WO2024053586A1 - 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 - Google Patents

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 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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PCT/JP2023/032151
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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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Priority to JP2023571225A priority Critical patent/JP7543575B2/ja
Priority to TW112133741A priority patent/TW202417843A/zh
Publication of WO2024053586A1 publication Critical patent/WO2024053586A1/ja

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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

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  • 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.

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PCT/JP2023/032151 2022-09-05 2023-09-01 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 WO2024053586A1 (ja)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59182367A (ja) * 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 糞便中の微量血液を検出するための方法および診断器具
JPH0324458A (ja) * 1989-06-21 1991-02-01 Daiso Co Ltd 尿中赤血球の免疫学的測定法
JPH05209879A (ja) * 1992-01-31 1993-08-20 Godo Shiyusei Kk ヘモグロビンの免疫学的検出方法
JPH10132824A (ja) * 1996-10-28 1998-05-22 Eiken Chem Co Ltd ヘモグロビンの安定化方法
JPH11201969A (ja) * 1998-01-14 1999-07-30 Wakamoto Pharmaceut Co Ltd 簡易遊離ヘモグロビン測定方法および測定用キット
JP2010518386A (ja) * 2007-02-12 2010-05-27 ハンソン，ステファン 子癇前症の診断及び治療
JP2017517753A (ja) * 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． クロマトグラフ検出パッドを使用する溶血検出

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59182367A (ja) * 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 糞便中の微量血液を検出するための方法および診断器具
JPH0324458A (ja) * 1989-06-21 1991-02-01 Daiso Co Ltd 尿中赤血球の免疫学的測定法
JPH05209879A (ja) * 1992-01-31 1993-08-20 Godo Shiyusei Kk ヘモグロビンの免疫学的検出方法
JPH10132824A (ja) * 1996-10-28 1998-05-22 Eiken Chem Co Ltd ヘモグロビンの安定化方法
JPH11201969A (ja) * 1998-01-14 1999-07-30 Wakamoto Pharmaceut Co Ltd 簡易遊離ヘモグロビン測定方法および測定用キット
JP2010518386A (ja) * 2007-02-12 2010-05-27 ハンソン，ステファン 子癇前症の診断及び治療
JP2017517753A (ja) * 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． クロマトグラフ検出パッドを使用する溶血検出

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