Classifications

• • 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

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.
• One example of a measurement method in the field of diagnostic drugs is an immunological measurement method that uses an antigen-antibody reaction to measure a substance to be measured that exists in a biological sample. Since the immunological measurement method uses an antigen-antibody reaction, it is a measurement method with very high specificity. Various substances exist in biological samples, and measurement errors occur when substances other than the substance being measured cause non-specific binding reactions or interfere with specific antigen-antibody reactions. Such phenomena are called non-specific reactions, and substances that cause non-specific reactions are called non-specific factors. As non-specific factors, the presence of heterophilic antibodies and rheumatoid factors (RF) has been clarified.
• RF rheumatoid factors
• Heterophilic antibodies are a general term for human antibodies that show reactivity to animal-derived antibodies that are the main reaction in immunological measurement methods, and human anti-mouse immunoglobulin antibodies (HAMA) are known as representative examples.
• Rheumatoid factors are glycoproteins that are frequently identified in patients with collagen diseases such as rheumatoid arthritis, chronic infectious diseases, and liver diseases, and share the characteristic of showing reactivity to animal-derived antibodies with HAMA. Both of these factors are known to be human immunoglobulin G or immunoglobulin M (hereinafter referred to as IgM) (Non-Patent Documents 1 and 2).
• Patent Document 1 discloses a method for suppressing non-specific 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 non-specific reactions by adding a polyclonal antibody against natural IgM antibodies prepared from the same animal species as the antibody used in the measurement.
• the anti-human IgM polyclonal antibody, anti-human IgG polyclonal antibody, and anti-human IgA polyclonal antibody shown in Patent Document 1 are currently in practical use as various reagents to suppress non-specific reactions caused by natural antibodies and RF, but it has become clear that this method is not sufficient to suppress non-specific reactions.
• the polyclonal antibodies against IgM-type natural antibodies shown in Patent Document 2 must be prepared from the same animal species as the antibodies used in the measurement, and there are limitations to the preparation method and the antibodies against the substance to be measured.
• the reducing agent shown in Patent Document 3 which is used to suppress non-specific reactions caused by interfering substances with a structure in which polypeptide chains are disulfide-bonded, may cleave disulfide bonds in the antigen or antibody that is the substance to be measured, or in the antigen or antibody contained in the reagent components, depending on the type and concentration, making it less versatile. Furthermore, in methods that use reducing agents, there is a possibility that IgG and IgA will be decomposed in addition to IgM. For this reason, this method could not be used, particularly when human IgG is the substance to be measured.
• the object of the present invention is to provide an immunological measurement method capable of suppressing non-specific reactions caused by IgM contained in the measurement sample.
• the inventors investigated the effect of various substances on suppressing non-specific reactions, and discovered that non-specific reactions can be suppressed by carrying out an antigen-antibody reaction in the presence of an enzyme that specifically degrades IgM, which led to the completion of the present invention.
• the present invention has the following configuration.
• An immunoassay method for measuring a substance to be measured in a sample comprising carrying out at least one antigen-antibody reaction in the presence of an enzyme that specifically degrades immunoglobulin M.
• the reagent for immunoassay according to [8], wherein the enzyme is a protease.
• a reagent kit for use in an immunological measurement method for measuring a substance to be measured in a sample comprising an enzyme that specifically degrades IgM.
• the enzyme is an enzyme that cleaves a part of the IgM constant region including CH ⁇ 1 to CH ⁇ 4.
• reagent kit for immunoassay according to any of [22] to [24], wherein the enzyme acts on IgM in the sample to degrade the IgM into an F(ab') 2 fragment and an Fc fragment.
• a non-specific reaction inhibitor comprising an enzyme that specifically degrades IgM.
• the present invention provides an immunological measurement method that can suppress non-specific reactions caused by IgM 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 to be 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 IgM.
• the method is a method in which an enzyme that specifically degrades IgM 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 measurement 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 IgM, and non-specific reactions in the reaction solution are suppressed during the reaction.
• 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 a 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 a B/F separation operation, washes away and removes excess components that were not involved in the binding reaction, and then allows the binding reaction to proceed to detect the substance to be measured.
• the heterogeneous method has the disadvantage that it requires many steps and takes a long time to measure because it requires a washing process, but it has the advantage that it is relatively less susceptible to the effects of non-specific reaction substances.
• the homogeneous method does not require a washing process and therefore has the disadvantage that it is more susceptible to the effects of non-specific reactions, but it has fewer steps, is simple, and requires less time to measure, so it is a method that is widely in demand in the field of clinical diagnosis.
• IA immunoagglutination assays
• TIA turbidimetric immunoassays
• immunochromatography lateral flow and flow-through
• IA immunoagglutination assays
• TIA is a method for qualitatively or quantitatively detecting an analyte in a sample based on the degree of agglutination of immune complexes formed by the cross-linking of the analyte (substance to be measured) by a specific binding partner such as an antibody.
• LTIA latex turbidimetric immunoassays
• 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 because they are expected to be more effective, and among homogeneous methods, latex immunoturbidimetric assay is the most preferred.
• One aspect of the present invention includes a method for performing an immunological measurement using an enzyme that specifically degrades IgM, or a non-specific reaction inhibitor containing the enzyme (hereinafter, the enzyme that specifically degrades IgM may be abbreviated as IgM-specific degrading enzyme).
• the IgM-specific degrading enzyme may be any enzyme that can specifically degrade IgM, and examples of such enzymes include proteases.
• proteases examples include proteinases (endopeptidases) and peptidases (exopeptidases).
• the enzyme that degrades IgM is preferably an enzyme that cleaves a part of the constant region of IgM including C H ⁇ 1 to C H ⁇ 4 (for example, the constant region of Ser124 to Tyr576 of human IgM: SEQ ID NO: 1), and more preferably capable of degrading the IgM into F(ab') 2 and Fc fragments.
• the IgM-specific degrading enzyme only needs to have the function of specifically degrading IgM 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 synonymously, 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 antigen immobilization ELISA, competitive ELISA, sandwich ELISA, etc., as well as by a method utilizing the principle of surface plasmon resonance (SPR method).
• SPR method can be performed using equipment, sensors, and reagents commercially available under the name Biacore (registered trademark).
• the IgM-specific degrading enzyme of the present invention specifically degrades IgM.
• the IgM-specific degrading enzyme can degrade IgM more efficiently than immunoglobulins other than IgM, such as IgG and IgA. Therefore, even if the substance to be measured is IgG, or even if the binding partner that specifically binds to the substance to be measured is IgG, the IgM-specific degrading enzyme can be used without affecting the specific measurement of the substance to be measured.
• the IgM-specific degrading enzyme can degrade IgM derived from organisms such as humans, mice, rats, rabbits, goats, ostriches, and pigs, and when it is desired to specifically degrade human IgM, it is desirable to use the human IgM-specific degrading enzyme.
• the IgM-specific degrading enzyme specifically degrades IgM means that, for example, after reacting IgG or IgM with the enzyme for 30 to 60 minutes at 37°C, the amount of IgM remaining is 50% or less, 40% or less, or 30% or less, preferably 20% or less, and more preferably 10% or less, compared to IgG.
• the IgM-specific degrading enzyme used in the present invention may be either a naturally occurring enzyme or a recombinant enzyme produced by genetic recombination technology.
• naturally occurring enzymes include enzymes produced by various bacteria and fungi, such as enzymes produced by streptococci, Legionella bacteria, Candida, etc.
• Preferred examples include immunoglobulin M-degrading enzyme of S. suis (Ig d Ssuis ) produced by streptococcus Streptococcus suis (Seele et al., J. Bacteriol., 2013; 195, 930-940) or amino acid mutants of said enzyme, IgMBRAZOR (Genovis) or amino acid mutants of said enzyme, etc.
• the above-mentioned enzyme may be a recombinant enzyme produced as a recombinant protein.
• recombinant enzyme variants having the desired enzyme activity can be produced (JP 2019-506866 A, EP 3148576 A). Therefore, in the present invention, various IgM-specific decomposition enzymes can be used to carry out a method for inhibiting nonspecific reactions in immunological assays, or to produce nonspecific reaction inhibitors.
• methods for making an enzyme that specifically degrades IgM present in an antigen-antibody reaction system include using it as one of the reagent components of an immunological measurement reagent, and 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 IgM-specific degrading enzyme may be used, or the IgM-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.
• the immunoassay reagent when the immunoassay reagent is a liquid reagent, it refers to the liquid phase where the sample and the liquid immunoassay reagent are mixed and the antigen-antibody reaction takes place.
• the sample may be mixed with an immunological measurement reagent containing an IgM-specific degrading enzyme, or the sample may be mixed in advance with a specimen pretreatment solution containing an IgM-specific degrading enzyme and then mixed with the LTIA measurement reagent.
• the sample may be mixed with an IgM-specific degrading enzyme beforehand and then dropped onto the microplate, or the sample may be mixed with a solution containing an IgM-specific degrading 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 IgM-specific degrading enzyme and then mixed with the immunoassay reagent, or the immunoassay reagent (e.g., a solution containing a detection antibody or detection antigen, and magnetic particles) may contain an IgM-specific degrading enzyme.
• 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 mixed in advance with a specimen pretreatment liquid containing an IgM-specific decomposition enzyme and then dropped onto the immunochromatography test piece, or the IgM-specific decomposition enzyme may be dried and held on a member such as a sample pad (sample supply site), and when the sample is dropped, the IgM-specific decomposition enzyme dissolves and develops the solid phase, becoming present in the reaction system.
• the concentration of the IgM-specific degrading enzyme may be any concentration that does not strongly affect the antigen-antibody reaction between the substance to be measured and the specific binding partner and that can exert the desired effect of suppressing non-specific reactions, and can be appropriately set by a person skilled in the art depending on the type of substance to be measured and the type of sample.
• the concentration of the IgM-specific degrading enzyme in the antigen-antibody reaction system varies depending on the reagent composition of the antigen-antibody reaction system. For example, when adding an IgM-specific degrading enzyme to a specimen pretreatment solution, 1 to 1000 U of enzyme can be added per 10 ⁇ L of specimen, preferably 5 to 800 U, more preferably 10 to 500 U, even more preferably 20 to 300 U, and most preferably 50 to 200 U.
• the concentration of the 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 10 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.
• the IgM-specific degrading enzyme may be used alone or in combination with other substances that have the effect of suppressing non-specific reactions.
• substances that have the effect of suppressing other non-specific reactions include anti-IgM 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. Note that substances that have the effect of suppressing other non-specific reactions are not limited to those listed here.
• the anti-IgM antibody and the enzyme may be directly or indirectly bound to each other.
• an IgM-specific degrading enzyme When an IgM-specific degrading enzyme is to be added to the immunoassay reagent of the present invention, it is preferable to add it to the assay reagent in advance so that the concentration is in the above-mentioned reaction system.
• LTIA Latex immunoturbidimetric assay
• the first method involves reacting the target substance with latex particles that have been immobilized with a specific binding partner for the target substance to form a sandwich-type immune complex, and measuring the target substance based on the degree of agglutination of the latex particles that accompanies the formation of the immune complex.
• the second method involves adding a protein or the like to which multiple substances to be measured or their analogs (including fragments of these) are immobilized to an immunoassay reagent, allowing these to compete with the substances to be measured in the sample, inhibiting the formation of immune complexes between the substances to be measured contained in the reagent and latex particles to which specific binding partners for the substances to be measured are immobilized, and measuring the substances to be measured (such as antigens) from the degree of inhibition of agglutination of the latex particles that accompanies the inhibition of immune complex formation.
• the substance to be measured and the specific binding partner for the substance to be measured may be any of proteins, peptides, sugar chains, lipids, glycoproteins, glycolipids, nucleic acids, low molecular weight compounds, and high molecular weight compounds, so long as they 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.
• step (1) a step of adding latex particles carrying a specific binding partner for the substance to be measured to the solution
• step (2) a step of 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 measurement target substance and the latex particles during or after step (2) without going through a washing/separation step.”
• the LTIA method can measure the target substance by optically observing the degree of agglutination that has occurred.
• optical observation methods include methods that use optical equipment to measure scattered light intensity, absorbance, or transmitted light intensity (endpoint method, rate method, etc.).
• the concentration (quantitative value) of the target substance contained in the sample is calculated by comparing the measured value of absorbance, etc. obtained by measuring a sample with the measured value of absorbance, etc. obtained by measuring a standard substance (a sample with a known concentration of the target substance).
• the measurement of absorbance, etc. 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 (dedicated machine).
• the measurement is performed by a method that involves 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 the latex particles by known methods such as physical adsorption, chemical binding, or a combination of these.
• the specific binding partner for the substance to be measured and the 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 the carrier, according to a known method.
• the chemical binding method it can be carried out according to known methods described in, for example, "Clinical Pathology Special Issue No.
• the synthetic polymers constituting 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: for example, styrene-methacrylic acid copolymers, styrene-acrylic acid copolymers, styrene-itaconic acid copolymers, and vinyl naphthalene polymers.
• styrene-methacrylic acid copolymers preferred are styrene-methacrylic acid copolymers, styrene-itaconic acid copolymers, and styrene and styrene-styrene sulfonate copolymers.
• the specific binding partners for the substance to be measured carried by the latex particles are preferably of multiple types in order to form a sandwich. If the substance to be measured has multiple antibody recognition sites, the specific binding partner may be of one type. For example, if the specific binding partner is a monoclonal antibody, multiple monoclonal antibodies with different recognition sites are used. Also, for example, if the specific binding partner is a polyclonal antibody, it may be a polyclonal antibody derived from one type of antiserum, or may be derived from multiple types of antisera. Also, a combination of monoclonal and polyclonal antibodies may be used.
• the surface of the latex particles may be blocked (masked) by a known method, such as by contacting and coating the surface 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.
• buffers such as acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonic acid, phthalic acid, succinic acid, maleic acid, imidazole, Good's buffer, and sodium salts, potassium salts, calcium salts, etc.
• polymers such as polyvinylpyrrolidone and phospholipid polymers may be contained as components for enhancing the agglutination formation of immunoassay particles.
• the concentration of the IgM-specific degrading enzyme in the constituent reagents may 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 varies depending on the type of reagent.
• the immunoassay reagent kit of the present invention is characterized in that it contains at least an IgM-specific degrading enzyme in its kit configuration. Therefore, the reagent kit of the present invention contains an IgM-specific degrading enzyme in one or more of the following components in addition to the reagents involved in the antigen-antibody reaction that constitute the kit: a sample dilution solution, a sample pretreatment solution including a sample extract, etc.
• the kit configuration may also include an instruction manual and a sample collection tool (a collection pipette, a syringe, a cotton swab, a filtration filter, etc.).
• the reagent compositions for each immunological measurement method will be described below.
• Latex immunoturbidimetric assay The following is an example of a reagent when the immunological measurement method is latex immunoturbidimetry (LTIA reagent), but is not limited thereto.
• a first reagent containing an IgM-specific decomposition enzyme (1) a first reagent containing an IgM-specific decomposition 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 IgM-specific degrading enzyme in the buffer solution may be adjusted to the preferred enzyme concentration in the mixed state of the reagent and sample at the time of measurement, and varies depending on the type of reagent.
• the enzyme may be contained in the second reagent in addition to the first reagent.
• the concentration of the IgM-specific degrading enzyme contained in the first reagent is generally 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 this concentration.
• the reaction temperature of the IgM-specific enzyme with the sample may be any temperature at which the IgM-specific enzyme is not inactivated and can specifically decompose IgM, and is in the range of 0 to 50°C, preferably 10 to 40°C, and more preferably 20 to 40°C, but is not limited to these temperatures.
• reaction time of the IgM-specific enzyme with the sample may be any reaction time that allows the IgM to be specifically and sufficiently decomposed by the IgM-specific enzyme, 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 IgM-specific degrading enzyme may be any pH at which the IgM-specific degrading enzyme functions sufficiently, and is in the range of pH 1 to 12, preferably 3 to 10, more preferably 5 to 9, and most preferably 6 to 8, but is not limited to this pH range.
• any known particles can be used as the particles for immunoassays in the present invention, so 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 immunological measurement can be appropriately selected from the range of 0.05 to 1 ⁇ m so as to obtain the desired measurement sensitivity and measurement range, taking into consideration the optical measurement method to be used (e.g., turbidimetry that measures transmitted light, nephelometry that measures scattered light, etc.). Note that, in optical measurements using automatic analyzers, an average particle size of 0.1 to 0.4 ⁇ m is generally used, but is not limited to this.
• the ELISA method is a method that utilizes a combination of various antigen-antibody reactions, and finally incorporates an enzyme-labeled antigen or antibody into the reaction system to detect enzyme activity.
• a substrate whose absorption spectrum changes with the reaction is used to detect enzyme activity, and the ELISA method includes the direct method, indirect method, sandwich method, competitive method, etc., depending on the combination of antigen-antibody reactions.
• immunoassay reagents when the immunoassay method of the present invention is a sandwich ELISA method.
• the labeling substance can be appropriately selected and used.
• the antibody immobilized on the insoluble carrier captures the substance to be measured in the solution containing the sample, and forms a complex on the insoluble carrier.
• the antibody labeled with the labeling substance binds to the captured substance to be measured and forms a sandwich with the complex.
• the substance to be measured in the sample can be measured by measuring the amount of the labeling substance by a method appropriate for the labeling substance. Specific methods such as a method for immobilizing the antibody on the insoluble carrier and a method for binding the antibody to the labeling substance can be any method known to those skilled in the art without any particular restrictions.
• the IgM-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 takes place.
• Immunochromatographic test strips When an antibody is used as the specific binding partner, the test piece is provided on a sheet-like insoluble carrier such as a porous membrane and is equipped with the following components in the order of the direction in which a solution containing a sample is developed: "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.
• the substance to be measured can then be detected by detecting the label by any method (for example, in the case of a visualized label such as gold colloid, its agglutination image, or in the case of an enzyme, a color reaction by adding a substrate).
• the IgM-specific degrading enzyme of the present invention can be present in the reaction system, for example, by adding it to a specimen pretreatment solution or by containing it in a sample supply site or a labeled antibody holding site and drying and holding it there.
• an enzyme it is called a chemiluminescent enzyme immunoassay (CLEIA method).
• a metal complex such as a ruthenium pyridine complex
• ELIA method electrochemiluminescent immunoassay
• a chemiluminescent substance is used as the label, it is called a chemiluminescent immunoassay (CLIA method).
• immunoassay reagents when the immunoassay method of the present invention is the CLEIA method.
• Magnetic particles to which an antibody (or antigen) that reacts with the substance to be measured is immobilized;
• Luminescent Reagent The antibody immobilized on the magnetic particles captures the analyte in the solution containing the sample to form a complex.
• the antibody labeled with the enzyme labeling substance binds to the captured analyte to form a sandwich with the complex.
• the analyte in the sample can be measured by reacting the enzyme labeling substance with the luminescent reagent and measuring the amount of luminescence.
• the IgM-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 takes place.
• the specific binding partner for the measured substance may be any substance capable of specifically binding to the measured substance, and examples of such specific binding partners include proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, haptens, low molecular weight compounds, and high molecular weight compounds. Furthermore, there are no particular limitations on the molecular weight and origin, such as natural or synthetic, and examples of specific binding partners include antibodies or antigens that can be used in immunological measurement methods that utilize antigen-antibody reactions.
• the antibody may be a polyclonal antibody or a monoclonal antibody. More preferably, it is 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, stool, or cell or tissue extracts.
• Blood is the most preferable sample containing a substance to be measured. It is generally known that the standard value of IgM contained in blood is 33 to 190 mg/dl for men and 46 to 260 mg/dl for women. A sample may be called a "specimen.”
• the immunoassay reagent of the present invention can measure various substances contained in the sample.
• the substances to be measured include proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, and haptens, but there is no particular limitation as long as they are theoretically measurable substances.
• C-reactive protein C-reactive protein
• lipoprotein a
• MMP3 matrix metalloproteinase 3
• antiphospholipid antibodies type IV collagen, prostate specific PSA, brain natriuretic peptide (BNP), insulin, albumin, cystatin C, rheumatoid factor (RF), KL-6, procalcitonin (PCT), fibrin and fibrinogen degradation products (FDP), D-dimer, soluble fibrin (SF), thrombin-antithrombin III complex (TAT), transferrin, haptoglobin, ⁇ 1-antitrypsin, ⁇ 1-acidoglycoprotein, ⁇ 2-macroglobulin, hemopexin, antithrombin- III, ⁇ -fetoprotein, carcinoembryonic antigen (CEA), ferritin, hepatitis B virus envelope s antigen (HBs-Ag), anti-hepatitis B virus envelope s antibody (Anti-HBs),
• the substances to be measured in the present invention include anti-Treponema pallidum antibodies, anti-cyclic citrullinated peptide (CCP) antibodies, anti-Helicobacter pylori antibodies, and antibodies such as IgG and IgA against viruses such as hepatitis, measles, and leukemia.
• these antibodies may also be decomposed by the reducing agent.
• the IgM-specific decomposing enzyme in the present invention does not decompose these antibodies. For this reason, the IgM-specific decomposing enzyme is particularly useful for the technology described in Patent Document 3 when the substance to be measured is an antibody.
• suppressing non-specific reactions means acting on factors (also called non-specific factors, non-specific causative substances, or non-specific reactive substances) that cause the above-mentioned non-specific reactions in biological samples and suppressing the influence of reactions other than antigen-antibody reactions on the measurement.
• factors also called non-specific factors, non-specific causative substances, or non-specific reactive substances
• whether a candidate substance as a non-specific reaction inhibitor has an effect of suppressing non-specific reactions can be determined by comparing the measured value (hereinafter referred to as the control method measured value) when measured using, for example, a measurement method with B/F separation (a method with a washing step and less susceptible to the influence of non-specific reactive substances (in the examples, the LBA method or the CLEIA method)) with and without the addition of the candidate substance and whether it approaches the control method measured value.
• a measurement method with B/F separation a method with a washing step and less susceptible to the influence of non-specific reactive substances (in the examples, the LBA method or the CLEIA method)
• the candidate substance has a non-specific reaction suppressing effect 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 so-called positive measurement errors, in which the measured substance is judged to have a higher content than its actual content, and factors that cause so-called negative measurement errors, in which the measured substance is judged to have a lower content than its actual value, due to some component contained in a biological sample.
• it is particularly effective against non-specific factors that cannot be suppressed by commercially available non-specific reaction inhibitors such as HBR-1. It is also effective against non-specific factors that cause positive measurement errors, in which the measured value becomes abnormally high, and negative measurement errors, in which the measured value becomes abnormally low, so-called deviation samples.
• various causative substances that cause non-specific reactions can be mentioned, but it is preferable to degrade causative substances that have a structure similar to at least part, all, or part or all of immunoglobulin M (IgM) with an IgM-specific degrading enzyme. Through this degradation reaction, the non-specific reaction inhibitor of the present invention can suppress non-specific reactions derived from the causative substances.
• IgM immunoglobulin M
• the non-specific reaction inhibitor of the present invention only needs to contain a substance capable of suppressing reactions caused by non-specific factors derived from a sample, as determined above, and contains at least an IgM-specific degrading enzyme as an active ingredient.
• the non-specific reaction inhibitor of the present invention can adopt a configuration in which an IgM-specific degrading enzyme is contained in the above-mentioned immunological measurement reagent as the configuration of the non-specific reaction inhibitor.
• 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., to the extent that the non-specific reaction inhibitory effect of the IgM-specific degrading enzyme is not hindered.
• Other non-specific reaction inhibitors may have a non-specific reaction inhibitory effect, and include, but are not limited to, anti-IgM 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 anti-IgM antibody and the enzyme may be directly or indirectly bound to each other.
• the method for suppressing non-specific reactions of the present invention is a method for suppressing non-specific reactions caused by a sample by carrying out an antigen-antibody reaction at least once in the presence of an IgM-specific degrading enzyme.
• the method for suppressing non-specific reactions can also be referred to as a method for reducing measurement errors.
• PCT measurement> The concentration of procalcitonin (PCT) contained in the sample (specimen) was measured by the LTIA method as follows. Human serum specimens 1 and 2 from multiple individuals (2 individuals) were used as samples. Specimen 1 (control specimen) is a specimen whose measured value by the LTIA method is close to the measured value by the LBA method (Liquid-phase Binding Assay) (Reference Example 1). Specimen 2 (deviation specimen) is a specimen that exhibits a non-specific reaction and whose measured value by the LTIA method is significantly deviated from the measured value by the LBA method in Reference Example 1. The IgM concentrations in each specimen were 62 mg/dL in specimen 1 and 88 mg/dL in specimen 2.
• First Reagent A first reagent base solution having the following composition was prepared. ⁇ 100mM Bis-Tris-HCl (pH 6.5) ⁇ 600mM NaCl 0.2% BSA
• Second reagent 5 mM MOPS-NaOH (pH 7.0) - Anti-human PCT monoclonal antibody sensitized latex (2 types)
• the anti-human PCT monoclonal antibody was obtained by a method known to those skilled in the art using a commercially available PCT antigen.
• the anti-human PCT monoclonal antibody-sensitized latex was prepared with reference to the method described in JP 2017-181377 A.
• an anti-human PCT monoclonal antibody solution diluted to 0.36 mg/mL with an equivalent amount of 5 mM Tris-HCl (pH 8.5) was added to a 1.0% latex solution (5 mM Tris buffer solution (hereinafter referred to as Tris-HCl or simply Tris) (pH 8.5)) having an average particle size of 0.3 ⁇ m, and the mixture was stirred. Then, an equivalent amount of 5 mM Tris-HCl (pH 8.5) containing 0.5% BSA was added and stirred to prepare an anti-human PCT monoclonal antibody-sensitized latex particle solution.
• the latex particle solution was diluted with 5 mM MOPS-NaOH (pH 7.0) so that the absorbance at 600 nm became about 5.5 OD, and used as the second reagent.
• Samples PBS buffer which is a sample pretreatment solution, was added to the specimens (serum) 1 and 2 described in Reference Example 1 at a volume ratio of 5:1, and the samples were reacted at 37° C. for 30 minutes and measured.
• Example 1 Measurement by LTIA method: Addition of IgM-specific degrading enzyme to sample 1. Measurement method Measurement was performed in the same manner as in Comparative Example 1, except that a PBS buffer solution containing 40 U/ ⁇ L IgM BRAZOR (Genovis), an IgM-specific degrading enzyme, was used as the sample pretreatment solution.
• Measurement Results The measurement results are shown in Table 1. The units of the values of the measurement results are ng/mL.
• control sample 1 (sample 1) was verified.
• the measured values were roughly equivalent in Reference Example 1, Example 1, and Comparative Example 1.
• sample 2 The measurement results of the deviation sample (sample 2) were verified.
• the measured value of sample 2 was 0.10 ng/mL in Reference Example 1.
• the measured value of Comparative Example 1 was 1.68 ng/mL, which deviated from the measured value of Reference Example 1.
• the measured value of Example 1 was 0.02 ng/mL, which showed a tendency to approach that of Reference Example 1. From the above, it was possible to suppress non-specific reactions originating from the sample by using an IgM-specific degrading enzyme in an immunological measurement method.
• Comparative Example 2 Measurement by LTIA method Addition of semi-alkaline protease to sample Measurement was performed in the same manner as in Example 1, except that proteinase from Aspergillus melleus, 5G (Sigma-Aldrich) diluted with PBS buffer to 0.4 mg/mL was used as the sample pretreatment solution. The measurement results are shown in Table 2.
• Comparative Example 3 Measurement by LTIA method: Addition of semi-alkaline protease to sample Measurement was performed in the same manner as in Comparative Example 2, except that the concentration of the enzyme contained in the specimen pretreatment solution was changed to 2.0 mg/mL. The measurement results are shown in Table 2.
• Comparative Example 4 Measurement by LTIA method: Addition of Proteinase K to sample The measurement was performed in the same manner as in Comparative Example 2, except that the enzyme used was changed to Proteinase K, recombinant, PCR grade, lyophilized from Pichia pastoris (manufactured by Roche) and diluted with PBS buffer to a concentration of 0.4 mg/mL. The measurement results are shown in Table 2.
• Comparative Example 5 Measurement by LTIA method: Proteinase K added to sample Measurement was performed in the same manner as in Comparative Example 4, except that the concentration of the enzyme contained in the sample pretreatment solution was changed to 2.0 mg/mL. The measurement results are shown in Table 2. The units of the measurement results shown in Table 2 are ng/mL.
• sIL-2R soluble interleukin-2 receptor
• the concentration of soluble interleukin-2 receptor (sIL-2R) contained in the sample (specimen) was measured by the LTIA method as follows. Human serum specimens 4 to 6 from multiple individuals (3 individuals) were used as samples. Specimen 4 (control specimen) is a specimen whose measured value by the LTIA method is close to the measured value by the chemiluminescence enzyme immunoassay (CLEIA method) (Reference Example 2). Specimens 5 and 6 (deviation specimens) are specimens that exhibit a non-specific reaction and whose measured value by the LTIA method is significantly different from the measured value by the CLEIA method in Reference Example 2. The IgM concentrations in each specimen were 76 mg/dL in specimen 4, 71 mg/dL in specimen 5, and 532 mg/dL in specimen 6.
• Measurement by CLEIA method 1 Measurement method 1-1. Measurement reagent Lumipulse Presto (registered trademark) IL-2R (Fujirebio Inc.) 1-2. Samples Specimens 4 to 6 1-3. Measurement procedure Measurements were performed using Lumipulse (registered trademark)-L2400 (Fujirebio Inc.) according to the instructions attached to the measurement reagent.
• the measurement results are shown in Table 3.
• the CLEIA method shown in Reference Example 2 includes a B/F separation step and a washing step, and is therefore a measurement method that is less susceptible to the effects of non-specific reactions derived from the sample.
• Measurement by LTIA method PBS buffer added to sample (no additives) 1.
• Measurement method 1-1 Measurement reagents The first and second reagents were prepared according to the method described in JP 2017-181377 A. 1-2. Sample pretreatment solution PBS buffer (pH 7.4) was used. 1-3. Samples Samples (serum) 4 to 6 described in Reference Example 2 were added with PBS buffer solution as a sample pretreatment solution at a volume ratio of 5:1, and the samples were reacted at 37° C. for 1 hour and measured. 1-4. Measurement procedure The sample, first reagent, and second reagent of each specimen were mixed, and the sIL-2R concentration in the sample was measured using a Hitachi 7180 automatic analyzer.
• the first reagent 120 ⁇ L was added to 5.6 ⁇ L of the sample, and the mixture was incubated at 37° C. for 5 minutes. Then, 40 ⁇ L of the second reagent was added and stirred. The change in absorbance associated with the formation of aggregates was measured over the next 5 minutes at a main wavelength of 570 nm and a sub-wavelength of 800 nm. The amount of change in absorbance was applied to a calibration curve obtained by measuring a standard substance of known concentration, and the measured value was calculated.
• Example 2 Measurement by LTIA method: Addition of IgM-specific decomposition enzyme to sample Measurement was performed in the same manner as in Comparative Example 6, except that a PBS buffer solution containing 40 U/ ⁇ L IgM BRAZOR (Genovis) was used as the sample pretreatment solution. The measurement results are shown in Table 3.
• Example 2 and Comparative Example 6 From the results of Reference Example 2, Example 2 and Comparative Example 6, the effect of the IgM-specific degrading enzyme in inhibiting non-specific reactions was discussed.
• the measurement results of the control sample (sample 4) were verified. In the control samples, the measured values were roughly equivalent in Reference Example 2, Example 2, and Comparative Example 6. These results demonstrate that the addition of IgM BRAZOR does not affect the measured values of samples that do not exhibit nonspecific reactions.
• the measurement results of the deviation samples (samples 5 and 6) were verified. The measured value of sample 5 was 306 U/mL in Reference Example 2. The measured value of Comparative Example 6 was 556 U/mL, which deviated from the measured value of Reference Example 2.
• Example 2 was 408 U/mL, which showed a tendency to approach the measured value of Reference Example 2. A similar tendency was obtained for sample 6.
• the measured value of sample 6 was 591 U/mL in Reference Example 2.
• the measured value of Comparative Example 6 was 905 U/mL, which deviated from the measured value of Reference Example 2.
• the measured value of Example 2 was 830 U/mL, which tended to approach the measured value of Reference Example 2. From the above, it was possible to suppress non-specific reactions derived from samples by using an IgM-specific degrading enzyme in an immunological measurement method.
• the degraded samples used in this test were samples for which existing non-specific reaction inhibitors such as anti-IgM antibodies were unable to provide a sufficient suppression effect, and it was only by the method of the present invention for suppressing non-specific reactions using an IgM-specific degrading enzyme that it was possible to suppress non-specific reactions.

Landscapes

• Health & Medical Sciences (AREA)
• Immunology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Hematology (AREA)
• Urology & Nephrology (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Cell Biology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Peptides Or Proteins (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Priority Applications (2)

Applications Claiming Priority (2)

Publications (1)

Family

ID=94819073

Family Applications (1)

Country Status (3)

Citations (16)

Family Cites Families (2)

• 2024
  • 2024-08-26 JP JP2025543445A patent/JPWO2025047641A1/ja active Pending
  • 2024-08-26 CN CN202480039860.8A patent/CN121311766A/zh active Pending
  • 2024-08-26 WO PCT/JP2024/030136 patent/WO2025047641A1/ja active Pending

Patent Citations (16)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events