、本発明の実施形態について説明する。 Hereinafter, embodiments of the present invention will be described.
〔用語〕
（ヘモグロビン）
　ヘモグロビンは、生体内中で赤血球中に含まれるタンパク質であり、酸素分子と結合する性質を持ち、酸素の運搬に関与している。
　ヘモグロビンは、１４１個のアミノ酸からなるα鎖（サブユニット）と、１４６個のアミノ酸からなるβ鎖（サブユニット）との２種類のサブユニットを二つずつ有する四量体構造［α２β２］となっている。α鎖の分子量は約１５，５００、β鎖の分子量は約１７，０００であり、後述するようにこれらは電気泳動等により分離が可能である。なお、ヘモグロビン全体の分子量は、約６４，５００である。 〔term〕
(hemoglobin)
Hemoglobin is a protein contained in red blood cells in living bodies, has the property of binding to oxygen molecules, and is involved in oxygen transport.
Hemoglobin has a tetrameric structure [α 2 β 2 ]. The molecular weight of the α chain is about 15,500, and the molecular weight of the β chain is about 17,000, and as described below, these can be separated by electrophoresis or the like. The molecular weight of hemoglobin as a whole is approximately 64,500.
（ハプトグロビン）
　ハプトグロビンは、糖タンパク質の１種であり、ヘモグロビンと特異的に結合しヘモグロビン－ハプトグロビン複合体を形成する。ヒトハプトグロビンには３種の血清型が存在し、Ｈｐ１－１型、Ｈｐ２－１型、Ｈｐ２－２型がある。ハプトグロビンは、最も単純な構造（Ｈｐ１－１型）では、２本のα鎖と２本のβ鎖とから構成され、これらはＳ－Ｓ結合により連結されている。
　分子量は、Ｈｐ１α鎖が約１００００、Ｈｐ２α鎖が約１８０００、β鎖が約３９０００である。ハプトグロビン全体の分子量は、例えばＨｐ１－１型で約９８０００である。 (haptoglobin)
Haptoglobin is a type of glycoprotein and specifically binds to hemoglobin to form a hemoglobin-haptoglobin complex. There are three serotypes of human haptoglobin, including Hp1-1 type, Hp2-1 type, and Hp2-2 type. In its simplest structure (Hp1-1 type), haptoglobin is composed of two α chains and two β chains, which are linked by SS bonds.
The molecular weights are approximately 10,000 for Hp1α chain, approximately 18,000 for Hp2α chain, and approximately 39,000 for β chain. The overall molecular weight of haptoglobin is, for example, Hp1-1 type, approximately 98,000.
（ヘモグロビン－ハプトグロビン複合体）
　ヘモグロビンとハプトグロビンとが複合体を形成する際、１分子の四量体ヘモグロビン［α２β２］は、２分子の二量体ヘモグロビン［αβ］へと解離して、それぞれの二量体ヘモグロビンがハプトグロビンと結合する。そして、Ｈｐ１－１型ハプトグロビンは、二量体ヘモグロビンとの結合部位を分子内に２か所有する。そのため、Ｈｐ１－１型ハプトグロビンと四量体ヘモグロビン［α２β２］とで複合体を形成すると、１分子のＨｐ１－１型ハプトグロビンに、１／２分子の二量体ヘモグロビン［αβ］が２つ結合した複合体となる。 (hemoglobin-haptoglobin complex)
When hemoglobin and haptoglobin form a complex, one molecule of tetrameric hemoglobin [α 2 β 2 ] dissociates into two molecules of dimeric hemoglobin [αβ], and each dimeric hemoglobin Combines with haptoglobin. Hp1-1 type haptoglobin has two binding sites with dimeric hemoglobin in its molecule. Therefore, when a complex is formed between Hp1-1 type haptoglobin and tetrameric hemoglobin [α 2 β 2 ], one molecule of Hp1-1 type haptoglobin contains two molecules of dimeric hemoglobin [αβ]. It becomes a complex with two bonds.
　四量体の遊離ヘモグロビン［α２β２］を抗原として得られた抗体は、ヘモグロビン－ハプトグロビン複合体に含まれる二量体ヘモグロビン［αβ］とも反応してしまう。そのため、ヘモグロビン-ハプトグロビン複合体と結合しない抗遊離ヘモグロビン抗体を、常法の動物免疫で取得することは、これまで非常に困難であった。
　これに対し、本実施形態においては、２種以上の抗ヘモグロビン抗体を選択して用いることで、ヘモグロビン-ハプトグロビン複合体と反応せず、遊離ヘモグロビンと反応することを見出したものである。 Antibodies obtained using tetrameric free hemoglobin [α 2 β 2 ] as an antigen also react with dimeric hemoglobin [αβ] contained in the hemoglobin-haptoglobin complex. Therefore, it has been extremely difficult to obtain anti-free hemoglobin antibodies that do not bind to hemoglobin-haptoglobin complexes through conventional animal immunization.
In contrast, in the present embodiment, it has been found that by selecting and using two or more types of anti-hemoglobin antibodies, the anti-hemoglobin antibodies do not react with the hemoglobin-haptoglobin complex, but react with free hemoglobin.
　ここで、後述する「ヘモグロビン－ハプトグロビン複合体に対する反応性」を評価する際に、抗原として用いるヘモグロビン－ハプトグロビン複合体は、ヘモグロビンとＨｐ１－１型ハプトグロビンとの複合体を意味する。ただし、かかる複合体に対する反応性が、遊離ヘモグロビンに対する反応性に比べて十分に低い場合には、その他のハプトグロビン（Ｈｐ２－１型，Ｈｐ２－２型）とヘモグロビンとの複合体に対する反応性も、遊離ヘモグロビンに対する反応性に比べて十分に低いものとなる。 Here, when evaluating the "reactivity to hemoglobin-haptoglobin complex" described below, the hemoglobin-haptoglobin complex used as an antigen means a complex of hemoglobin and Hp1-1 type haptoglobin. However, if the reactivity to such complexes is sufficiently lower than the reactivity to free hemoglobin, the reactivity to other complexes of haptoglobin (Hp2-1 type, Hp2-2 type) and hemoglobin will also be This is sufficiently lower than the reactivity to free hemoglobin.
〔遊離ヘモグロビン測定方法：第一の実施形態〕
　本発明の一実施形態に係る、遊離ヘモグロビン測定方法は、ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせを含む２種以上の抗ヘモグロビン抗体を用い、遊離ヘモグロビンを測定するため抗原抗体反応を行うものである。
　なお、以下にここで述べる実施形態を、遊離ヘモグロビン測定方法の第一の実施形態ということがある。 [Free hemoglobin measurement method: first embodiment]
A method for measuring free hemoglobin according to an embodiment of the present invention uses two or more types of anti-hemoglobin antibodies, including a combination of an antibody specific to hemoglobin α chain and an antibody specific to hemoglobin β chain, to measure free hemoglobin. The antigen-antibody reaction is carried out to measure the
Note that the embodiment described here below may be referred to as the first embodiment of the free hemoglobin measurement method.
（免疫学的手法）
　本実施形態の遊離ヘモグロビン測定方法は、抗原抗体反応を利用する方法、すなわち免疫学的手法であれば特に限定されず、例えば、免疫凝集法（例えば、ラテックス凝集法、金コロイド凝集法等）、ＥＬＩＳＡ法、イムノクロマトグラフ法等が挙げられる。本実施形態の測定方法は、これらの中でも免疫凝集法、より好ましくはラテックス凝集法に好適に用いられる。 (immunological method)
The free hemoglobin measuring method of the present embodiment is not particularly limited as long as it is a method that utilizes an antigen-antibody reaction, that is, an immunological method, and includes, for example, an immunoagglutination method (for example, a latex agglutination method, a colloidal gold agglutination method, etc.), Examples include ELISA method and immunochromatography method. Among these, the measurement method of the present embodiment is suitably used for immunoagglutination methods, more preferably for latex agglutination methods.
（抗体）
　本実施形態で用いる抗ヘモグロビン抗体は、ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせを含む２種以上の抗ヘモグロビン抗体である。
　ここで、「ヘモグロビンα鎖に特異的な抗体」とは、ヘモグロビンα鎖に対する反応が、β鎖に対する反応より十分に高い、抗ヘモグロビン抗体を意味する。なお、本明細書において、抗ヘモグロビン抗体が有するこのような性質を、抗ヘモグロビン抗体の特異性ということがある。 (antibody)
The anti-hemoglobin antibodies used in this embodiment are two or more types of anti-hemoglobin antibodies including a combination of an antibody specific to hemoglobin α chain and an antibody specific to hemoglobin β chain.
Here, the term "antibody specific for hemoglobin α chain" means an anti-hemoglobin antibody whose reaction against hemoglobin α chain is sufficiently higher than that against β chain. In addition, in this specification, such a property that an anti-hemoglobin antibody has is sometimes referred to as the specificity of the anti-hemoglobin antibody.
　抗ヘモグロビン抗体の特異性は、例えば、ヘモグロビンα鎖に対する反応とβ鎖に対する反応との合計に対し、ヘモグロビンα鎖に対する反応が７５％以上、さらには８０％以上である性質とすることができる。「ヘモグロビンβ鎖に特異的な抗体」も同様である。
　ヘモグロビンα鎖に対する特異性は、後述する実施例にて示すように：ヘモグロビンに対し、評価しようとする抗ヘモグロビン抗体を一次抗体として用いてウエスタンブロットを実施し；メンブレン上のα鎖が存在する領域のバンド輝度と、β鎖が存在する領域のバンド輝度とをそれぞれ積算し；両積算値の合計量に対するα鎖積算値の割合を算出する；ことで評価することができる。ヘモグロビンβ鎖に対する特異性も同様である。 The specificity of the anti-hemoglobin antibody can be such that, for example, the reaction against hemoglobin α chain is 75% or more, or even 80% or more, of the total reaction against hemoglobin α chain and β chain. The same applies to "antibodies specific to hemoglobin β chain."
The specificity for the hemoglobin α chain is determined as shown in the example below: Western blotting is performed using the anti-hemoglobin antibody to be evaluated as the primary antibody for hemoglobin; the region on the membrane where the α chain is present. The evaluation can be made by integrating the band brightness of the region where the β chain is present and the band brightness of the region where the β chain is present; and calculating the ratio of the α chain integrated value to the total amount of both integrated values. The specificity for hemoglobin β chain is also similar.
　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせにより、遊離ヘモグロビンを特異的に測定できる作用機序について、本発明者らは以下のように考えている。
　ヘモグロビン－ハプトグロビン複合体のヘモグロビン部分、すなわち、ハプトグロビンと複合体を形成した、αβの二量体構造のヘモグロビンに対しては、立体障害等により、α鎖特異的抗体とβ鎖特異的抗体とが同時に結合できないものと考えられる。
　これに対し、遊離ヘモグロビンは、α２β２の四量体構造のヘモグロビンであり、かつハプトグロビンと複合体を形成していないため、α鎖特異的抗体とβ鎖特異的抗体とが同時に結合できるものと考えられる。
　ただし、α鎖特異的抗体とβ鎖特異的抗体との組み合わせにより遊離ヘモグロビンを特異的に測定できる作用機序は、必ずしも上記の作用機序に限定されない。 The present inventors believe that the mechanism of action by which free hemoglobin can be specifically measured by a combination of an antibody specific to the hemoglobin α chain and an antibody specific to the hemoglobin β chain is as follows.
For the hemoglobin part of the hemoglobin-haptoglobin complex, that is, for the αβ dimeric hemoglobin that has formed a complex with haptoglobin, α chain-specific antibodies and β chain-specific antibodies cannot be used due to steric hindrance. It is considered that they cannot be combined at the same time.
On the other hand, free hemoglobin is hemoglobin with a tetrameric structure of α 2 β 2 and does not form a complex with haptoglobin, so that α chain-specific antibodies and β chain-specific antibodies can bind at the same time. considered to be a thing.
However, the mechanism of action by which free hemoglobin can be specifically measured by the combination of an α-chain-specific antibody and a β-chain-specific antibody is not necessarily limited to the above-mentioned mechanism of action.
　本実施形態で使用し得る抗ヘモグロビン抗体の種類は、前述した特異性を満たすものであれば特に限定されない。例えば、抗体が由来する動物種は特に限定されず、例えば、ウサギ、ヤギ、マウス、ラット、ウマ、ヒツジ等の動物に由来する抗体が挙げられ、公知の方法により、測定対象物を免疫した動物の血清から得られるポリクローナル、測定対象物を免疫した動物の脾臓をミエローマ細胞と細胞融合して得られるモノクローナル抗体のいずれを用いてもよい。また、それらの断片［例えば、Ｆ（ａｂ’）２、Ｆａｂ、Ｆａｂ’、またはＦｖ］を用いてもよい。 The type of anti-hemoglobin antibody that can be used in this embodiment is not particularly limited as long as it satisfies the specificity described above. 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, and animals that have been immunized with the measurement target by a known method. Either a polyclonal antibody obtained from the serum of an animal to be measured or a monoclonal antibody obtained by cell fusion of the spleen of an animal immunized with the object to be measured with myeloma cells may be used. Further, fragments thereof [eg, F(ab')2, Fab, Fab', or Fv] may be used.
（不溶性担体）
　本実施形態で用いる上記２種以上の抗ヘモグロビン抗体は、それらの少なくとも１種が不溶性担体に担持されていてもよく、２種以上が不溶性担体に担持されていてもよい。
　かかる不溶性担体は、抗体を担持できるものであれば特に限定されず、前述した免疫学的手法の種類に応じて適宜選択することができる。例えば、不溶性担体としては、例えば、免疫学的手法に使用できる不溶性粒子が挙げられ、不溶性粒子としては、一般的に使用される金コロイド粒子等の金属コロイド粒子、ラテックス粒子、シリカ粒子、磁性粒子、蛍光粒子、赤血球等が挙げられる。不溶性粒子としては、ラテックス粒子が好ましく、ポリスチレン系ラテックス粒子がさらに好ましい。不溶性担体は、好ましくは粒子状であり、その平均粒子径としては５～１,０００nmが好ましく、３０～５００nmがより好ましく、７５～３５０nmがさらに好ましいが、特にこの範囲に捉われることなく、使用することが可能である。 (Insoluble carrier)
At least one of the two or more anti-hemoglobin antibodies used in this embodiment may be supported on an insoluble carrier, or two or more of them may be supported on an insoluble carrier.
Such an insoluble carrier is not particularly limited as long as it can support antibodies, and can be appropriately selected depending on the type of immunological technique described above. For example, examples of insoluble carriers include insoluble particles that can be used in immunological techniques, and examples of insoluble particles include commonly used metal colloid particles such as colloidal gold particles, latex particles, silica particles, and magnetic particles. , fluorescent particles, red blood cells, etc. As the insoluble particles, latex particles are preferred, and polystyrene latex particles are more preferred. The insoluble carrier is preferably in the form of particles, and its average particle diameter is preferably 5 to 1,000 nm, more preferably 30 to 500 nm, and even more preferably 75 to 350 nm, but it can be used without being particularly limited to this range. It is possible to do so.
　抗体を担持しているとは、抗体が不溶性担体の表面に物理吸着または化学結合していることによって固定化されることをいう。担持方法（固定化方法）としては、例えば、公知の技術である、抗体と不溶性担体粒子とを混合して、不溶性担体粒子の表面に抗体を物理的に吸着させることにより不溶性担体粒子への抗体の固定化が可能である。また、表面にアミノ基やカルボキシル基を導入した不溶性担体粒子を用いる場合には、グルタルアルデヒドやカルボキシイミド試薬を用いた化学結合によって不溶性担体粒子の表面への抗体の固定化が可能である。
　抗体の担持量は、特に限定されないが、０．５～２,０００μg/mgラテックスであればよく、１～１,０００μg/mgラテックス、または２～５００μg/mgラテックスであってもよい。抗体の担持量は、不溶性担体に固定化する前の抗体量から固定化した後の抗体量を引いた量で算出することができる。 Carrying an antibody means that the antibody is immobilized on the surface of an insoluble carrier by physical adsorption or chemical bonding. The supporting method (immobilization method) includes, for example, a known technique in which the antibody and insoluble carrier particles are mixed and the antibody is physically adsorbed onto the surface of the insoluble carrier particles. can be immobilized. Furthermore, when insoluble carrier particles having amino groups or carboxyl groups introduced onto their surfaces are used, antibodies can be immobilized on the surface of the insoluble carrier particles by chemical bonding using glutaraldehyde or carboximide reagents.
The amount of antibody supported is not particularly limited, but may be 0.5 to 2,000 μg/mg latex, 1 to 1,000 μg/mg latex, or 2 to 500 μg/mg latex. The amount of antibody supported can be calculated by subtracting the amount of antibody after immobilization from the amount of antibody before immobilization on the insoluble carrier.
　また、不溶性担体に２種以上の抗ヘモグロビン抗体が担持されている場合、ともに同じ不溶性担体に担持されてもよい。また、１種の不溶性担体に１種の抗ヘモグロビン抗体を担持させた不溶性担体を複数種混合して用いてもよい。この場合において、異なる種の抗ヘモグロビン抗体を担持するために用いられる不溶性担体は、同種の不溶性担体であってもよく、材質または粒径等が異なる異種の不溶性担体であってもよい。 Furthermore, when two or more types of anti-hemoglobin antibodies are supported on an insoluble carrier, both may be supported on the same insoluble carrier. Alternatively, a mixture of a plurality of insoluble carriers in which one type of anti-hemoglobin antibody is supported on one type of insoluble carrier may be used. In this case, the insoluble carriers used to support different types of anti-hemoglobin antibodies may be the same type of insoluble carriers, or may be different types of insoluble carriers with different materials, particle sizes, etc.
（反応性）
　本実施形態においては、ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下となる条件で、遊離ヘモグロビンを測定するため抗原抗体反応を行うことが好ましい。
　「遊離ヘモグロビンに対する反応性」とは、抗原として遊離ヘモグロビンを単独で添加した場合の反応性をいう。「反応性」は、上記免疫学的手法において抗原を定量するための指標により評価される。例えば、免疫凝集法であれば、所定時間における濁度変化量、ＥＬＩＳＡ法であれば標識化抗体の標識に起因して生じる発色・吸光量等が挙げられる。遊離ヘモグロビンに対する反応性は、例えば、抗原抗体反応の反応液において遊離ヘモグロビンが２．７pmol/mL（１μg/mL）、２７pmol/mL（１０μg/mL）、２７０pmol/mL（１００μg/mL）となる条件にて評価することができる。 (reactivity)
In this embodiment, it is preferable to perform an antigen-antibody reaction to measure free hemoglobin under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
"Reactivity to free hemoglobin" refers to reactivity when free hemoglobin is added alone as an antigen. "Reactivity" is evaluated by an index for quantifying the antigen in the above-mentioned immunological method. For example, in the case of an immunoagglutination method, the amount of change in turbidity over a predetermined time, and in the case of an ELISA method, the amount of color development/light absorption caused by the labeling of a labeled antibody, etc. can be mentioned. Reactivity to free hemoglobin is determined, for example, by conditions under which free hemoglobin is 2.7 pmol/mL (1 μg/mL), 27 pmol/mL (10 μg/mL), or 270 pmol/mL (100 μg/mL) in the antigen-antibody reaction reaction solution. It can be evaluated by
　また、「ヘモグロビン－ハプトグロビン複合体に対する反応性」は、抗原としてヘモグロビン－ハプトグロビン複合体を添加した場合の反応性をいう。ここで、ヘモグロビン－ハプトグロビン複合体を添加するとは、あらかじめヘモグロビン－ハプトグロビン複合体を形成させたものを添加する態様のほか、遊離ヘモグロビンと、それと等モルのハプトグロビンとをそれぞれ添加し、混合液中でヘモグロビン－ハプトグロビン複合体を形成させる態様を包含する。 Furthermore, "reactivity to hemoglobin-haptoglobin complex" refers to reactivity when a hemoglobin-haptoglobin complex is added as an antigen. Here, adding a hemoglobin-haptoglobin complex means not only adding a hemoglobin-haptoglobin complex formed in advance, but also adding free hemoglobin and haptoglobin in an equimolar amount to each other in a mixed solution. Includes embodiments in which a hemoglobin-haptoglobin complex is formed.
　ヘモグロビン－ハプトグロビン複合体に対する反応性は、抗原を等モルのヘモグロビン－ハプトグロビン複合体に変更する以外は、前述した遊離ヘモグロビンに対する反応性と同一の反応条件にて評価される。例えば、遊離ヘモグロビンに対する反応性を遊離ヘモグロビンが２．７pmol/mLとなる条件にて評価する場合には、ヘモグロビン－ハプトグロビン複合体に対する反応性は、当該複合体が２．７pmol/mLとなる条件にて評価される。
　ここで、同一の反応条件であるとは、抗原抗体反応に用いる抗ヘモグロビン抗体、当該抗体の濃度、抗原抗体反応の反応液の組成（ｐＨ緩衝剤，塩，添加剤の種類・濃度）およびｐＨ、抗原抗体反応の反応時間および温度が同一であることをいう。なお、抗ヘモグロビン抗体が後述するように不溶性担体に担持されている場合は、同種の抗体担持担体を用いるものとする。 Reactivity to hemoglobin-haptoglobin complexes is evaluated under the same reaction conditions as the reactivity to free hemoglobin described above, except that the antigen is changed to equimolar hemoglobin-haptoglobin complexes. For example, when evaluating reactivity to free hemoglobin under conditions where free hemoglobin is 2.7 pmol/mL, reactivity to a hemoglobin-haptoglobin complex is evaluated under conditions where the complex is 2.7 pmol/mL. will be evaluated.
Here, the same reaction conditions refer to the anti-hemoglobin antibody used for the antigen-antibody reaction, the concentration of the antibody, the composition of the reaction solution for the antigen-antibody reaction (types and concentrations of pH buffer, salt, and additives), and the pH. , meaning that the reaction time and temperature of the antigen-antibody reaction are the same. In addition, when the anti-hemoglobin antibody is supported on an insoluble carrier as described below, the same type of antibody-supported carrier shall be used.
　「遊離ヘモグロビンを測定するため抗原抗体反応」は、遊離ヘモグロビンを測定しようとする測定対象（例えば、検体等）を添加して行われる抗原抗体反応であり、前述した反応性を評価するための抗原抗体反応とは区別される。上記測定対象には、遊離ヘモグロビンの他に、ヘモグロビン－ハプトグロビン複合体が存在する場合が多い。換言すると、遊離ヘモグロビンを測定するための抗原抗体反応は、ヘモグロビン－ハプトグロビン複合体の存在し得る環境下で行われることが多い。 "Antigen-antibody reaction for measuring free hemoglobin" is an antigen-antibody reaction performed by adding a measurement target (e.g., a specimen) to which free hemoglobin is to be measured. Distinguished from antibody reactions. In addition to free hemoglobin, a hemoglobin-haptoglobin complex is often present in the above measurement target. In other words, antigen-antibody reactions for measuring free hemoglobin are often performed in an environment where hemoglobin-haptoglobin complexes can exist.
　本実施形態において、遊離ヘモグロビンを測定するため抗原抗体反応は、ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下となる条件で行われることが好ましい。かかる反応性の比率は、１０％以下であってよく、さらには５％以下であってもよい。
　遊離ヘモグロビンを測定するため抗原抗体反応が当該条件で行われるとは、前述した「遊離ヘモグロビンに対する反応性」および「ヘモグロビン－ハプトグロビン複合体に対する反応性」を評価したのと同一の反応条件によって行われることを意味する。同一の反応条件の定義は前述したとおりである。 In this embodiment, in order to measure free hemoglobin, the antigen-antibody reaction is preferably performed under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin. Such a reactivity ratio may be 10% or less, or even 5% or less.
The antigen-antibody reaction is carried out under the conditions for measuring free hemoglobin, which means that it is carried out under the same reaction conditions as those used to evaluate the above-mentioned "reactivity to free hemoglobin" and "reactivity to hemoglobin-haptoglobin complex." It means that. The definition of the same reaction conditions is as described above.
（測定）
　本実施形態においては、遊離ヘモグロビンを測定するための抗原抗体反応を行った後、公知の方法により測定される。測定する対象は、採用した免疫学的手法に応じて適宜設定することができ、例えば、免疫凝集法であれば、所定時間における濁度変化量、ＥＬＩＳＡ法であれば標識化抗体の標識に起因して生じる発色・吸光量等が挙げられる。そのため、これらを測定する方法も、公知の方法、例えば光学的手法を採用することができ、汎用の光学的測定装置を用いることができる。 (measurement)
In this embodiment, free hemoglobin is measured by a known method after performing an antigen-antibody reaction. The target to be measured can be set as appropriate depending on the immunological method employed. For example, in the case of immunoagglutination, the amount of turbidity change over a given time, and in the case of ELISA, the amount of change in turbidity due to the labeling of the labeled antibody. Examples include the color development and amount of light absorption that occur. Therefore, as a method for measuring these, a known method such as an optical method can be adopted, and a general-purpose optical measuring device can be used.
〔遊離ヘモグロビン測定方法：第二の実施形態〕
　本発明の遊離ヘモグロビン測定方法は、第二の実施形態として：２種以上の抗ヘモグロビン抗体を用い；ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下となる条件で、遊離ヘモグロビンを測定するため抗原抗体反応を行う；ものとすることができる。 [Free hemoglobin measurement method: second embodiment]
The method for measuring free hemoglobin of the present invention, as a second embodiment, uses two or more types of anti-hemoglobin antibodies; Antigen-antibody reactions can be performed to measure hemoglobin;
　「遊離ヘモグロビンに対する反応性」、「ヘモグロビン－ハプトグロビン複合体に対する反応性」の定義は、第一の実施形態で説明したとおりである。 The definitions of "reactivity to free hemoglobin" and "reactivity to hemoglobin-haptoglobin complex" are as explained in the first embodiment.
　本実施形態で用いる２種以上の抗ヘモグロビン抗体は、上記反応性を満たす組み合わせであれば、特に限定されない。例えば、ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせ；ヘモグロビンα鎖とβ鎖との境界近傍に特異的な抗体の組み合わせ；等を用いることができる。
　これらの中でも、ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせが特に好ましい。 The two or more types of anti-hemoglobin antibodies used in this embodiment are not particularly limited as long as the combination satisfies the above reactivity. For example, a combination of an antibody specific to hemoglobin α chain and an antibody specific to hemoglobin β chain; a combination of antibodies specific to the vicinity of the boundary between hemoglobin α chain and β chain; and the like can be used.
Among these, a combination of an antibody specific for hemoglobin α chain and an antibody specific for hemoglobin β chain is particularly preferred.
　本実施形態においては、２種以上の抗ヘモグロビン抗体を用いるため、上記抗原抗体反応は、同一の抗原（本実施形態においては、ヘモグロビン）中の２以上の異なるエピトープに対する抗原抗体反応であることが好ましい。換言すると、本実施形態の免疫学的手法は、当該２以上の抗原抗体反応を利用する方法であることが好ましい。 In this embodiment, two or more types of anti-hemoglobin antibodies are used, so the antigen-antibody reaction described above is an antigen-antibody reaction against two or more different epitopes in the same antigen (hemoglobin in this embodiment). preferable. In other words, the immunological method of this embodiment is preferably a method that utilizes the two or more antigen-antibody reactions.
　本実施形態に係るその他の構成（不溶性担体等）は、第一の実施形態と同様である。 Other configurations (insoluble carrier, etc.) according to this embodiment are the same as those of the first embodiment.
　上記実施形態に係る測定方法は、遊離ヘモグロビンを測定するため抗原抗体反応を：
　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせを含む２種以上の抗ヘモグロビン抗体を用いて行う；または、
　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下となる条件で行う。
　これにより、例えばヘモグロビン－ハプトグロビン複合体の存在し得る環境下で行われる場合であっても、ヘモグロビン－ハプトグロビン複合体の量に左右されず、遊離ヘモグロビンの量を測定することができる。
　本実施形態の測定方法によれば、遊離ヘモグロビンを測定するため抗原抗体反応の前に、あらかじめヘモグロビン－ハプトグロビン複合体を除去する等の工程を必要としない。そのため、操作が簡便でありながら、遊離ヘモグロビンを特異的に測定することができる。 The measurement method according to the above embodiment uses an antigen-antibody reaction to measure free hemoglobin:
carried out using two or more anti-hemoglobin antibodies, including a combination of an antibody specific for hemoglobin α chain and an antibody specific for hemoglobin β chain; or
The test is carried out under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
Thereby, even when the measurement is carried out in an environment where hemoglobin-haptoglobin complexes may exist, the amount of free hemoglobin can be measured regardless of the amount of hemoglobin-haptoglobin complexes.
According to the measurement method of this embodiment, in order to measure free hemoglobin, there is no need for a step such as removing the hemoglobin-haptoglobin complex before the antigen-antibody reaction. Therefore, free hemoglobin can be specifically measured while the operation is simple.
〔遊離ヘモグロビン測定試薬：第一の実施形態〕
　本発明の一実施形態に係る遊離ヘモグロビン測定試薬は、ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせを含む２種以上の抗ヘモグロビン抗体を含むものである。
　なお、以下にここで述べる実施形態を、遊離ヘモグロビン測定試薬の第一の実施形態ということがある。 [Free hemoglobin measurement reagent: first embodiment]
A reagent for measuring free hemoglobin according to an embodiment of the present invention contains two or more types of anti-hemoglobin antibodies, including a combination of an antibody specific to hemoglobin α chain and an antibody specific to hemoglobin β chain.
Note that the embodiment described here below may be referred to as the first embodiment of the free hemoglobin measurement reagent.
　本実施形態の試薬で用いる抗ヘモグロビン抗体は、前述した遊離ヘモグロビン測定方法の第一の実施形態において説明した抗体を用いることができる。 As the anti-hemoglobin antibody used in the reagent of this embodiment, the antibody described in the first embodiment of the method for measuring free hemoglobin mentioned above can be used.
　本実施形態の試薬で用いる上記２種以上の抗ヘモグロビン抗体は、それらの少なくとも１種が不溶性担体に担持されていてもよく、２種以上が不溶性担体に担持されていてもよい。かかる不溶性担体は、不溶性粒子であってよい。不溶性担体や不溶性粒子の種類、抗体の担持方法等については、前述した測定方法で説明したとおりである。 At least one of the two or more anti-hemoglobin antibodies used in the reagent of this embodiment may be supported on an insoluble carrier, or two or more of them may be supported on an insoluble carrier. Such insoluble carriers may be insoluble particles. The types of insoluble carriers and insoluble particles, the method of supporting antibodies, etc. are as explained in the measurement method described above.
　本実施形態の試薬は、ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下であることが好ましい。「遊離ヘモグロビンに対する反応性」、「ヘモグロビン－ハプトグロビン複合体に対する反応性」は、前述した測定方法で説明したとおりである。 It is preferable that the reactivity of the reagent of this embodiment with respect to the hemoglobin-haptoglobin complex is 15% or less of the reactivity with respect to free hemoglobin. "Reactivity to free hemoglobin" and "reactivity to hemoglobin-haptoglobin complex" are as explained in the measurement method described above.
　本実施形態に係る測定試薬は、後述する試薬の構成（反応液の組成等）のほか、通常、取扱説明書等の添付文書を備えており、遊離ヘモグロビンを測定するための反応条件が、試薬の構成および添付文書により特定される。より具体的には、測定試薬の添付文書には、２種以上の抗ヘモグロビン抗体（これらが不溶性担体に担持されている態様を含む）の添加量、反応液の合計液量、添加手順、反応時間および反応温度などが特定されており、これらに沿って本実施形態の測定試薬を使用することにより、遊離ヘモグロビンを測定するための反応条件が特定されることとなる。
　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下である試薬、とは、当該試薬について特定された反応条件において、「遊離ヘモグロビンに対する反応性」と「ヘモグロビン－ハプトグロビン複合体に対する反応性」とをそれぞれ評価して対比したときに、１５％以下であればよい。ここで述べた反応性は、１０％以下であってよく、さらには５％以下であってもよい。また、「遊離ヘモグロビンに対する反応性」は、所定濃度の遊離ヘモグロビン（例えば、２．７pmol/mL、２７pmol/mL、２７０pmol/mL）に対する反応性で評価することができ、同様に「ヘモグロビン－ハプトグロビン複合体に対する反応性」は遊離ヘモグロビンの所定濃度と等モルのヘモグロビン－ハプトグロビン複合体に対する反応性で評価することができる。 In addition to the reagent composition (composition of the reaction solution, etc.) described later, the measurement reagent according to this embodiment usually includes an attached document such as an instruction manual, and the reaction conditions for measuring free hemoglobin are specified in the reagent. identified by its structure and attached documents. More specifically, the package insert of the measurement reagent includes the amount of two or more anti-hemoglobin antibodies (including embodiments in which these are supported on an insoluble carrier), the total volume of the reaction solution, the addition procedure, and the reaction. The time, reaction temperature, etc. are specified, and by using the measurement reagent of this embodiment in accordance with these, the reaction conditions for measuring free hemoglobin will be specified.
A reagent whose reactivity to hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin is defined as "reactivity to free hemoglobin" and "reactivity to hemoglobin-haptoglobin complex" under the reaction conditions specified for the reagent. It is sufficient if it is 15% or less when evaluating and comparing the "reactivity" respectively. The reactivity mentioned here may be 10% or less, or even 5% or less. In addition, "reactivity to free hemoglobin" can be evaluated by reactivity to a predetermined concentration of free hemoglobin (for example, 2.7 pmol/mL, 27 pmol/mL, 270 pmol/mL), and similarly "reactivity to hemoglobin-haptoglobin complex" The "reactivity to the body" can be evaluated by the reactivity to a hemoglobin-haptoglobin complex that is equimolar to a predetermined concentration of free hemoglobin.
（測定試薬の形態等）
　本実施形態の測定試薬は、前述した構成を備え、免疫学的手法を利用して遊離ヘモグロビンを測定する試薬である。これらの要件を満たすものであれば、測定試薬の形態は特に限定されず、例えば、免疫凝集法（例えば、ラテックス凝集法、金コロイド凝集法等）、ＥＬＩＳＡ法、イムノクロマトグラフ法等を利用した試薬とすることができる。これらの中でも、免疫凝集法の試薬であることが好ましく、ラテックス凝集法の試薬であることがより好ましい。 (Form of measurement reagent, etc.)
The measurement reagent of this embodiment has the above-described configuration and is a reagent for measuring free hemoglobin using an immunological technique. The form of the measurement reagent is not particularly limited as long as it satisfies these requirements; for example, reagents using immunoagglutination methods (e.g., latex agglutination method, colloidal gold agglutination method, etc.), ELISA method, immunochromatography method, etc. It can be done. Among these, reagents for immunoagglutination are preferred, and reagents for latex agglutination are more preferred.
　本実施形態の測定試薬は、例えば、不溶性担体を含有しない試薬（第１試薬）および抗体を担持する不溶性担体（抗体担持不溶性担体）を含有する試薬（第２試薬）の二試薬系で構成されてもよく、抗体担持不溶性担体を含有する試薬のみの一試薬系で構成されるようにしてもよい。
　ここで、第１試薬は、反応系における測定対象物や夾雑物の濃度の調整や反応速度の調整等の点から希釈液として用いるなど、測定環境を調整するよう用いることができる。第２試薬は、抗体担持不溶性担体を含有しており、第１試薬および試料と混合されて、免疫凝集反応を生じる。第１試薬および第２試薬は、ｐＨ緩衝剤、塩、界面活性剤、凝集促進剤、防腐剤などを、適宜含むことができる。凝集反応時のｐＨとしては５～９が好ましい。 The measurement reagent of this embodiment is composed of a two-reagent system, for example, a reagent that does not contain an insoluble carrier (first reagent) and a reagent that contains an insoluble carrier that supports an antibody (antibody-supported insoluble carrier) (second reagent). Alternatively, the reagent system may consist of only one reagent containing an antibody-supported insoluble carrier.
Here, the first reagent can be used to adjust the measurement environment, such as by being used as a diluent for adjusting the concentration of the object to be measured or impurities in the reaction system, adjusting the reaction rate, etc. The second reagent contains an antibody-supported insoluble carrier and is mixed with the first reagent and sample to produce an immunoagglutination reaction. The first reagent and the second reagent can appropriately contain a pH buffer, a salt, a surfactant, an aggregation promoter, a preservative, and the like. The pH during the aggregation reaction is preferably 5 to 9.
　また、測定試薬は試料と混合して反応液を得るが、不溶性担体の反応液中の濃度は、使用する不溶性担体の粒径や測定系全体の設計にあわせ、例えば０．０００１mg/mL～１０mg/mLの範囲から適宜選択をすることができる。測定試薬における抗ヘモグロビン抗体を担持する不溶性担体の濃度は、０．０１～５mg/mL、または０．０５～１mg/mLであってよい。なお、不溶性担体の第二試薬中の濃度は、使用時には第一試薬や試料等と混合することにより希釈されるため、その第二試薬中の不溶性担体の濃度は、希釈倍率に応じて適宜選択することができ、例えば、２倍希釈して用いる場合は、０．０００２mg/mL～２０mg/mL、３倍希釈して用いる場合は、０．０００３mg/mL～３０mg/mLとなるように適宜調整することができる。 In addition, the measurement reagent is mixed with the sample to obtain a reaction solution, and the concentration of the insoluble carrier in the reaction solution is determined depending on the particle size of the insoluble carrier used and the overall design of the measurement system, for example, 0.0001 mg/mL to 10 mg/mL. An appropriate selection can be made from the range of /mL. The concentration of the insoluble carrier carrying the anti-hemoglobin antibody in the measurement reagent may be 0.01 to 5 mg/mL, or 0.05 to 1 mg/mL. Note that the concentration of the insoluble carrier in the second reagent is diluted by mixing with the first reagent, sample, etc. during use, so the concentration of the insoluble carrier in the second reagent should be selected appropriately depending on the dilution ratio. For example, when diluting 2 times and using it, it is 0.0002 mg/mL to 20 mg/mL, and when diluting 3 times and using it, it can be adjusted appropriately so that it is 0.0003 mg/mL to 30 mg/mL. can do.
〔遊離ヘモグロビン測定試薬：第二の実施形態〕
　本発明の遊離ヘモグロビン測定試薬は、第二の実施形態として：２種以上の抗ヘモグロビン抗体を含み；ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下である；ものとしてもよい。 [Free hemoglobin measurement reagent: second embodiment]
As a second embodiment, the free hemoglobin measurement reagent of the present invention: contains two or more types of anti-hemoglobin antibodies; has reactivity with hemoglobin-haptoglobin complexes that is 15% or less of the reactivity with free hemoglobin; good.
　本実施形態の測定試薬で用いる抗体は、前述した遊離ヘモグロビン測定方法における第二の実施形態において説明したとおりである。
　また、「遊離ヘモグロビンに対する反応性」、「ヘモグロビン－ハプトグロビン複合体に対する反応性」は、遊離ヘモグロビン測定試薬に係る第一の実施形態で説明したとおりである。本実施形態の測定試薬におけるその他の構成（不溶性担体，試薬の形態等）も、遊離ヘモグロビン測定試薬に係る第一の実施形態で説明したとおりである。 The antibody used in the measurement reagent of this embodiment is as described in the second embodiment of the method for measuring free hemoglobin described above.
Furthermore, "reactivity to free hemoglobin" and "reactivity to hemoglobin-haptoglobin complex" are as described in the first embodiment regarding the reagent for measuring free hemoglobin. Other configurations of the measurement reagent of this embodiment (insoluble carrier, form of reagent, etc.) are also as explained in the first embodiment related to the free hemoglobin measurement reagent.
　以上述べた遊離ヘモグロビン測定試薬は：
　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせを含む２種以上の抗ヘモグロビン抗体を含む；または、
　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下である。
　これにより、ヘモグロビン－ハプトグロビン複合体の量に左右されず、遊離ヘモグロビンの量を測定することができる。そのため、遊離ヘモグロビンを測定するための抗原抗体反応が、ヘモグロビン－ハプトグロビン複合体の存在し得る環境下で行われる場合であっても、遊離ヘモグロビンを特異的に測定することができる。これにより、遊離ヘモグロビンを測定するため抗原抗体反応の前に、あらかじめヘモグロビン－ハプトグロビン複合体を除去する等の工程を必要とせず、操作が簡便でありながら、遊離ヘモグロビンを特異的に測定することができる。 The free hemoglobin measuring reagents mentioned above are:
comprising two or more anti-hemoglobin antibodies, including a combination of an antibody specific for hemoglobin alpha chain and an antibody specific for hemoglobin beta chain; or
The reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
This allows the amount of free hemoglobin to be measured regardless of the amount of hemoglobin-haptoglobin complex. Therefore, even when the antigen-antibody reaction for measuring free hemoglobin is performed in an environment where a hemoglobin-haptoglobin complex may exist, free hemoglobin can be specifically measured. This eliminates the need for steps such as removing the hemoglobin-haptoglobin complex before the antigen-antibody reaction to measure free hemoglobin, making it possible to specifically measure free hemoglobin while simplifying the operation. can.
〔そのほかの実施形態〕
　本発明は、ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせをさらに提供する。
　「ヘモグロビンα鎖に特異的に反応する」、「ヘモグロビンβ鎖に特異的に反応する」の定義は前述したとおりである。
　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせは、遊離ヘモグロビンを特異的に測定するために特に好適である。 [Other embodiments]
The invention further provides a combination of an antibody specific for hemoglobin alpha chain and an antibody specific for hemoglobin beta chain.
The definitions of "reacts specifically with hemoglobin α chain" and "reacts specifically with hemoglobin β chain" are as described above.
A combination of an antibody specific for hemoglobin α chain and an antibody specific for hemoglobin β chain is particularly suitable for specifically measuring free hemoglobin.
　また、本発明は、上記の遊離ヘモグロビン測定試薬を含む、遊離ヘモグロビン測定キットをさらに提供する。
　当該キットに含まれる測定試薬は、例えば、抗ヘモグロビン抗体担持不溶性担体を含むものとすることができる。また、上記遊離ヘモグロビン測定キットは、測定試薬の他に、キャリブレータ、およびコントロールなどの構成を含んでもよく、また、検体を採取するための器具および容器、検体を保存するための保存溶液などの構成を含んでいてもよい。 Moreover, the present invention further provides a free hemoglobin measurement kit containing the above-mentioned free hemoglobin measurement reagent.
The measurement reagent included in the kit may include, for example, an anti-hemoglobin antibody-supported insoluble carrier. In addition to the measurement reagent, the free hemoglobin measurement kit may also include components such as a calibrator and a control, and also includes components such as an instrument and container for collecting a specimen, and a storage solution for preserving the specimen. May contain.
　以上説明した実施形態は、本発明の理解を容易にするために記載されたものであって、本発明を限定するために記載されたものではない。したがって、上記実施形態に開示された各要素は、本発明の技術的範囲に属する全ての設計変更や均等物をも含む趣旨である。 The embodiments described above are described to facilitate understanding of the present invention, and are not described to limit the present invention. Therefore, each element disclosed in the above embodiments is intended to include all design changes and equivalents that fall within the technical scope of the present invention.
　以下、製造例、試験例等を示すことにより本発明をさらに詳細に説明するが、本発明は下記の製造例、試験例等に何ら限定されるものではない。 Hereinafter, the present invention will be explained in more detail by showing production examples, test examples, etc., but the present invention is not limited to the following production examples, test examples, etc.
実施例１：抗ヒ卜ヘモグロビンモノクローナル抗体の作製
（１）マウスへの免疫
　マウスに対してヘモグロビンで免疫した。各々の免疫後に、マウスの抗体価は、１２５Ｉで標識したヘモグロビンを用いた２抗体法のＲＩＡ法で測定した。その結果、高い抗血清価が得られたマウスを選択した。
（２）細胞融合
　選択したマウスより脾臓を摘出し、脾細胞を調製した。調製した脾細胞とマウスミエローマ細胞とを電気融合法にて細胞融合を行い、融合細胞選択培地に懸濁して９６穴マイクロプレートに播種した。
（３）モノクローナル抗体産生細胞株のスクリーニング
　細胞融合の１０日後に、１２５Ｉで標識したヘモグロビンを用いた２抗体法のＲＩＡ法で、抗ヒトヘモグロビンモノクローナル抗体産生細胞をスクリーニングし、１０クローンを得た。 Example 1: Preparation of anti-human hemoglobin monoclonal antibody (1) Immunization of mice Mice were immunized with hemoglobin. After each immunization, the antibody titer of the mice was measured by the two-antibody RIA method using 125 I-labeled hemoglobin. As a result, mice with high antiserum titers were selected.
(2) Cell fusion The spleen was removed from the selected mouse, and splenocytes were prepared. The prepared splenocytes and mouse myeloma cells were fused by electrofusion method, suspended in a fused cell selection medium, and seeded in a 96-well microplate.
(3) Screening for monoclonal antibody-producing cell lines Ten days after cell fusion, anti-human hemoglobin monoclonal antibody-producing cells were screened by the two-antibody RIA method using 125 I-labeled hemoglobin, and 10 clones were obtained. .
実施例２：抗体の特異性確認
　ヒトＯ型血液より精製したヒトヘモグロビン（栄研化学社製）を、電気泳動用ポリアクリルアミドプレキャストゲル（ATTO社製）およびＡＥ－６５３０ラピダス・ミニスラブ電気泳動槽（ATTO社製）を用いて泳動した。泳動したタンパク質を、トランスブロットＳＤセル（Bio-Rad社製）を用いてＰＶＤＦ膜へ転写した。
　一次抗体として実施例１で得られた抗体（１μg/mL）、二次抗体としてＨＲＰで標識した抗マウスＩｇＧ抗体認識抗体（ウサギ由来ポリクローナル抗体，Cappel社製）を用いてウエスタンブロットを実施し、Western Lightning ECL Pro（PerkinElmer社製）を用いてＨＲＰを発光させ、バンド画像を取得した。バンド画像を、CS Analyzer ver.3.0（ATTO社製）を用いて解析し、計測モード：指定領域ゾーンデンシトメトリーにて、泳動レーンのバンド輝度を積算値として算出した（図１）。
　次に、ウエスタンブロットのバンド輝度よりα鎖、β鎖のバンド輝度積算値を求めた。なお、バンド輝度の補正のため、後述する方法により算出した係数０．７８３をα鎖のバンド積算値に乗じた。
　α鎖積算値＋β鎖積算値を１００％とした場合の各サブユニットのバンド輝度割合を求めた。この結果より、α鎖バンドの積算値割合が７５％以上を示した抗体をヘモグロビンα鎖特異的抗体、β鎖バンドの積算値割合が７５％以上を示した抗体をヘモグロビンβ鎖特異的抗体と判断した。結果を表１に示す。 Example 2: Confirmation of antibody specificity Human hemoglobin purified from human type O blood (manufactured by Eiken Kagaku Co., Ltd.) was transferred to a polyacrylamide precast gel for electrophoresis (manufactured by ATTO) and an AE-6530 Rapidus mini-slab electrophoresis tank (manufactured by ATTO). (manufactured by ATTO). The migrated proteins were transferred to a PVDF membrane using a Transblot SD cell (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 polyclonal antibody, manufactured by Cappel) as the secondary antibody. HRP was caused to emit light using Western Lightning ECL Pro (manufactured by PerkinElmer), and a band image was obtained. The band image was analyzed using CS Analyzer ver. 3.0 (manufactured by ATTO), and the band brightness of the electrophoresis lane was calculated as an integrated value in measurement mode: designated area zone densitometry (FIG. 1).
Next, the integrated value of the band brightness of the α chain and β chain was determined from the band brightness of the Western blot. In addition, in order to correct the band luminance, the band integrated value of the α chain was multiplied by a coefficient of 0.783 calculated by the method described later.
The band brightness ratio of each subunit was determined when α chain integrated value + β chain integrated value was set as 100%. From this result, antibodies that showed an integrated value ratio of α chain band of 75% or more were considered hemoglobin α chain-specific antibodies, and antibodies that showed an integrated value ratio of β chain bands of 75% or more were considered hemoglobin β chain-specific antibodies. It was judged. The results are shown in Table 1.
　なお、バンド輝度を補正するため、ヒトヘモグロビンを、電気泳動用ポリアクリルアミドプレキャストゲル（ATTO社製）およびＡＥ－６５３０ラピダス・ミニスラブ電気泳動槽（ATTO社製）を用いて上記と同様に泳動し、ＣＢＢ染色を行った。得られた泳動画像のα鎖、β鎖のバンド輝度の積算値を算出し、α鎖積算値＋β鎖積算値を１００％とした場合の各サブユニットのバンド輝度割合を求めた。この結果より、β鎖とα鎖のバンド輝度は４３．９％：５６．１％であり、バンド輝度のノーマライズのため、α鎖のバンド積算値に係数０．７８３をかけることとした（図２）。 In addition, in order to correct the band brightness, human hemoglobin was electrophoresed in the same manner as above using a polyacrylamide precast gel for electrophoresis (manufactured by ATTO) and an AE-6530 Rapidus mini-slab electrophoresis tank (manufactured by ATTO). CBB staining was performed. The integrated value of the band brightness of α chain and β chain in the obtained electrophoretic image was calculated, and the band brightness ratio of each subunit was determined when α chain integrated value + β chain integrated value was set as 100%. From this result, the band brightness of β chain and α chain is 43.9%:56.1%, and in order to normalize the band brightness, it was decided to multiply the band integrated value of α chain by a coefficient of 0.783 (Fig. 2).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１に示すように、実施例１で得られた抗体のうち、No.１、８、１２および１６は、α鎖積算値とβ鎖積算値との合計に対し、α鎖積算値の割合が７５％以上であったことから、α鎖特異的抗体であると認められた。
　これらに対し、No.４、１１、２１、２３、２４および３２は、α鎖積算値とβ鎖積算値との合計に対し、β鎖積算値の割合が７５％以上であったことから、β鎖特異的抗体であると認められた。 As shown in Table 1, among the antibodies obtained in Example 1, Nos. 1, 8, 12, and 16 have a ratio of α chain integrated value to the total α chain integrated value and β chain integrated value. was 75% or more, so it was recognized as an α chain-specific antibody.
On the other hand, in Nos. 4, 11, 21, 23, 24, and 32, the ratio of the β-chain integrated value to the total of the α-chain integrated value and the β-chain integrated value was 75% or more, so It was recognized that it is a β chain-specific antibody.
実施例３：モノクローナル抗体の反応性－１
　実施例１で得られた抗体を用い、遊離ヘモグロビンに対する反応性と、ヘモグロビン－ハプトグロビン複合体に対する反応性とを評価した。以下に述べる方法により、各々の抗ヒトヘモグロビンモノクローナル抗体をポリスチレンラテックス粒子に固定化し、ヘモグロビンまたはヘモグロビン－ハプトグロビン複合体を含む試料と反応させたときの凝集の度合いを比較した。 Example 3: Monoclonal antibody reactivity-1
Using the antibody obtained in Example 1, reactivity to free hemoglobin and reactivity to hemoglobin-haptoglobin complex was evaluated. By the method described below, each anti-human hemoglobin monoclonal antibody was immobilized on polystyrene latex particles, and the degree of aggregation when reacted with a sample containing hemoglobin or hemoglobin-haptoglobin complex was compared.
（１）モノクローナル抗体のポリスチレンラテックス粒子への固定化
　ポリスチレンラテックス粒子への抗体の固定化は、公知の技術を利用して実施した。各抗ヘモグロビンモノクローナル抗体とポリスチレンラテックス粒子（粒径２００nm）を混合してそれぞれ固定化し、ポリスチレンラテックス粒子表面に抗ヘモグロビンモノクローナル抗体を担持することにより、抗体担持ポリスチレンラテックス粒子溶液を調製した。 (1) Immobilization of monoclonal antibodies onto polystyrene latex particles Antibodies were immobilized onto polystyrene latex particles using a known technique. Each anti-hemoglobin monoclonal antibody and polystyrene latex particles (particle size 200 nm) were mixed and immobilized, and the anti-hemoglobin monoclonal antibody was supported on the surface of the polystyrene latex particles to prepare an antibody-supported polystyrene latex particle solution.
（２）試料の調製
　ヘモグロビン－ハプトグロビン複合体の試料は、５０ｍＭ ＨＥＰＥＳ緩衝液（ｐＨ７．４）に３２．３pmol/mLのヘモグロビンと、それと等モルの３２．３pmol/mLのハプトグロビンを等液量で混和して調製した。ヘモグロビンのみの試料は、５０ｍＭ ＨＥＰＥＳ緩衝液（ｐＨ７．４）に１６．１pmol/mLのヘモグロビンを含むものを使用した。 (2) Preparation of sample For a sample of hemoglobin-haptoglobin complex, 32.3 pmol/mL hemoglobin and the same molar amount of 32.3 pmol/mL haptoglobin were prepared in equal volumes in 50 mM HEPES buffer (pH 7.4). Prepared by mixing. The hemoglobin-only sample contained 16.1 pmol/mL hemoglobin in 50 mM HEPES buffer (pH 7.4).
（３）ラテックス凝集の測定方法
　９６穴平底マイクロプレートのウェルを用いて凝集反応を行なった。具体的な方法は、５０ｍＭ ＨＥＰＥＳ緩衝液（ｐＨ７．４）をマイクロプレートの各ウェルに１００μL分注し、各々の抗体を固定化したポリスチレンラテックス粒子溶液を５０μL添加した後に、（２）で調製した試料を３０μL添加した。吸光マイクロプレートリーダー（テカンジャパン社）を用いて試料の添加１０秒後および５分１０秒後に波長６６０nmで吸光度を測定し、その差を凝集の指標とした。さらに、ヘモグロビンに対する凝集反応性と、ヘモグロビン－ハプトグロビン複合体に対する凝集反応性との比を求めた。
　結果を表２に示す。 (3) Method for measuring latex aggregation Agglutination reaction was performed using wells of a 96-well flat bottom microplate. The specific method was to dispense 100 μL of 50 mM HEPES buffer (pH 7.4) into each well of a microplate, add 50 μL of each antibody-immobilized polystyrene latex particle solution, and then prepare in (2). 30 μL of sample was added. Using an absorption microplate reader (Tecan Japan), the absorbance was measured at a wavelength of 660 nm 10 seconds and 5 minutes and 10 seconds after the addition of the sample, and the difference was used as an index of aggregation. Furthermore, the ratio of the agglutination reactivity to hemoglobin and the agglutination reactivity to the hemoglobin-haptoglobin complex was determined.
The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２に示すように、用いた抗体の組み合わせにおいては、遊離ヘモグロビンに対し、特異的反応が認められた。ヘモグロビン－ハプトグロビン複合体に対する反応性は、遊離ヘモグロビンに対する反応性の１５％以下であった。 As shown in Table 2, specific reactions against free hemoglobin were observed in the combinations of antibodies used. Reactivity to hemoglobin-haptoglobin complex was less than 15% of the reactivity to free hemoglobin.
実施例４：モノクローナル抗体の反応性－２
　実施例１で得られた１０種の抗体の全ての組み合わせについて、遊離ヘモグロビンに対する反応性と、ヘモグロビン－ハプトグロビン複合体に対する反応性とを評価した。方法は、実施例３で用いたのと同様の方法で確認した。 Example 4: Monoclonal antibody reactivity-2
All combinations of the 10 types of antibodies obtained in Example 1 were evaluated for reactivity to free hemoglobin and reactivity to hemoglobin-haptoglobin complex. The method was confirmed using the same method as used in Example 3.
　以下の基準にて、反応性を評価した。
Ａ：ヘモグロビン－ハプトグロビン複合体と反応させたときの凝集の度合い（ΔＯＤ×10,000，以下同様）が、遊離ヘモグロビンと反応させたときの凝集の度合いの１５％以下だったもの
Ｂ：ヘモグロビン－ハプトグロビン複合体と反応させたときの凝集の度合いが、遊離ヘモグロビンと反応させたときの凝集の度合いの５０％以上だったもの（ただし、次の「－」を除く）
－：遊離ヘモグロビン、ヘモグロビン－ハプトグロビン複合体の両方で凝集の度合いが著しく低かった（ΔＯＤ×10,000がいずれも５００以下）もの Reactivity was evaluated based on the following criteria.
A: The degree of aggregation when reacted with hemoglobin-haptoglobin complex (ΔOD x 10,000, the same applies hereinafter) was 15% or less of the degree of aggregation when reacted with free hemoglobin B: Hemoglobin-haptoglobin complex Those whose degree of aggregation when reacting with the body was 50% or more of the degree of aggregation when reacting with free hemoglobin (excluding the following "-")
-: The degree of aggregation of both free hemoglobin and hemoglobin-haptoglobin complex was extremely low (ΔOD×10,000 was 500 or less for both)
　上記Ｂは、遊離ヘモグロビン、ヘモグロビン－ハプトグロビン複合体の両方の凝集の度合いが装置の測定限界を超えたもの（ΔＯＤ×10,000がいずれも３０００超）となった結果、上記比率になったものを含む。
　結果を表３に示す。なお、本試験においては、ヘモグロビン－ハプトグロビン複合体と反応させたときの凝集の度合いが、遊離ヘモグロビンと反応させたときの凝集の度合いの１５％超５０％未満の組み合わせは得られなかった。 B above includes cases in which the degree of aggregation of both free hemoglobin and hemoglobin-haptoglobin complex exceeds the measurement limit of the device (ΔOD x 10,000 exceeds 3000 in both cases), resulting in the above ratio. .
The results are shown in Table 3. In this test, no combination was obtained in which the degree of aggregation when reacted with a hemoglobin-haptoglobin complex was more than 15% and less than 50% of the degree of aggregation when reacted with free hemoglobin.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表３に示すように、α鎖特異的抗体とβ鎖特異的抗体を組み合わせることにより、遊離ヘモグロビンに対する特異的な反応が認められた。 As shown in Table 3, a specific reaction against free hemoglobin was observed by combining an α chain-specific antibody and a β chain-specific antibody.
実施例５：遊離ヘモグロビン測定試薬
　実施例１で得られた抗体を用いて免疫凝集反応測定試薬を作製した。 Example 5: Free hemoglobin measurement reagent The antibody obtained in Example 1 was used to prepare an immunoagglutination reaction measurement reagent.
（１）試薬の調製
　測定試薬として、第１試薬および第２試薬を調製した。第１試薬として、５０ｍＭ ＨＥＰＥＳ緩衝液（ｐＨ７．４）を用いた。第２試薬として、実施例２の抗ヘモグロビンモノクローナル抗体（抗Ｈｂ抗体）のそれぞれを担持する各ポリスチレンラテックスを混合してラテックス濃度１．０mg/mLに調整し、免疫凝集反応測定試薬を作製した。
　抗体担持不溶性担体は、各抗ヘモグロビンモノクローナル抗体とポリスチレンラテックス粒子（平均粒径１００nm）とを混合して、ポリスチレンラテックス粒子表面に抗ヘモグロビンモノクローナル抗体を担持することにより調製した。本試薬では、α鎖特異的抗ヘモグロビンモノクローナル抗体としてNo.１６を用い、β鎖特異的抗ヘモグロビンモノクローナル抗体としてNo.１１を用いた。 (1) Preparation of reagents A first reagent and a second reagent were prepared as measurement reagents. As the first reagent, 50 mM HEPES buffer (pH 7.4) was used. As a second reagent, each polystyrene latex carrying each of the anti-hemoglobin monoclonal antibodies (anti-Hb antibody) of Example 2 was mixed and the latex concentration was adjusted to 1.0 mg/mL to prepare an immunoagglutination reaction measurement reagent.
The antibody-supported insoluble carrier was prepared by mixing each anti-hemoglobin monoclonal antibody with polystyrene latex particles (average particle size: 100 nm) and supporting the anti-hemoglobin monoclonal antibody on the surface of the polystyrene latex particles. In this reagent, No. 16 was used as the α-chain-specific anti-hemoglobin monoclonal antibody, and No. 11 was used as the β-chain-specific anti-hemoglobin monoclonal antibody.
（２）試料調製、測定条件
　総ヒトヘモグロビン常用参照標準物質ＪＣＣＲＭ９１２－３（検査医学標準物質機構製）を、Ｈｂキャリブレータ希釈液‘栄研’（栄研化学社製）を用い、表４～表５に示す目的濃度となるように混合し、上記（１）で作成した測定試薬と、生化学自動分析装置ＪＣＡ－ＢＭ６０７０とを用いて測定した。
　低濃度域（０～１０μg/mL，ＦＨＢ＿Ｌ）と高濃度域（０～１００μg/mL，ＦＨＢ＿Ｈ）について、直線性試験を行った。 (2) Sample preparation and measurement conditions Total human hemoglobin regular reference standard material JCCRM912-3 (manufactured by Japan Institute for Medical Reference Materials) was used in Hb calibrator diluent 'Eiken' (manufactured by Eiken Chemical Co., Ltd.) in Tables 4 to 4. The mixture was mixed to the desired concentration shown in 5, and measured using the measurement reagent prepared in (1) above and an automatic biochemical analyzer JCA-BM6070.
Linearity tests were conducted in the low concentration range (0 to 10 μg/mL, FHB_L) and the high concentration range (0 to 100 μg/mL, FHB_H).
＝低濃度域（ＦＨＢ＿Ｌ）の測定条件＝
　検体量：５．０μL　第１試薬：５０μL　第２試薬：２５μL　測定波長：６５８nm
＝高濃度域（ＦＨＢ＿Ｈ）の測定条件＝
　検体量：１．０μL　第１試薬：５０μL　第２試薬：５０μL　測定波長：６５８nm
　結果を表４～表５および図３～図４に示す。 =Measurement conditions for low concentration range (FHB_L)=
Sample amount: 5.0μL First reagent: 50μL Second reagent: 25μL Measurement wavelength: 658nm
=Measurement conditions for high concentration area (FHB_H)=
Sample amount: 1.0μL First reagent: 50μL Second reagent: 50μL Measurement wavelength: 658nm
The results are shown in Tables 4 and 5 and FIGS. 3 and 4.
（３）遊離ヘモグロビンおよびヘモグロビン－ハプトグロビン複合体に対する反応性
　総ヒトヘモグロビン常用参照標準物質ＪＣＣＲＭ９１２－３（検査医学標準物質機構製）、およびヒトプール血漿由来ハプトグロビン（SIGMA-ALDRICH社製）を、Ｈｂキャリブレータ希釈液‘栄研’（栄研化学社製）を用いて混合し、遊離ヘモグロビンおよびヘモグロビン－ハプトグロビン複合体の測定用試料を調製した。
　遊離ヘモグロビンの測定用試料は、それぞれ１μg/mL（２．７pmol/mL）、１０μg/mL（２７pmol/mL）、および１００μg/mL（２７０pmol/mL）となるよう調製した。また、ヘモグロビン－ハプトグロビン複合体の測定用試料は、等モルのハプトグロビンを添加することで、測定用試料中で複合体を形成させた。 (3) Reactivity to free hemoglobin and hemoglobin-haptoglobin complex Total human hemoglobin routine reference standard JCCRM912-3 (manufactured by Japan Institute for Medical Research Standards) and human pool plasma-derived haptoglobin (manufactured by SIGMA-ALDRICH) were diluted with Hb calibrator. The mixture was mixed using Eiken liquid (manufactured by Eiken Kagaku Co., Ltd.) to prepare samples for measurement of free hemoglobin and hemoglobin-haptoglobin complex.
Samples for measuring free hemoglobin were prepared to have concentrations of 1 μg/mL (2.7 pmol/mL), 10 μg/mL (27 pmol/mL), and 100 μg/mL (270 pmol/mL), respectively. Further, in the measurement sample of the hemoglobin-haptoglobin complex, an equimolar amount of haptoglobin was added to form a complex in the measurement sample.
　調製した試料を、上記（１）で作成した測定試薬と、生化学自動分析装置ＪＣＡ－ＢＭ６０７０とを用いて測定した。
　結果を表６に示す。 The prepared sample was measured using the measurement reagent prepared in (1) above and an automatic biochemical analyzer JCA-BM6070.
The results are shown in Table 6.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　表４～表５および図３～図４に示すように、本実施例の免疫凝集反応測定試薬によれば、濃度に比例した遊離ヘモグロビンの測定が可能であった。
　また、表６に示すように、本実施例の免疫凝集反応測定試薬は、２．７pmol/mL、２７pmol/mLおよび２７０pmol/mL（＝遊離ヘモグロビンでそれぞれ１μg/mL、１０μg/mLおよび１００μg/mL）において、ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下であった。 As shown in Tables 4 and 5 and FIGS. 3 and 4, the immunoagglutination reaction measurement reagent of this example enabled measurement of free hemoglobin in proportion to concentration.
In addition, as shown in Table 6, the immunoagglutination reaction measurement reagent of this example was 2.7 pmol/mL, 27 pmol/mL, and 270 pmol/mL (=1 μg/mL, 10 μg/mL, and 100 μg/mL free hemoglobin, respectively). ), the reactivity to the hemoglobin-haptoglobin complex was 15% or less of the reactivity to free hemoglobin.
実施例６：他の測定法との比較
　遊離ヘモグロビンとヘモグロビン－ハプトグロビン複合体とを混合した検体で、測定法の比較を実施した。 Example 6: Comparison with other measurement methods A comparison of measurement methods was carried out using a sample containing a mixture of free hemoglobin and hemoglobin-haptoglobin complex.
（１）精製遊離ヘモグロビンとヘモグロビン－ハプトグロビン複合体の作製
　総ヒトヘモグロビン常用参照標準物質ＪＣＣＲＭ９１２－３Ｈ（検査医学標準物質機構製）を、生理食塩水を用いて２．０mg/mLに希釈した。
　また、ヒトプール血漿由来ハプトグロビン（SIGMA-ALDRICH社製）凍結乾燥品１mgを、生理食塩水３０３μLに溶解し、ハプトグロビン溶液（３．３mg/mL）を調製した。 (1) Preparation of purified free hemoglobin and hemoglobin-haptoglobin complex Total human hemoglobin routine reference standard JCCRM912-3H (manufactured by Japan Institute for Medical Research and Standards) was diluted to 2.0 mg/mL using physiological saline.
In addition, 1 mg of lyophilized human pool plasma-derived haptoglobin (manufactured by SIGMA-ALDRICH) was dissolved in 303 μL of physiological saline to prepare a haptoglobin solution (3.3 mg/mL).
（２）遊離ヘモグロビンとヘモグロビン－ハプトグロビン複合体の混合
　２．０mg/mLに調製した遊離ヘモグロビン（free-Ｈｂ）は一定量とし、これに３．３mg/mLに調製したハプトグロビン（Ｈｐｔ）溶液を表７の容量で混合し、混合比の異なる溶液（検体）を調製した。なお、表７中のfree-Complexは、遊離ヘモグロビンとヘモグロビン－ハプトグロビン複合体とのモル比を表す。 (2) Mixing free hemoglobin and hemoglobin-haptoglobin complex A fixed amount of free hemoglobin (free-Hb) prepared at 2.0 mg/mL is added to a haptoglobin (Hpt) solution prepared at 3.3 mg/mL. 7 volumes to prepare solutions (specimens) with different mixing ratios. Note that free-Complex in Table 7 represents the molar ratio of free hemoglobin and hemoglobin-haptoglobin complex.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
（３）遊離ヘモグロビン／ヘモグロビン－ハプトグロビン複合体混合液の測定
　市販のヘモグロビン比色測定試薬を使用し、実施例５で作製した免疫凝集反応測定試薬と比較した。
　ヘモグロビン比色測定試薬は、ラウリル硫酸ナトリウムで赤血球膜を溶解し、溶出したヘモグロビンを吸光度測定することにより定量する試薬である。ただし本試験では溶血のステップを省略した。上記（２）で調製した遊離ヘモグロビン（free-Ｈｂ）／ヘモグロビン－ハプトグロビン（Ｈｂ－Ｈｐ）複合体の各混合液（検体）を、ヘモグロビン比色測定試薬と混合し、分光光度計（島津社製、ＵＶ－１９００）を用いて５４０nmで測定した。 (3) Measurement of free hemoglobin/hemoglobin-haptoglobin complex mixture A commercially available hemoglobin colorimetric measurement reagent was used and compared with the immunoagglutination reaction measurement reagent prepared in Example 5.
A hemoglobin colorimetric measurement reagent is a reagent that dissolves red blood cell membranes with sodium lauryl sulfate and quantifies the eluted hemoglobin by measuring its absorbance. However, in this study, the hemolysis step was omitted. Each mixture (sample) of free hemoglobin (free-Hb)/hemoglobin-haptoglobin (Hb-Hp) complex prepared in (2) above was mixed with a hemoglobin colorimetric measurement reagent, and a spectrophotometer (manufactured by Shimadzu Corporation) was used. , UV-1900) at 540 nm.
　一方、実施例５で作製した免疫凝集反応測定試薬（Ｌａｔｅｘ試薬）は、上記（２）で調製した各混合液（検体）を、それぞれ生理食塩水で５０倍に希釈し、下記の条件にて、生化学自動分析装置ＪＣＡ－ＢＭ６０７０を用いて測定した。
　検体量：１．０μL，第１試薬：５０μL，第２試薬：５０μL，測定波長：６５８nm
　結果を表８および図５に示す。 On the other hand, the immunoagglutination reaction measurement reagent (Latex reagent) prepared in Example 5 was prepared by diluting each mixed solution (sample) prepared in (2) above 50 times with physiological saline and using the following conditions. , was measured using an automatic biochemical analyzer JCA-BM6070.
Sample amount: 1.0 μL, 1st reagent: 50 μL, 2nd reagent: 50 μL, measurement wavelength: 658 nm
The results are shown in Table 8 and FIG.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　市販のヘモグロビン比色定量試薬は、遊離ヘモグロビンとヘモグロビン－ハプトグロビン複合体の比率が変わっても測定値が変化せず、両者を識別して測定することができなかった。これに対し、本発明の免疫凝集反応測定試薬（ラテックス試薬）は、ヘモグロビン－ハプトグロビン複合体の量に影響されず、遊離ヘモグロビン濃度に比例した測定値が得られた。
　以上の結果から、本発明のラテックス試薬は、遊離ヘモグロビンを特異的に測定することが示された。 With commercially available hemoglobin colorimetric reagents, the measured value does not change even if the ratio of free hemoglobin and hemoglobin-haptoglobin complex changes, and it was not possible to distinguish between the two. On the other hand, the immunoagglutination reaction measurement reagent (latex reagent) of the present invention was not affected by the amount of hemoglobin-haptoglobin complex, and a measurement value proportional to the free hemoglobin concentration was obtained.
The above results showed that the latex reagent of the present invention specifically measures free hemoglobin.

Claims (16)

  1. 　遊離ヘモグロビンを測定する方法であって、
    　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせを含む２種以上の抗ヘモグロビン抗体を用い、前記遊離ヘモグロビンを測定するための抗原抗体反応を行う、
    遊離ヘモグロビン測定方法。     A method for measuring free hemoglobin, the method comprising:
    Performing an antigen-antibody reaction for measuring the free hemoglobin using two or more types of anti-hemoglobin antibodies including a combination of an antibody specific for hemoglobin α chain and an antibody specific for hemoglobin β chain,
    Free hemoglobin measurement method.
  2. 　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下となる条件で、前記遊離ヘモグロビンを測定するための前記抗原抗体反応を行う、請求項１に記載の遊離ヘモグロビン測定方法。 The method for measuring free hemoglobin according to claim 1, wherein the antigen-antibody reaction for measuring the free hemoglobin is performed under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
  3. 　前記遊離ヘモグロビンを測定するための前記抗原抗体反応を、ヘモグロビン－ハプトグロビン複合体の存在し得る環境下で行う、請求項１に記載の遊離ヘモグロビン測定方法。 The method for measuring free hemoglobin according to claim 1, wherein the antigen-antibody reaction for measuring the free hemoglobin is performed in an environment where a hemoglobin-haptoglobin complex can exist.
  4. 　前記２種以上の抗ヘモグロビン抗体の少なくとも１種が不溶性担体に担持されている、請求項１に記載の遊離ヘモグロビン測定方法。 The method for measuring free hemoglobin according to claim 1, wherein at least one of the two or more anti-hemoglobin antibodies is supported on an insoluble carrier.
  5. 　前記不溶性担体が不溶性粒子である、請求項４に記載の遊離ヘモグロビン測定方法。 The method for measuring free hemoglobin according to claim 4, wherein the insoluble carrier is an insoluble particle.
  6. 　免疫凝集法である、請求項５に記載の遊離ヘモグロビン測定方法。 The method for measuring free hemoglobin according to claim 5, which is an immunoagglutination method.
  7. 　遊離ヘモグロビンを測定する方法であって、
    　２種以上の抗ヘモグロビン抗体を用い、
    　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下となる条件で、前記遊離ヘモグロビンを測定するための抗原抗体反応を行う、
    遊離ヘモグロビン測定方法。     A method for measuring free hemoglobin, the method comprising:
    Using two or more types of anti-hemoglobin antibodies,
    Performing the antigen-antibody reaction for measuring the free hemoglobin under conditions such that the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
    Free hemoglobin measurement method.
  8. 　遊離ヘモグロビンを測定するための試薬であって、
    　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体との組み合わせを含む２種以上の抗ヘモグロビン抗体を含む、
    遊離ヘモグロビン測定試薬。     A reagent for measuring free hemoglobin, comprising:
    Two or more types of anti-hemoglobin antibodies, including a combination of an antibody specific for hemoglobin α chain and an antibody specific for hemoglobin β chain,
    Free hemoglobin measurement reagent.
  9. 　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下である、請求項８に記載の遊離ヘモグロビン測定試薬。 The reagent for measuring free hemoglobin according to claim 8, wherein the reactivity to the hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin.
  10. 　遊離ヘモグロビンを測定するための抗原抗体反応が、ヘモグロビン－ハプトグロビン複合体の存在し得る環境下で行われる、請求項８に記載の遊離ヘモグロビン測定試薬。 The reagent for measuring free hemoglobin according to claim 8, wherein the antigen-antibody reaction for measuring free hemoglobin is performed in an environment where a hemoglobin-haptoglobin complex can exist.
  11. 　前記２種以上の抗ヘモグロビン抗体の少なくとも１種が不溶性担体に担持されている、請求項８に記載の遊離ヘモグロビン測定試薬。 The reagent for measuring free hemoglobin according to claim 8, wherein at least one of the two or more anti-hemoglobin antibodies is supported on an insoluble carrier.
  12. 　前記不溶性担体が不溶性粒子である、請求項１１に記載の遊離ヘモグロビン測定試薬。 The free hemoglobin measurement reagent according to claim 11, wherein the insoluble carrier is an insoluble particle.
  13. 　免疫凝集法の試薬である、請求項１２に記載の遊離ヘモグロビン測定試薬。 The free hemoglobin measuring reagent according to claim 12, which is a reagent for immunoagglutination.
  14. 　遊離ヘモグロビンを測定するための試薬であって、
    　２種以上の抗ヘモグロビン抗体を含み、
    　ヘモグロビン－ハプトグロビン複合体に対する反応性が遊離ヘモグロビンに対する反応性の１５％以下である、
    遊離ヘモグロビン測定試薬。     A reagent for measuring free hemoglobin, comprising:
    Contains two or more types of anti-hemoglobin antibodies,
    The reactivity to hemoglobin-haptoglobin complex is 15% or less of the reactivity to free hemoglobin,
    Free hemoglobin measurement reagent.
  15. 　ヘモグロビンα鎖に特異的な抗体と、ヘモグロビンβ鎖に特異的な抗体とを組み合わせてなる、抗ヘモグロビン抗体の組み合わせ。 A combination of anti-hemoglobin antibodies, consisting of an antibody specific to the hemoglobin α chain and an antibody specific to the hemoglobin β chain.
  16. 　請求項８～１４のいずれか一項に記載の遊離ヘモグロビン測定試薬を含む、遊離ヘモグロビン測定キット。 A free hemoglobin measurement kit comprising the free hemoglobin measurement reagent according to any one of claims 8 to 14.
PCT/JP2023/032151 2022-09-05 2023-09-01 Free hemoglobin measurement reagent, free hemoglobin measurement method, and anti-hemoglobin antibody WO2024053586A1 (en)

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JPH0324458A (en) * 1989-06-21 1991-02-01 Daiso Co Ltd Immunological measuring method of erythrocyte in urine
JPH05209879A (en) * 1992-01-31 1993-08-20 Godo Shiyusei Kk Immunological method for detecting hemoglobin
JPH10132824A (en) * 1996-10-28 1998-05-22 Eiken Chem Co Ltd Hemoglobin stabilizing method
JPH11201969A (en) * 1998-01-14 1999-07-30 Wakamoto Pharmaceut Co Ltd Simple free hemoglobin measuring method and kit for measurement
JP2010518386A (en) * 2007-02-12 2010-05-27 ハンソン，ステファン Diagnosis and treatment of preeclampsia
JP2017517753A (en) * 2014-06-13 2017-06-29 シーメンス・ヘルスケア・ダイアグノスティックス・インコーポレーテッドＳｉｅｍｅｎｓ Ｈｅａｌｔｈｃａｒｅ Ｄｉａｇｎｏｓｔｉｃｓ Ｉｎｃ． Hemolysis detection using a chromatographic detection pad

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59182367A (en) * 1983-02-02 1984-10-17 オ−ストレイリアン・モノクロ−ナル・デベロプメント・プロプライエタリ−・リミテツド Method and diagnostic appliance for detecting very small amount of blood in excrement
JPH0324458A (en) * 1989-06-21 1991-02-01 Daiso Co Ltd Immunological measuring method of erythrocyte in urine
JPH05209879A (en) * 1992-01-31 1993-08-20 Godo Shiyusei Kk Immunological method for detecting hemoglobin
JPH10132824A (en) * 1996-10-28 1998-05-22 Eiken Chem Co Ltd Hemoglobin stabilizing method
JPH11201969A (en) * 1998-01-14 1999-07-30 Wakamoto Pharmaceut Co Ltd Simple free hemoglobin measuring method and kit for measurement
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