WO2025183146A1 - Procédé de mesure immunologique, réactif pour mesure immunologique, liquide de prétraitement d'échantillon pour mesure immunologique, kit de réactif pour mesure immunologique et inhibiteur de réaction non spécifique - Google Patents

Procédé de mesure immunologique, réactif pour mesure immunologique, liquide de prétraitement d'échantillon pour mesure immunologique, kit de réactif pour mesure immunologique et inhibiteur de réaction non spécifique

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Publication number
WO2025183146A1
WO2025183146A1 PCT/JP2025/007073 JP2025007073W WO2025183146A1 WO 2025183146 A1 WO2025183146 A1 WO 2025183146A1 JP 2025007073 W JP2025007073 W JP 2025007073W WO 2025183146 A1 WO2025183146 A1 WO 2025183146A1
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Prior art keywords
iga
enzyme
sample
specific
reagent
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PCT/JP2025/007073
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English (en)
Japanese (ja)
Inventor
建午 藤村
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Sekisui Medical Co Ltd
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Sekisui Medical Co Ltd
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Priority to JP2025531141A priority Critical patent/JP7736395B1/ja
Priority to JP2025133646A priority patent/JP2025156581A/ja
Publication of WO2025183146A1 publication Critical patent/WO2025183146A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G01N33/546—Synthetic resin as water suspendable particles

Definitions

  • the present invention relates to an immunoassay method, an immunoassay reagent, a sample pretreatment solution for immunoassay, an immunoassay reagent kit, and a non-specific reaction inhibitor.
  • the immunological measurement method measures the target substance present in a biological sample by utilizing an antigen-antibody reaction. Because the immunological measurement method utilizes an antigen-antibody reaction, it is a measurement method with extremely high specificity. Various substances exist in biological samples, and measurement errors occur when substances other than the substance being measured cause nonspecific binding reactions or interfere with specific antigen-antibody reactions. Such phenomena are called nonspecific reactions, and the substances that cause nonspecific reactions are called nonspecific factors. The existence of heterophilic antibodies and rheumatoid factors (RF) has been elucidated as non-specific factors.
  • RF rheumatoid factors
  • Heterophilic antibodies are a general term for human antibodies that show reactivity to animal-derived antibodies, which are the main reaction in immunological assays, and human anti-mouse immunoglobulin antibodies (HAMA) are known as representative examples.
  • Rheumatoid factors are glycoproteins that are frequently detected in patients with collagen diseases such as rheumatoid arthritis, chronic infections, and liver diseases, and share the characteristic of showing reactivity to animal-derived antibodies with HAMA.
  • IgM-RF of the IgM class is the most frequently detected RF, but the existence of IgA-RF has also been confirmed (Non-Patent Documents 1 and 3).
  • IgA immunoglobulin A
  • IgA causes non-specific reactions in immunological assays
  • Patent Document 1 discloses a method for suppressing nonspecific reactions caused by RF by treating a sample in advance with a sufficient amount of animal-derived antibodies capable of binding to the antigen-binding site (Fab) of human rheumatoid factor.
  • animal-derived antibodies include anti-human immunoglobulin Fab antibodies, anti-human IgG antibodies (Fab-specific), anti-human IgA antibodies (Fab-specific), and anti-human IgM antibodies (Fab-specific).
  • Patent Document 2 discloses a method for suppressing nonspecific reactions caused by interfering substances, such as rheumatoid factors, which have a structure in which polypeptide chains are bonded together via disulfide bonds, by using a reducing agent to cleave the disulfide bonds and thereby decompose the interfering substance.
  • Patent Document 3 discloses a method for reducing or eliminating interference from non-target proteins by mixing a liquid sample containing a target protein and one or more other non-target proteins with a protein digesting agent, and carrying out protein digestion under conditions that provide antigenic determinants of the target protein in the mixture.
  • the anti-human IgM polyclonal antibody, anti-human IgG polyclonal antibody, and anti-human IgA polyclonal antibody shown in Patent Document 1 are currently used in various reagents to suppress non-specific reactions caused by natural antibodies and RF, but it has become clear that this method is unable to sufficiently suppress non-specific reactions.
  • the reducing agent shown in Patent Document 2 which is used to suppress non-specific reactions caused by interfering substances with a structure in which polypeptide chains are disulfide-bonded, has the potential to cleave disulfide bonds in the antigens or antibodies being measured or in the antigens or antibodies contained in the reagent components, depending on the type and concentration, making it less versatile. Furthermore, methods that use reducing agents have the potential to decompose molecules with disulfide bonds, such as IgG and IgA, without distinguishing between them. For this reason, this method could not be used, particularly when human IgG was being measured.
  • Patent Document 3 discloses protein digestive agents such as pepsin, papain, and trypsin.
  • Pepsin exhibits enzymatic activity at an optimal pH of 1 to 3, which is in the acidic range, and therefore there is a possibility that the target substance to be measured and/or substances that specifically bind to the target substance may be denatured or decomposed.
  • Papain and trypsin exhibit enzymatic activity against many targets, and therefore there is a possibility that the target substance to be measured and/or substances that specifically bind to the target substance may be denatured or decomposed.
  • the object of the present invention is to provide an immunological measurement method that can suppress non-specific reactions caused by IgA contained in the measurement sample.
  • the inventors investigated the effects of various substances on inhibiting non-specific reactions, and discovered that non-specific reactions can be inhibited by carrying out antigen-antibody reactions in the presence of an enzyme that specifically degrades IgA, leading to the completion of the present invention.
  • the present invention has the following features:
  • An immunological assay method for measuring a substance to be measured in a sample in which an antigen-antibody reaction is carried out at least once in the presence of an enzyme that specifically degrades immunoglobulin A (IgA).
  • the immunological assay method described in [1], wherein the enzyme is a protease.
  • a reagent for immunoassay used in an immunoassay method for measuring a substance to be measured in a sample comprising an enzyme that specifically decomposes IgA.
  • the reagent for immunoassay according to [9], wherein the enzyme is a protease.
  • the immunoassay reagent according to any one of [9] to [15], wherein the substance to be measured is an antigen or an antibody in the immunoassay method.
  • a sample pretreatment solution for immunological assays comprising an enzyme that specifically degrades IgA.
  • a reagent kit for immunological measurement used in an immunological measurement method for measuring a substance to be measured in a sample comprising an enzyme that specifically degrades IgA.
  • a non-specific reaction inhibitor comprising an enzyme that specifically degrades IgA.
  • the present invention provides an immunological measurement method that can suppress non-specific reactions caused by IgA contained in the measurement sample.
  • the immunological measurement method of the present invention can suppress non-specific reactions that could not be suppressed even with conventional non-specific reaction inhibitors. As a result, it is now possible to accurately measure the substance being measured.
  • the immunoassay method of the present invention is a method for immunologically measuring a substance to be measured in a sample, characterized in that an antigen-antibody reaction is carried out at least once in the presence of an enzyme that specifically degrades IgA.
  • the method is a method in which an enzyme that specifically degrades IgA is reacted with the sample as a non-specific reaction inhibitor, and the substance to be measured in the sample is immunologically measured using a specific binding partner in the presence of the enzyme.
  • One aspect of the immunological assay method of the present invention is a method for suppressing non-specific reactions, in which an antigen-antibody reaction is carried out at least once in the presence of an enzyme that specifically degrades IgA, and non-specific reactions in the reaction solution are suppressed during this process.
  • Immunological assay methods are broadly divided into homogeneous and heterogeneous methods.
  • the homogeneous method is a measurement method that specifically detects the binding reaction that occurs between the substance to be measured and its specific binding partner in a mixed solution (reaction solution) of sample and reagent solution, without performing B/F (bound/unbound) separation.
  • the heterogeneous method is a measurement method that performs B/F separation, washes away and removes excess components that were not involved in the binding reaction, and then allows the binding reaction to proceed, detecting the substance to be measured.
  • the heterogeneous method has the drawback of requiring many steps and time for measurement due to the need for washing steps, but has the advantage of being relatively unaffected by non-specific reaction substances.
  • the homogeneous method does not require a washing step and therefore has the drawback of being susceptible to the effects of non-specific reactions, but it is simple with few steps and requires short measurement times, making it a method that is widely sought after in the field of clinical diagnostics.
  • IA immunoagglutination assays
  • TIA turbidimetric immunoassays
  • lateral flow and flow-through immunochromatography
  • LTIA latex turbidimetric immunoassays
  • Heterogeneous methods include ELISA using well-shaped plates and chemiluminescence.
  • the present invention can be used with any of the above immunological measurement methods, but homogeneous methods, which are relatively susceptible to non-specific reactions, are preferred as they are expected to be more effective, and of the homogeneous methods, latex immunoturbidimetry is the most preferred.
  • One aspect of the present invention is a method for performing immunoassays using an enzyme that specifically degrades IgA, or a non-specific reaction inhibitor containing the enzyme (hereinafter, an enzyme that specifically degrades IgA may be abbreviated as an IgA-specific degrading enzyme).
  • the IgA-specific degrading enzyme may be any enzyme that can specifically degrade IgA, such as a protease.
  • proteases include proteinases (endopeptidases), peptidases (exopeptidases), metalloproteases, serine proteases, and cysteine proteases.
  • the IgA-degrading enzyme of the present invention is an enzyme that cleaves the amino acid sequence of the IgA constant region including CH ⁇ 1 to CH ⁇ 3, preferably an enzyme that cleaves a portion of the amino acid sequence of the hinge region between CH ⁇ 1 and CH ⁇ 2 (e.g., VPSSTPPTPSPST of human IgA: SEQ ID NO: 1), and more preferably an enzyme that can degrade IgA into Fab fragments and Fc fragments.
  • the IgA-specific degrading enzyme only needs to have the function of specifically degrading IgA when it is brought into contact with a sample such as a specimen, and the enzyme may be inactivated by the time the subsequent antigen-antibody reaction is carried out.
  • the terms "reacting" with an antigen and “recognizing” an antigen are used interchangeably, but are not limited to these examples and should be interpreted in the broadest sense. Whether an antibody “reacts” with an antigen can be confirmed by methods such as antigen-immobilized ELISA, competitive ELISA, and sandwich ELISA, as well as methods utilizing the principle of surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the SPR method can be performed using equipment, sensors, and reagents commercially available under the name Biacore (registered trademark).
  • the IgA-specific enzyme of the present invention specifically degrades IgA.
  • IgA-specific enzymes can degrade IgA more efficiently than immunoglobulins other than IgA, such as IgG and IgM. Therefore, even when the substance to be measured is IgG, or when the binding partner that specifically binds to the substance to be measured is IgG, IgA-specific enzymes can be used without affecting the specific measurement of the substance to be measured.
  • IgA-specific enzymes can degrade IgA derived from organisms such as humans, mice, rats, rabbits, goats, ostriches, and pigs. When specifically degrading human IgA, it is preferable to use human IgA-specific enzymes.
  • Human IgA in particular, has two subclasses, IgA1 and IgA2.
  • Human IgA-specific enzymes may specifically degrade human IgA1 and human IgA2, but it is more preferable for them to specifically degrade human IgA1.
  • IgA is present in both blood and secretory fluids; in blood it exists mainly as a monomer, while in secretory fluids it exists as a dimer or multimer. It is preferable for the IgA-specific degrading enzyme to degrade both the IgA present in blood and the IgA present in secretory fluids, but it may also degrade only one of them.
  • the IgA-specific degrading enzyme specifically degrades IgA means that, for example, after reacting IgG or IgA with the enzyme at 37°C for 30 to 60 minutes, the amount of IgA remaining is 50% or less, 40% or less, 30% or less, preferably 20% or less, and more preferably 10% or less compared to IgG.
  • the IgA-specific degrading enzyme used in the present invention may be either a naturally occurring enzyme or a recombinant enzyme produced by genetic engineering.
  • naturally occurring enzymes include enzymes produced by various bacteria and fungi, such as those produced by bacteria that cause oral infections (Streptococcus sanguis, Streptococcus mitis, and Streptococcus oralis), bacteria that cause genital tract infections such as gonorrhea (Neisseria gonorrhoeae), and bacteria that cause meningitis (Haemophilus influenza, Neisseria meningitidis, and Streptococcus pneumoniae). (Patricia de Sousa-Pereira and Jenny M. Woof. (2019).
  • IgA Structure, Function, and Developability. Antibodies, 8, 57.
  • Preferred examples include IgASAP (manufactured by Genovis) and amino acid variants of this enzyme.
  • the enzyme may also be a recombinant enzyme produced as a recombinant protein. Modified recombinant enzymes with desired enzymatic activity can be produced by artificially introducing amino acid residue substitutions into the recombinant enzyme and screening using various known assay systems (JP 2019-506866, EP 3148576). Therefore, in the present invention, various IgA-specific degrading enzymes can be used to suppress nonspecific reactions in immunological assays or to produce nonspecific reaction inhibitors.
  • methods for making an enzyme that specifically degrades IgA present in an antigen-antibody reaction system include using it as one of the reagent components of an immunoassay reagent, or adding it to a specimen dilution solution, specimen extract, specimen pretreatment solution, etc.
  • a specimen dilution solution, specimen extract, or specimen pretreatment solution that already contains an IgA-specific degrading enzyme may be used, or the IgA-specific degrading enzyme may be added to a specimen dilution solution, specimen extract, or specimen pretreatment solution.
  • specimen dilution solutions and specimen extracts are both referred to as specimen pretreatment solutions.
  • inside the antigen-antibody reaction system refers to the liquid phase in which, for example, when the immunoassay reagent is a liquid reagent, the sample and the liquid immunoassay reagent are mixed and the antigen-antibody reaction takes place.
  • the sample may be mixed with an immunoassay reagent containing an IgA-specific degrading enzyme, or the sample may be mixed in advance with a specimen pretreatment solution containing an IgA-specific degrading enzyme and then mixed with the LTIA reagent.
  • the sample may be mixed with an IgA-specific enzyme beforehand and then dropped onto the microplate, or the sample may be mixed with a solution containing an IgA-specific enzyme and a detection antibody before being dropped onto the microplate.
  • the sample may be mixed in advance with a specimen pretreatment solution containing an IgA-specific degrading enzyme and then mixed with the immunoassay reagent, or the immunoassay reagent (e.g., a solution containing a detection antibody or antigen and magnetic particles) may contain the IgA-specific degrading enzyme.
  • the immunoassay reagent e.g., a solution containing a detection antibody or antigen and magnetic particles
  • the term "inside the antigen-antibody reaction system” refers to the solid phase where the antigen-antibody reaction between the liquid sample and the binding partner takes place.
  • the sample may be pre-mixed with a specimen pretreatment solution containing an IgA-specific enzyme and then dropped onto the immunochromatographic test piece, or the IgA-specific enzyme may be dried and held on a component such as a sample pad (sample supply site), and when the sample is dropped, the IgA-specific enzyme dissolves and develops the solid phase, becoming present in the reaction system.
  • the concentration of the IgA-specific degrading enzyme may be any concentration that does not strongly affect the antigen-antibody reaction between the substance to be measured and its specific binding partner, and that can exert the desired effect of suppressing non-specific reactions.
  • This concentration can be appropriately determined by a person skilled in the art depending on the type of substance to be measured and the type of sample.
  • the concentration of IgA-specific enzyme in an antigen-antibody reaction system varies depending on the reagent composition of the antigen-antibody reaction system.
  • 1 to 1000 U of enzyme can be added per 10 ⁇ L of sample, preferably 5 to 800 U, more preferably 10 to 500 U, even more preferably 15 to 300 U, and most preferably 20 to 200 U.
  • the concentration of enzyme in the pretreatment solution can be 1 to 1000 U/ ⁇ L, preferably 5 to 800 U/ ⁇ L, more preferably 10 to 500 U/ ⁇ L, even more preferably 15 to 300 U/ ⁇ L, and most preferably 20 to 200 U/ ⁇ L.
  • the mixing ratio of the sample to the sample pretreatment solution can be 1:100 to 100:1, preferably 1:50 to 50:1, more preferably 1:20 to 20:1, and most preferably 1:10 to 10:1.
  • IgA-specific enzymes may be used alone or in combination with other substances that have the effect of inhibiting non-specific reactions.
  • examples of other substances that have the effect of inhibiting non-specific reactions include anti-IgA antibodies, polymeric compounds, Heteroblocks (manufactured by OMEGA Biologicals), and modified antibodies in which part or all of the variable region of the L chain or H chain of a specific antibody has been modified. It should be noted that other substances that have the effect of inhibiting non-specific reactions are not limited to those listed here.
  • the effect of inhibiting non-specific reactions is expected to be enhanced by using IgA-specific enzymes in combination with other substances that have the effect of inhibiting non-specific reactions.
  • anti-IgA antibodies and IgA-specific enzymes may be bound directly or indirectly.
  • an IgA-specific degrading enzyme When an IgA-specific degrading enzyme is to be added to the immunoassay reagent of the present invention in advance, it is preferable to add it to the assay reagent in advance so that it has a concentration that will be present in the reaction system described above.
  • LTIA Latex immunoturbidimetric assay
  • the first method involves reacting the target substance with latex particles onto which a specific binding partner for the target substance has been immobilized, forming a sandwich-type immune complex, and measuring the target substance based on the degree of agglutination of the latex particles that accompanies immune complex formation.
  • the second method involves adding proteins or the like to which multiple analytes or their analogs (including fragments thereof) are immobilized to an immunoassay reagent, causing them to compete with the analytes in the sample, inhibiting the formation of immune complexes between the analytes contained in the reagent and latex particles to which specific binding partners for the analytes are immobilized, and measuring the analyte (such as an antigen) based on the degree of inhibition of agglutination of the latex particles that accompanies the inhibition of immune complex formation.
  • an immunoassay reagent such as an antigen
  • the substance to be measured and its specific binding partner can be any protein, peptide, sugar chain, lipid, glycoprotein, glycolipid, nucleic acid, low molecular weight compound, or high molecular weight compound, as long as it can specifically bind to the substance to be measured, and any substance can be selected depending on the purpose.
  • any antibody such as a polyclonal antibody or a monoclonal antibody (including monoclonal antibodies produced from hybridomas, recombinant antibodies, and functional fragments of each antibody)
  • an antigen such as a natural or recombinant antigen, can be selected as the specific binding partner for the substance to be measured.
  • the present invention can be used in any of the above methods, and specific examples include, but are not limited to, the following steps.
  • a step of contacting a sample containing the substance to be measured with an IgA-specific degrading enzyme in a solution (1) A step of contacting a sample containing the substance to be measured with an IgA-specific degrading enzyme in a solution.
  • step (1) adding latex particles carrying a specific binding partner for the substance to be measured to the solution.
  • step (2) optically detecting the degree of aggregation of the latex particles in the solution.
  • step (3) means "a step of measuring the agglutination reaction between the substance to be measured and the latex particles during or after step (2) without going through a washing or separation step.”
  • the LTIA method can measure the target substance by optically observing the degree of agglutination that occurs.
  • Optical observation methods include measuring scattered light intensity, absorbance, or transmitted light intensity with optical equipment (endpoint method, rate method, etc.). The change in absorbance measured in this way is used to calculate the concentration (quantitative value) of the target substance contained in the sample, using a calibration curve obtained by measuring a standard substance with a known concentration of the target substance.
  • measurement of absorbance of transmitted light or scattered light may be a one-wavelength measurement or a two-wavelength measurement (the difference or ratio of two wavelengths).
  • the measurement wavelength is generally selected from the range of 500 nm to 900 nm.
  • the measurement of the substance to be measured in the sample may be performed manually or using an apparatus such as a measuring device.
  • the measuring device may be a general-purpose automatic analyzer or a dedicated measuring device (specialized machine).
  • this measurement be performed using a method involving multiple operational steps, such as a two-step method (two-reagent method).
  • the specific binding partner for the substance to be measured can be immobilized and supported on latex particles by known methods such as physical adsorption, chemical binding, or a combination of these.
  • physical adsorption the specific binding partner for the substance to be measured and latex particles can be mixed and contacted in a solution such as a buffer solution, or the specific binding partner for the substance to be measured dissolved in a buffer solution or the like can be contacted with a carrier, according to known methods.
  • the chemical binding method it can be carried out according to known methods described in, for example, "Clinical Pathology Extra Special Issue No.
  • the synthetic polymers that make up the latex particles are not particularly limited, but examples include polystyrene, styrene-styrene sulfonate copolymers, methacrylic acid polymers, acrylic acid polymers, itaconic acid polymers, and styrene-hydrophilic carboxy monomer copolymers such as styrene-methacrylic acid copolymers, styrene-acrylic acid copolymers, and styrene-itaconic acid copolymers.
  • styrene-methacrylic acid copolymers preferred are styrene-methacrylic acid copolymers, styrene-itaconic acid copolymers, and styrene and styrene-styrene sulfonate copolymers. Particularly preferred are styrene and styrene-(meth)acrylic acid copolymers.
  • the latex particles carry multiple types of specific binding partners for the substance to be measured in order to form a sandwich.
  • a single type of specific binding partner may be used.
  • the specific binding partner is a monoclonal antibody
  • multiple monoclonal antibodies with different recognition sites are used.
  • the specific binding partner is a polyclonal antibody, it may be a polyclonal antibody derived from a single type of antiserum, or may be derived from multiple types of antisera. It is also possible to use a combination of monoclonal and polyclonal antibodies.
  • the surface of the latex particles may be blocked (masked) by known methods, such as by contacting and coating with a protein such as bovine serum albumin (BSA), casein, gelatin, egg albumin or a salt thereof, a surfactant, or skim milk powder.
  • a protein such as bovine serum albumin (BSA), casein, gelatin, egg albumin or a salt thereof, a surfactant, or skim milk powder.
  • the immunoassay method of the present invention can be carried out using an immunoassay reagent.
  • the immunoassay reagent is characterized by containing the above-mentioned IgA-specific degrading enzyme in addition to the main components of an antigen-antibody reaction.
  • the main components include a binding partner specific to the substance to be measured, as well as insoluble carriers such as immunoassay particles, immunochromatographic test strips, and microplates.
  • the immunoassay reagent of the present invention may contain buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, sugars, glycoproteins, glycolipids, inorganic salts, polymeric compounds, surfactants, other non-specific reaction inhibitors, preservatives, etc., to the extent that the non-specific reaction inhibitory effect of the IgA-specific degrading enzyme is not impaired.
  • buffers such as acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonic acid, phthalic acid, succinic acid, maleic acid, and imidazole, as well as Good's buffers and their sodium salts, potassium salts, and calcium salts.
  • Polymers such as polyvinylpyrrolidone and phospholipid polymers may also be included as components that enhance the agglutination of particles for immunoassays.
  • the concentration of IgA-specific enzyme in the constituent reagents should be such that it can be adjusted to the concentration in the antigen-antibody reaction system when the reagent and sample are mixed together at the time of measurement, and this concentration will vary depending on the type of reagent.
  • the immunoassay reagent kit of the present invention is characterized by including at least an IgA-specific enzyme in its components. Therefore, the reagent kit of the present invention includes the IgA-specific enzyme in one or more of the following components: a sample dilution solution, a sample pretreatment solution including a sample extract, and the like, in addition to the reagents involved in the antigen-antibody reaction that make up the kit.
  • the kit also includes instructions for use and sample collection tools (such as a collection pipette, syringe, cotton swab, and filter).
  • sample collection tools such as a collection pipette, syringe, cotton swab, and filter.
  • the reagent (LTIA reagent) when the immunological assay is latex immunoturbidimetry is exemplified, but is not limited to this.
  • a first reagent containing an IgA-specific degrading enzyme (1) a first reagent containing an IgA-specific degrading enzyme; and (2) a second reagent containing latex particles carrying a specific binding partner for the substance to be measured.
  • the first reagent typically contains a buffer solution, and the concentration of the IgA-specific degrading enzyme in the buffer solution may be adjusted to the preferred enzyme concentration when the reagent and sample are mixed together at the time of measurement, and this concentration will vary depending on the type of reagent.
  • the enzyme may also be contained in the second reagent in addition to the first reagent.
  • the concentration of the IgA-specific protease contained in the first reagent is typically 1 to 1000 ⁇ g/mL, preferably 5 to 500 ⁇ g/mL, more preferably 10 to 100 ⁇ g/mL, and even more preferably 20 to 50 ⁇ g/mL, but is not limited to these concentrations.
  • the IgA-specific degrading enzyme may be contained in the sample pretreatment solution, and the enzyme concentration in the treatment solution is 1 to 1000 ⁇ g/mL, preferably 5 to 500 ⁇ g/mL, more preferably 10 to 300 ⁇ g/mL, and even more preferably 20 to 200 ⁇ g/mL, but is not limited to these concentrations.
  • the reaction temperature of the IgA-specific enzyme with the sample may be any temperature at which the IgA-specific enzyme is not inactivated and can specifically decompose IgA, and is in the range of 0 to 60°C, preferably 10 to 50°C, and more preferably 20 to 40°C, but is not limited to these temperatures.
  • reaction time The reaction time of the IgA-specific enzyme with the sample may be any reaction time that allows the IgA-specific enzyme to specifically and sufficiently decompose IgA, and is in the range of 30 seconds to 24 hours, preferably 1 minute to 120 minutes, but is not limited to this reaction time.
  • the pH of the solution containing the IgA-specific degrading enzyme may be any pH at which the IgA-specific degrading enzyme functions sufficiently, and is not limited to a pH range of 1 to 12, preferably 3 to 10, more preferably 5 to 9, and most preferably 6 to 8.
  • protease enzymes such as papain, pepsin, and trypsin
  • the enzymatic activity of the IgA-specific degrading enzyme is enhanced over a wider pH range.
  • any known particles can be used as the particles for immunoassays in the present invention as long as they can carry a specific binding partner for the substance to be measured.
  • inorganic particles such as metal colloids, silica, carbon, and magnetic particles can also be used as the particles for immunoassays in the present invention.
  • the size of particles for immunoassays can be selected appropriately from the range of 0.05 to 1 ⁇ m to obtain the desired measurement sensitivity and measurement range, taking into consideration the optical measurement method used (for example, turbidimetry, which measures transmitted light, or nephelometry, which measures scattered light). Note that, for optical measurements using automated analyzers, an average particle size of 0.1 to 0.4 ⁇ m is commonly used, but is not limited to this.
  • the ELISA method utilizes a combination of various antigen-antibody reactions, ultimately incorporating an enzyme-labeled antigen or antibody into the reaction system to detect enzyme activity.
  • a substrate whose absorption spectrum changes upon reaction is used, and methods include direct, indirect, sandwich, and competitive methods depending on the combination of antigen-antibody reactions.
  • the following provides examples of immunoassay reagents when the immunoassay method of the present invention is a sandwich ELISA method.
  • the insoluble carrier is preferably a plate, and the labeling substance can be selected appropriately.
  • the antibody immobilized on the insoluble carrier captures the analyte in the solution containing the sample, forming a complex on the insoluble carrier.
  • the antibody labeled with the labeling substance binds to the captured analyte and forms a sandwich with the complex.
  • the analyte in the sample can be measured by measuring the amount of the labeling substance using a method appropriate for the labeling substance.
  • Specific methods such as the method of immobilizing the antibody on the insoluble carrier and the method of binding the antibody to the labeling substance can be any method known to those skilled in the art, and can be used without particular limitation.
  • the IgA-specific degrading enzyme of the present invention can be present in the immune reaction system, for example, by adding it to a sample pretreatment solution or to a solution in which an antigen-antibody reaction occurs.
  • test strip constitution when the immunoassay method of the present invention is an immunochromatography method will be described below.
  • Immunochromatographic test strips When an antibody is used as the specific binding partner, the test piece comprises, on a sheet-like insoluble carrier such as a porous membrane, the following components arranged in the direction of development of a solution containing a sample: "1. Sample supply site,”"2. Site for holding a labeled antibody (labeled antibody holding site)," and "3.
  • capture antibody site Site for immobilizing an antibody to capture a complex formed by the labeled antibody and the substance to be measured.
  • capture antibody site Site for immobilizing an antibody to capture a complex formed by the labeled antibody and the substance to be measured.
  • immunochromatography when a predetermined amount of a sample containing at least the above-described test strip and a target substance is added to a sample supply site, the sample enters the labeled antibody retention site by capillary action, where the target substance binds to the labeled antibody to form a complex.
  • the complex spreads across the membrane and enters the capture antibody site, where it is captured by an antibody (capture antibody) immobilized on the membrane, forming a capture antibody-target substance-labeled antibody complex.
  • the target substance can then be detected by detecting the label using any method (e.g., by its agglutination in the case of a visible label such as gold colloid, or by a color reaction resulting from the addition of a substrate in the case of an enzyme).
  • the IgA-specific degrading enzyme of the present invention can be present in the reaction system, for example, by adding it to a sample pretreatment solution or by containing it in a sample supply site or a labeled antibody holding site and drying and holding it there.
  • chemiluminescent immunoassay When a chemiluminescent substance is used as the label, it is called a chemiluminescent immunoassay (CLIA).
  • CLIA chemiluminescent immunoassay
  • the following is an example of an immunoassay reagent when the immunoassay method of the present invention is the CLEIA method.
  • An enzyme-labeled antibody (or antigen) that reacts with the substance to be measured.
  • Luminescent Reagent The antibody immobilized on the magnetic particles captures the analyte in the solution containing the sample, forming a complex.
  • the antibody labeled with the enzyme-labeled substance binds to the captured analyte and forms a sandwich with the complex.
  • the analyte in the sample can be measured by reacting the enzyme-labeled substance with the luminescent reagent and measuring the amount of luminescence.
  • the IgA-specific degrading enzyme of the present invention can be present in the immune reaction system, for example, by adding it to a sample pretreatment solution or to a solution in which an antigen-antibody reaction occurs.
  • the specific binding partner for the analyte may be any substance capable of specifically binding to the analyte, including proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, haptens, low molecular weight compounds, high molecular weight compounds, etc.
  • examples include antibodies or antigens that can be used in immunological assays that utilize antigen-antibody reactions.
  • the antibody may be a polyclonal antibody or a monoclonal antibody. It is more preferably a monoclonal antibody.
  • samples containing a substance to be measured include human or animal blood, serum, plasma, culture supernatant, urine, cerebrospinal fluid, saliva, sweat, ascites, nasal discharge, feces, or cell or tissue extracts.
  • Blood is the most preferred sample containing a substance to be measured. It is generally known that the reference value for IgA contained in blood is 110 to 410 mg/dl. The sample is sometimes called a "specimen.”
  • the immunoassay reagent of the present invention can measure various substances contained in the sample.
  • substances to be measured include proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, and haptens, but there are no particular limitations as long as they are theoretically measurable.
  • C-reactive protein C-reactive protein
  • Lp(a) lipoprotein(a)
  • MMP3 matrix metalloproteinase 3
  • antiphospholipid antibodies type IV collagen, and prostate specific antibodies.
  • PSA molecular weight: 34,000
  • brain natriuretic peptide BNP
  • N-terminal pro-brain natriuretic peptide N-proBNP
  • insulin molecular weight: 5,800
  • albumin cystatin C
  • RF rheumatoid factor
  • KL-6 procalcitonin
  • PCT procalcitonin
  • FDP fibrin and fibrinogen degradation products
  • D-dimer D-dimer
  • thrombin-antithrombin III complex TAT
  • transferrin haptoglobin, ⁇ 1-antitrypsin, ⁇ 1-acid Glycoprotein, ⁇ 2-macroglobulin, hemopexin, antithrombin-III, ⁇ -fetoprotein, carcinoembryonic antigen (CEA), ferritin, hepatitis B virus envelope s antigen (HBs-Ag)
  • Substances to be measured in the present invention include anti-Treponema (Treponema pallidum) antibodies, anti-cyclic citrullinated peptide (CCP) antibodies, anti-Helicobacter pylori antibodies, and antibodies such as IgG and IgM against viruses such as hepatitis, measles, and leukemia.
  • Treponema pallidum anti-cyclic citrullinated peptide
  • CCP citrullinated peptide
  • anti-Helicobacter pylori antibodies anti-Helicobacter pylori antibodies
  • antibodies such as IgG and IgM against viruses such as hepatitis, measles, and leukemia.
  • the IgA-specific degrading enzyme of the present invention does not degrade these antibodies. Therefore, the IgA-specific degrading enzyme is particularly useful for the technology described in Patent Document 2 when the substance to be measured is an antibody.
  • Non-specific reaction inhibitor refers to acting on factors (also referred to as non-specific factors, non-specific causative substances, or non-specific reaction substances) that cause the above-mentioned non-specific reactions in biological samples, thereby suppressing the influence of reactions other than antigen-antibody reactions on the measurement.
  • a candidate substance as a non-specific reaction inhibitor has the effect of inhibiting non-specific reactions can be determined by comparing the measured value (hereinafter referred to as the control measured value) obtained by measuring using, for example, a measurement method with B/F separation (a method that has a washing step and is less susceptible to the influence of non-specific reaction substances (in the examples, the CLEIA method)) with and without the addition of the candidate substance and determining whether the measured value approaches the control measured value.
  • the control measured value obtained by measuring using, for example, a measurement method with B/F separation (a method that has a washing step and is less susceptible to the influence of non-specific reaction substances (in the examples, the CLEIA method)
  • the candidate substance in the target measurement method is closer to the control measured value than the measured value obtained by not adding the candidate substance, it can be determined that the candidate substance has the effect of inhibiting non-specific reactions in the measurement method, and that the candidate substance can be a non-specific reaction inhibitor.
  • the non-specific reaction inhibitor of the present invention targets both factors that cause positive measurement errors, in which a substance to be measured is determined to be higher than its actual content, due to some component contained in a biological sample, and factors that cause negative measurement errors, in which a substance to be measured is determined to be lower than its actual value.
  • the inhibitor is particularly effective against non-specific factors that cannot be suppressed by commercially available non-specific reaction inhibitors such as HBR-1 and Heteroblock. It is also effective against non-specific factors that cause positive measurement errors, in which measured values become abnormally high, and negative measurement errors, in which measured values become abnormally low, so-called deviation samples.
  • various causative substances that cause non-specific reactions can be cited, but it is preferable to degrade causative substances that have a structure similar to at least part, all, or part or all of IgA with an IgA-specific degrading enzyme. Through this degradation reaction, the non-specific reaction inhibitor of the present invention can suppress non-specific reactions resulting from the causative substance.
  • the non-specific reaction inhibitor of the present invention may contain a substance capable of inhibiting reactions caused by non-specific factors derived from a sample, as determined above, and may contain at least an IgA-specific degrading enzyme as an active ingredient.
  • the non-specific reaction inhibitor of the present invention may be configured such that the above-mentioned immunological assay reagent contains an IgA-specific degrading enzyme.
  • the non-specific reaction inhibitor of the present invention may contain buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, sugars, glycoproteins, glycolipids, inorganic salts, polymeric compounds, surfactants, other non-specific reaction inhibitors, preservatives, etc., as long as they do not interfere with the non-specific reaction inhibitory effect of the IgA-specific degrading enzyme.
  • Other non-specific reaction inhibitors may be those that have the non-specific reaction inhibitory effect, and include, but are not limited to, anti-IgA antibodies, polymeric compounds, and modified antibodies in which part or all of the variable region of the L chain or H chain of a specific antibody has been modified.
  • the non-specific reaction inhibitory effect is expected to be enhanced by using the IgA-specific degrading enzyme in combination with other substances that have the non-specific reaction inhibitory effect. Furthermore, when an anti-IgA antibody and an IgA-specific degrading enzyme are used in combination, the anti-IgA antibody and the enzyme may be bound directly or indirectly.
  • the method for inhibiting non-specific reactions of the present invention is a method for inhibiting non-specific reactions caused by a sample by performing an antigen-antibody reaction at least once in the presence of an IgA-specific protease.
  • the method for inhibiting non-specific reactions can also be described as a method for reducing measurement errors.
  • ⁇ Non-specific reaction inhibition in LTIA method measurement of sIL-2R> The concentration of soluble interleukin-2 receptor (sIL-2R) contained in the sample (specimen) was measured by the LTIA method as follows. Human serum samples 1 to 3 from multiple individuals (3 individuals) were used as samples. Sample 1 (control sample) is a sample whose LTIA measurement value is close to the chemiluminescent enzyme immunoassay (CLEIA) measurement value (Reference Example 1). Samples 2 and 3 (deviant samples) are samples that exhibit a nonspecific reaction, and whose LTIA measurement value deviates significantly from the CLEIA measurement value in Reference Example 1.
  • CLIA chemiluminescent enzyme immunoassay
  • the IgA concentrations in each sample were 692 mg/dl for Sample 1, 387 mg/dl for Sample 2, and 247 mg/dl for Sample 3.
  • the IgA concentrations of these samples were measured using a Hitachi 7180 automatic analyzer using N-Assay TIA IgA-SH Nittobo reagent.
  • Measurement Method 1-1 Measurement Reagent Determiner CL (registered trademark) IL-2R NX (Minaris Medical Co., Ltd.) 1-2. Samples: Samples 1 to 3 1-3. Measurement Procedure Measurement was performed using CL-JACK NX (registered trademark) (Minaris Medical Co., Ltd.) according to the instructions attached to the measurement reagent.
  • the measurement results are shown in Table 1.
  • the CLEIA method shown in Reference Example 1 includes a B/F separation step and a washing step, making the CLEIA method less susceptible to the influence of non-specific reactions originating from the sample.
  • the sample, first reagent, and second reagent for each specimen were mixed, and the sIL-2R concentration in the sample was measured using a Hitachi 7180 automatic analyzer. Specifically, 120 ⁇ L of the first reagent was added to 5.6 ⁇ L of the sample, and the mixture was incubated at 37°C for 5 minutes. 40 ⁇ L of the second reagent was then added and stirred. The change in absorbance associated with aggregate formation was measured over the next 5 minutes at a dominant wavelength of 570 nm and a sub-wavelength of 800 nm. The absorbance change was applied to a calibration curve obtained by measuring a standard substance of known concentration, and a measured value was calculated. Furthermore, a value equivalent to the original solution was calculated, taking into account the dilution factor of the sample.
  • Example 1 Measurement by LTIA method: Addition of IgA-specific degrading enzyme to sample Measurement was carried out in the same manner as in Comparative Example 1, except that a PBS buffer solution containing 40 U/ ⁇ L of IgASAP (manufactured by Genovis) was used as the sample pretreatment solution. The measurement results are shown in Table 1.
  • Example 1 was 810 U/mL, which tended to approach the measured value of Reference Example 1. A similar trend was observed for sample 3.
  • the measured value for sample 3 in Reference Example 1 was 630 U/mL.
  • the measured value for Comparative Example 1 was 1766 U/mL, which deviated from the measured value in Reference Example 1.
  • the measured value for Example 1 was 756 U/mL, which tended to approach that of Reference Example 1.
  • the use of an IgA-specific degrading enzyme in an immunological assay method was able to suppress non-specific reactions originating from the sample.
  • the degraded samples used in this test were samples for which sufficient non-specific reaction suppression effects could not be obtained by using existing non-specific reaction inhibitors such as anti-IgA antibodies alone, and non-specific reactions were first suppressed by the non-specific reaction suppression method of the present invention using an IgA-specific degrading enzyme.
  • Example 2 ⁇ Non-specific reaction inhibition in LTIA method: measurement of sIL-2R> The concentration of soluble interleukin-2 receptor (sIL-2R) contained in the sample (specimen) was measured by the LTIA method in the same manner as in Example 1. Human serum specimen 2 was used as the sample.
  • sIL-2R soluble interleukin-2 receptor
  • Measurement by LTIA method PBS buffer solution added to sample (no additives) 1. Measurement Method 1-1. Measurement Reagents A first reagent and a second reagent were prepared according to the method described in JP 2017-181377 A. 1-2. Sample pretreatment solution PBS buffer solution (pH 7.4) was used. 1-3. Samples PBS buffer solution, which is a sample pretreatment solution, was added to the sample (serum) 2 described in Reference Example 1 at a volume ratio of 4.5:1.5, and the sample was reacted at 37°C for 1 hour and measured. 1-4.
  • the specimen, first reagent, and second reagent were mixed, and the sIL-2R concentration in the sample was measured using a Hitachi 7180 automatic analyzer. Specifically, 120 ⁇ L of the first reagent was added to 5.6 ⁇ L of the sample, and the mixture was incubated at 37°C for 5 minutes. 40 ⁇ L of the second reagent was then added and stirred. The change in absorbance associated with aggregate formation was measured over the next 5 minutes at a dominant wavelength of 570 nm and a sub-wavelength of 800 nm. The absorbance change was applied to a calibration curve obtained by measuring a standard substance of known concentration, and a measured value was calculated. Furthermore, a value equivalent to the original solution was calculated, taking into account the dilution factor of the sample.
  • Measurement Results The measurement results converted to per mL of serum before the addition of the sample pretreatment solution are shown in Table 2. The unit of the measurement results is U/mL.
  • Reference Example 2 Measurement by LTIA method Addition of commercially available nonspecific reaction inhibitor (Heteroblock) to sample Measurement was carried out in the same manner as in Comparative Example 2, except that a PBS buffer solution containing 3.3 mg/mL Heteroblock (manufactured by OMEGA Biologicals) was used as the sample pretreatment solution. The measurement results are shown in Table 2.
  • Example 2 Measurement by LTIA method: Addition of IgA-specific degrading enzyme to sample Measurement was carried out in the same manner as in Comparative Example 2, except that a PBS buffer solution containing 26.7 U/ ⁇ L of IgASAP (manufactured by Genovis) was used as the sample pretreatment solution. The measurement results are shown in Table 2.
  • Comparative Example 3 Measurement by LTIA Method: Addition of Pepsin to Sample Measurement was carried out in the same manner as in Comparative Example 2, except that a PBS buffer solution containing 3.3 mg/mL Pepsin (manufactured by Roche, dissolved in 0.2 M citrate buffer, pH 3.0) was used as the sample pretreatment solution. The measurement results are shown in Table 2.
  • Example 3 Measurement by LTIA method: Addition of IgA-specific degrading enzyme and commercially available non-specific reaction inhibitor (Heteroblock) to sample Measurement was performed in the same manner as in Comparative Example 2, except that a PBS buffer solution containing 26.7 U/ ⁇ L IgASAP (manufactured by Genovis) and 3.3 mg/mL Heteroblock (manufactured by OMEGA Biologicals) was used as the sample pretreatment solution. The measurement results are shown in Table 2.
  • Comparative Example 4 Measurement by LTIA Method Addition of Pepsin and Commercially Available Nonspecific Reaction Inhibitor (Heteroblock) to Sample Measurement was performed in the same manner as in Comparative Example 2, except that a PBS buffer solution containing 3.3 mg/mL Pepsin (manufactured by Roche, dissolved in 0.2 M citrate buffer, pH 3.0) and 3.3 mg/mL Heteroblock (manufactured by OMEGA Biologicals) was used as the sample pretreatment solution. The measurement results are shown in Table 2.
  • IgA-specific enzyme in immunoassays could suppress nonspecific reactions originating from samples. Furthermore, it showed a nonspecific reaction suppression effect even for divergent samples for which the addition of Pepsin was not sufficiently effective.
  • IgA-specific enzyme can also be used in combination with commercially available nonspecific reaction inhibitors, and is found to be extremely useful compared to Pepsin.
  • the divergent samples used in this test were samples for which existing non-specific reaction inhibitors such as anti-IgA antibodies could not provide sufficient inhibitory effects, and non-specific reactions were successfully suppressed for the first time by the non-specific reaction inhibition method of the present invention using an IgA-specific degrading enzyme.

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Abstract

L'invention concerne un procédé de mesure immunologique permettant de mesurer une substance cible dans un échantillon, une réaction antigène-anticorps étant mise en oeuvre au moins une fois en présence d'une enzyme qui décompose spécifiquement l'immunoglobuline A, abrégée en IgA.
PCT/JP2025/007073 2024-02-29 2025-02-28 Procédé de mesure immunologique, réactif pour mesure immunologique, liquide de prétraitement d'échantillon pour mesure immunologique, kit de réactif pour mesure immunologique et inhibiteur de réaction non spécifique Pending WO2025183146A1 (fr)

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JP2025531141A JP7736395B1 (ja) 2024-02-29 2025-02-28 免疫学的測定方法、免疫学的測定用試薬、免疫学的測定用検体前処理液、免疫学的測定用試薬キット、及び非特異反応抑制剤
JP2025133646A JP2025156581A (ja) 2024-02-29 2025-08-08 免疫学的測定方法、免疫学的測定用試薬、免疫学的測定用検体前処理液、免疫学的測定用試薬キット、及び非特異反応抑制剤

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