Classifications

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  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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  • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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  • 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
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
• • 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
  • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• • 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
  • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• • 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
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
• • 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
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/322—2'-R Modification
• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/323—Chemical structure of the sugar modified ring structure
  • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

• the present invention relates to a non-specific reaction inhibitor for suppressing non-specific reactions that hinder accurate detection and quantification of trace substances in immunoassay methods.
• Japanese Patent Application Laid-Open No. 11-287801 discloses that as a method for suppressing non-specific reactions, antibodies against non-specific reactive substances are allowed to coexist in measurement reagents to reduce non-specific reactions caused by non-specific reactive substances in the sample.
• IgG or IgM is used as an antibody against a non-specific reaction substance
• immune complex formation with the non-specific reaction substance causes an immune nephelometric reaction
• latex immunonephelometry is used for accurate measurement.
• the value may not be indicated.
• As a countermeasure against this problem if the amount of IgG or IgM added to the non-specific reaction substance is reduced, there are cases where a sufficient non-specific suppression effect cannot be obtained.
• IgG is fragmented into F(ab′) 2 to reduce the hydrophobicity, allowing a large amount to be added to the reagent and making the immunological nephelometric reaction less likely to occur. can.
• the cause was presumed to be the attenuation of the non-specific inhibitory effect due to the decomposition of F(ab') 2 into Fab'.
• the low non-specific inhibitory effect of Fab' indicates the possibility that the molecular size is insufficient.
• the inventors discovered that even a very small peptide of about 1 kDa can be efficiently inhibited against non-specificity by binding a PEG of about 5 kDa to a peptide of about 5 kDa, even though the molecular weight is smaller than that of Fab'. effect.
• a nonspecific reaction inhibitor for immunoassay comprising a complex of a peptide or nucleic acid molecule that specifically binds to a nonspecific reaction substance and a polymer compound.
• the nonspecific reaction inhibitor of [1] wherein the substance that specifically binds to the nonspecific reaction substance is a substance that can be chemically synthesized.
• the nonspecific reaction inhibitor according to [1] or [2], wherein the complex is 50 kDa or less.
• the nonspecific reaction inhibitor according to any one of [1] to [4], wherein the nonspecific reaction substance is immunoglobulin.
• An immunoassay method characterized by using a complex of a peptide or nucleic acid molecule that specifically binds to a non-specific reactant and a polymer compound.
• the immunoassay is latex agglutination optical assay, immunonephelometry, immunoturbidimetry, chemiluminescence immunoassay, enzyme immunoassay, fluorescence immunoassay, or radioimmunoassay , the immunological assay method of [6].
• the cost is lower than that of biological raw materials, and because chemical synthesis is possible, lot differences can be reduced.
• the present invention since it has a lower molecular weight, it has high solubility, and it is possible to secure a sufficient amount to be added.
• FIG. 4 is an explanatory diagram explaining each step of measurement by Octet (trademark) R2.
• the nonspecific reaction inhibitor of the present invention includes a complex of a peptide or nucleic acid molecule (hereinafter sometimes referred to as a binding partner) that specifically binds to a nonspecific reaction substance and a polymer compound.
• a binding partner a peptide or nucleic acid molecule
• non-specific reactive substance refers to a substance that induces a non-specific reaction in an immunoassay method that utilizes an antigen-antibody reaction.
• Specific factors include human IgM, human IgG, human IgA, human IgE, human IgD, and factors that bind to the antibody, such as complement, rheumatoid factor, Fc receptor, etc., when human body fluid is used as a sample. Examples include IgM, IgG, IgA, and IgE of the animal, and factors that bind to the antibody when the body fluid of an animal other than human is used as the sample.
• immunoassay examples include latex agglutination optical assay, immunonephelometry, immunoturbidimetry, chemiluminescence immunoassay, enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, and the like. mentioned. Assays without a B/F separation step, such as latex agglutination optical assays, immunonephelometry, and immunoturbidimetric assays, are particularly susceptible to non-specific reactants. In such a measurement method, the concentration of the test sample during the antigen-antibody reaction is often about several percent.
• the nonspecific reaction inhibitor of the present invention ensures a sufficient amount to be added. be able to. All of them utilize an antigen-antibody reaction, but antibodies that detect target antigens include polyclonal antibodies and monoclonal antibodies. In any measurement method, the nonspecific reaction inhibitor of the present invention may be added to the test sample before or during the antigen-antibody reaction, preferably before the antigen-antibody reaction. should be.
• Binding partners that specifically bind to non-specific reactive substances used in the present invention include peptides or nucleic acid molecules (e.g., aptamers).
• antibodies or Fab' or higher antibody fragments are used. It is characterized by not doing it.
• the molecular weight of said binding partner is typically 0.5-47 kDa, preferably 0.8-15 kDa, more preferably 1-8 kDa.
• the solubility is high and a sufficient addition amount can be ensured. According to the present invention, it is possible to increase the amount of the non-specific reaction inhibitor added in the reaction system in which immune complexes are formed. It is possible, and it is also possible to increase the concentration of the test sample in the reaction system.
• the binding partner used in the present invention is preferably a substance that can be chemically synthesized. Since the present invention does not use an antibody or an antibody fragment, it is possible to provide a non-specific reaction inhibitor that is inexpensive and has little lot variation.
• the binding partner can appropriately contain functional groups for polymer compound modification, biotin, spacers, and the like.
• the nonspecific reaction inhibitor of the present invention (that is, a complex of a binding partner of a nonspecific reaction substance and a polymer compound) includes, for example, a chemically modified peptide or nucleic acid molecule chemically modified with a polymer compound, or , a complex of a peptide or nucleic acid molecule and a macromolecular compound bound by a biotin-avidin bond.
• a thiol group, an amino group, a hydroxyl group, or a carboxyl group can be targeted and bound via a "reactive derivative.”
• "Reactive derivatives" used in thiol-targeted modifications include, for example, thiol-selective reactive groups such as maleimides and vinyl sulfones.
• a polymer in which a reactive derivative is directly bound may be used, or a cross-linking agent containing a reactive derivative may be used.
• Reactive derivatives used in modification targeting amino groups include, for example, N-hydroxysuccinimide (NHS) esters, N-hydroxysulfosuccinimide (Sulfo-NHS) esters, and the like. Further, there are compounds containing an aldehyde group (for example, glutaraldehyde) and polymers that contain an aldehyde group in advance. Carboxyl-targeted modifications include, for example, conjugates obtained by catalyzed reaction of carbodiimide (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride) with amino groups.
• target hydroxyl groups can be prepared, for example, using compounds containing isocyanate derivatives.
• the polymer into which the reactive derivative has been introduced may be obtained as a commercial product (for example, NOF CORPORATION), or may be prepared using an ordinary chemical method.
• the present invention also includes a method that utilizes a binding mode with high affinity, such as biotin and avidin, where the binding mode between the binding partner and the polymer is not a covalent bond.
• polysaccharides include dextran, dextrin, agarose, carboxymethyl (CM)-cellulose, heparin, soluble starch, and the like.
• Polysaccharides may be linear or branched.
• For modification with polysaccharides conventionally known periodate oxidation method, cyanogen bromide method, carbodiimide method, cyanuric chloride method, epichlorohydrin method, SPDP (N-Succinimidyle 3-[2-pyridyldithio]propionate ) reagent method, active ester method, etc. can be used.
• Polysaccharides into which reactive derivatives have been introduced may be obtained as commercial products, or may be prepared using ordinary chemical methods.
• the above protein is a complex in which a plurality of amino acids are bound by peptide bonds, and may be purified from animals, artificially prepared by genetic engineering, or synthetic peptides. It may be prepared by chemical synthesis such as Proteins include, for example, casein, milk casein, gelatin, recombinant albumin, and the like. Polyamino acids include homopolymers such as arginine, lysine and glutamic acid, and random polymers such as lysine and glycine and lysine and serine.
• a protein binding method there is a method in which amino groups, carboxyl groups, sulfide groups, or the like of a protein are targeted to be bound with a cross-linking agent, and the protein is bound to a binding partner via the cross-linking agent. There is also a method of binding a binding partner to a protein using carbodiimide as a catalyst.
• the amino group of the protein is reacted with a cross-linking agent such as EMCS [N-(6-Maleimidocaproyloxy) succinimide; Dojin] or SMCC [succinimdyl 4-(N-maleimidomethyl)cyclohexane carbonate;
• EMCS N-(6-Maleimidocaproyloxy) succinimide
• Dojin or SMCC
• a method of binding the cross-linking agent to a binding partner into which a sulfide group has been introduced is preferred.
• the protein into which a reactive derivative has been introduced, which is used for preparing the product of the present invention may be obtained as a commercial product, or may be prepared using a conventional chemical method.
• organic polymers examples include polyethylene glycol, polyvinyl alcohol, polyacrylic alcohol, polyethyleneimine, polymethyl methacrylate, polyacrylic acid, polyallylamine, and polysaccharide.
• the organic high molecular weight polymer may be linear or branched, or may be a random copolymer of multiple types.
• a synthetic polymer having a spherical structure such as a dendrimer may be used.
• the polymer may be synthetic or naturally derived.
• Polyethylene glycol is a polymer compound based on a structure in which ethylene glycol is polymerized. Another functional group can be introduced into the hydroxyl group of polyethylene glycol, and the functional group can be used to bind to the antibody. Activation performed when binding polyethylene glycol to an antibody includes, for example, a method using cyanuric chloride, carbodiimidazole, N-hydroxysuccinimide, carbodiimide, and the like. A commercially available product can be used as polyethylene glycol into which a functional group has been introduced. Efficient preparation can be achieved by using commercially available products in which a maleimide group, a succimido group, an amino group, a sulfide group, or the like is introduced into polyethylene glycol.
• Polyethylene glycol into which a maleimide group or a succimido group has been introduced is more preferable because it has good binding efficiency with a binding partner having a terminal sulfide group or amino group.
• polyethylene glycol may be straight chain or branched chain, polyethylene glycol partially substituted with other chemical structure, or polyethylene glycol modified with other polymer or compound. including those that were
• a method of chemical modification with an organic high molecular weight polymer other than polyethylene glycol there is a method of introducing a functional group into an organic high molecular weight polymer and binding it to an antibody, similar to the above method of chemical modification of polyethylene glycol.
• an organic high molecular weight polymer that already contains a reactive derivative it may not be necessary to introduce a new functional group into the organic high molecular weight polymer in the preparation stage.
• the organic high molecular weight polymer into which a reactive derivative has been introduced may be obtained as a commercial product, or may be prepared using a normal chemical method.
• the molecular size of the polymer compound is not particularly limited, but generally the average molecular weight is about 200Da to 1000kDa, for example 1kDa to 1000kDa, preferably 10kDa to 100kDa.
• polyethylene glycol it is preferably between 3 kDa and 49.5 kDa.
• the molecular size can be appropriately selected in consideration of hydrophilicity, steric structure, non-specific suppression effect, and the like.
• the non-specific reaction inhibitor of the present invention can be used by adding a complex of its active ingredient, a binding partner and a polymer compound, to an immunoassay system. Specifically, before preparing a solution of a binding partner modified with a polymer compound and reacting an antibody against an antigen to be measured with the antigen, the solution is added to the sample in advance to obtain a non-specific reaction substance. By reacting with a binding partner against it, a non-specific reaction by a non-specific reaction substance may be suppressed, or a binding property modified with a polymer compound may be added to a solution of an antibody against the antigen to be measured. A non-specific reaction due to a non-specific reaction substance may be suppressed by adding a partner and adding this solution to the sample to allow the non-specific reaction substance and its binding partner to react.
• the immunological measurement reagent of the present invention may be a conventional immunological measurement reagent that utilizes an antigen-antibody reaction and further contains the non-specific reaction inhibitor of the present invention. It consists of the above. When it consists of one liquid, it consists of a reaction reagent containing at least a support carrying an antigen or an antibody for forming an immunological complex. A sample is reacted with the reagent and a signal is detected according to known methods.
• the non-specific reaction inhibitor of the present invention is preferably added to the reagent before the reaction between the sample and the reagent, but is preferably supplied in the state of being added to the reagent.
• the stabilizing reagent is a reagent that dilutes the sample to an appropriate concentration or performs pretreatment, and can be prepared according to a known method.
• the sample is reacted with a stabilizing reagent, followed by a reaction reagent, and the signal is detected according to known methods.
• the non-specific reaction inhibitor of the present invention is preferably added to the stabilizing reagent and/or the reaction reagent before the reaction between the sample and the reaction reagent, but preferably the stabilizing reagent and/or Alternatively, it may be supplied in a state of being added to the reaction reagent. Particularly preferably, it is supplied in a state of being added to the stabilizing reagent.
• Measurement items of immunological measurement reagents include elastase, cystatin C, sEs (soluble E selectin), SF (soluble fibrin), PC (protein C), PPI (plasmin plasmin inhibitor), cTn (thrombomodulin), myoglobin.
• CK-MB BNP (B-type natriuretic peptide), NT-proBNP (N-terminal proBNP), cTnI (cardiac troponin I), AFP ( ⁇ -fetoprotein), ⁇ 2m ( ⁇ -2-microglobulin), CEA ( carcinoembryonic antigen), ferritin, CA19-9 (carbohydrate antigen 19-9), PAP (prostatic acid phosphatase), PSA (prostate specific antigen), CRP (C-reactive protein), Mb (myoglobin), PCT (procalcitonin), preseptin, IL-2R (interleukin-2 receptor), RF (rheumatoid factor), ASO (antistreptolysin-O), FDP (fibrin degradation product), ATIII (antithrombin III), TAT (thrombin antithrombin III complex), plasminogen, ⁇ 2PI ( ⁇ -2-plasmin inhibitor), D-dimer (fibrin degradation product
• a chemically synthesized peptide or nucleic acid aptamer was used as a small molecule that binds to a non-specific reaction substance, and polyethylene glycol (hereinafter referred to as PEG) was used as a polymer compound that modifies it.
• PEG polyethylene glycol
• peptides include a known human IgG-binding peptide of about 1.5 kDa (amino acid sequence: DCAWHLGELVWCT: SEQ ID NO: 1) for the purpose of purifying human IgG, and a reported about 1.5 kDa peptide for the purpose of purifying human IgA.
• a .8 kDa human IgA-binding peptide (amino acid sequence: HMVCLSYRGRPVCFSL: SEQ ID NO: 2) was used, and PEG having a succinimide group at one end (molecular weight: 20 kDa; manufactured by NOF CORPORATION) was used.
• Example 2 Confirmation of the effect of suppressing reaction to human IgG or human IgA with or without PEG modification>>
• As a model system to confirm the non-specific inhibitory effect using anti-human IgG antibody and human IgG, or anti-human IgA antibody and human IgA, IgG-binding peptide or IgA-binding peptide having a smaller molecular weight than the antibody fragment, depending on the presence or absence of PEG modification A reaction suppression effect was confirmed.
• an anti-human IgG polyclonal rabbit antibody (manufactured by Dako) was diluted with PBS( ⁇ ) to 25 ⁇ g/mL to prepare an anti-human IgG antibody solution.
• the sample solution was human IgG adjusted to 1 mg/mL in PBS(-), and 20 ⁇ L of this test sample was added with an IgG-binding peptide or a 20 kDa PEG-modified IgG-binding peptide at a final concentration of 25 ⁇ g/mL or 50 ⁇ g.
• a sample solution and an anti-human IgG antibody solution were added to predetermined wells in a 96-well black plate (manufactured by Greiner Bio-One) at 200 ⁇ L/well. 200 ⁇ L/well of PBS(-) was added to the baseline, dissociation and washing wells.
• This plate and a biosensor (Biosensor/ProL: manufactured by SARTRIUS) were placed at predetermined positions of OctetR2. After acquiring data by operating OctetR2 under the conditions shown in Table 1 below, the value of change in wavelength shift (Response) was calculated using analysis software attached to OctetR2. In addition, the measurement was implemented at the temperature of 30 degreeC.
• an anti-human IgA polyclonal rabbit antibody (manufactured by Dako) was diluted with PBS( ⁇ ) to 25 ⁇ g/mL to prepare an anti-human IgA antibody solution.
• the sample solution is human IgA adjusted to 250 ⁇ g / mL with PBS (-) as a test sample, and 20 ⁇ L of this test sample is added with an IgA-binding peptide or an IgA-binding peptide modified with 20 kDa PEG at a final concentration of 25 ⁇ g / mL or 50 ⁇ g. /mL, and prepared so that the total amount of R1 was 200 ⁇ L was used.
• Measurement with OctetR2 was performed in the same manner as confirmation of the effect of suppressing the response to human IgG.
• Table 2 shows the inhibitory effect on human IgG
• Table 3 shows the inhibitory effect on human IgA.
• the reason why the measured value increased by adding a peptide not modified with PEG was that the peptide bound to human IgG or human IgA, but the binding of anti-human IgG antibody and human IgG or anti-human IgA antibody and human IgA increased.
• human IgG bound with IgG-binding peptide binds to anti-human IgG antibody
• human IgA bound with IgA-binding peptide binds to anti-human IgA antibody, non-specific reaction inhibitor It was considered that the thickness of the layer at the tip of the sensor increased and the wavelength shift changed more than when only human IgG or human IgA was bound to anti-human IgG antibody or anti-human IgA antibody when not added.
• Example 3 Confirmation of non-specific reaction suppression effect with or without PEG modification>> Using human plasma exhibiting non-specific reactions, the effect of suppressing non-specific reactions with or without PEG modification was confirmed for IgG-binding peptides with molecular weights smaller than those of antibody fragments.
• a sample A which is human plasma exhibiting a non-specific reaction in LPIA Genesis TAT (manufactured by LSI Rulece) and whose specific reactant is IgG, was used. Measurements were performed under the same assay conditions as described in Example 2. In this example, the sample A and the non-specific reaction inhibitor coexist in the measurement sample, and the non-specific reaction substance is absorbed in the sample solution.
• the antibody solution used in the second reagent of LPIA genesis TAT (hereinafter referred to as R2) is characterized by containing latex particles on which a monoclonal antibody having a specific binding ability to TAT is immobilized.
• An anti-TAT antibody solution prepared by diluting the anti-TAT antibody described in the above with PBS( ⁇ ) to a concentration of 25 ⁇ g/mL was used.
• 20 ⁇ L of a test sample diluted 10-fold with PBS( ⁇ ) is added with an IgG-binding peptide or a 20-kDa PEG-modified IgG-binding peptide as a non-specific reaction inhibitor at a final concentration of 25 ⁇ g/mL or 50 ⁇ g/mL.
• the total amount was adjusted to 200 ⁇ L with R1 and used.
• Table 4 shows the results.
• the PEG-modified IgG-binding peptide exhibited a greater effect of suppressing non-specific reactions than the non-modified IgG-binding peptide.
• the PEG-modified IgG-binding peptide although having a smaller molecular weight than the antibody fragment, exhibited a non-specific reaction suppressing effect.
• the reason why the measured value increased by adding the peptide not modified with PEG is that the IgG-binding peptide binds to the non-specific reaction substance, but the stericity of the IgG-binding peptide is such that it prevents the binding of the non-specific reaction substance and the anti-TAT antibody. No damage occurs, and the IgG-binding peptide-bound non-specific reaction substance binds to the anti-TAT antibody, so that only the non-specific reaction substance binds to the anti-TAT antibody when the non-specific reaction inhibitor is not added. , it was thought that the thickness of the layer at the tip of the sensor increased and the change in the wavelength shift increased.
• Example 4 Comparison of effects of PEG-modified peptide and antibody fragment>> From the results of Examples 2 and 3, it was clarified that the PEG-modified peptide exhibits an effect of suppressing non-specific reaction even if the molecular weight is smaller than that of the antibody fragment. This overturns the report in Japanese Patent No. 5189098 that the antibody fragment has a small non-specific reaction suppressing effect due to its small molecular weight, and that the non-specific reaction suppressing effect is exhibited by modifying and enlarging it with PEG. It can be said that it is a great discovery. Therefore, the effect of suppressing the non-specific reaction was compared between the case of modifying the peptide that binds to the non-specific reaction substance with PEG and the case of modifying the antibody fragment that binds to the non-specific reaction substance with PEG.
• Fab' that reacts with human IgG
• a PEG-modified antibody fragment was prepared by chemically modifying a 20 kDa PEG containing a maleimide group at the end.
• Goat anti-human IgG polyclonal IgG purified from antiserum manufactured by International Immunology Corporation
• F(ab') was pepsin-digested to prepare F(ab')
• TCEP-HCL at 37°C for 210 minutes.
• Fab' were prepared.
• 20 kDa PEG (manufactured by NOF CORPORATION) having a maleimide group attached thereto was added to a 5 mg/mL Fab' solution, and reacted at 37° C. for 30 minutes.
• the reaction solution was concentrated to about 5 mg/mL by ultrafiltration and buffer-substituted with 0.2 mol/L TBS to obtain PEG-modified anti-human IgG Fab'.
• the PEG-modified peptides described in Examples 2-3 and the prepared PEG-modified antibody fragments were compared for their non-specific reaction inhibitory effects.
• the amount of the non-specific reaction inhibitor added was 25 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL, 200 ⁇ g/mL or 400 ⁇ g/mL, and the same sample A as in Example 3 was used as the test sample.
• PEG-modified IgG-binding peptides were prepared by the method described in Example 1, chemically modified with 5 kDa, 10 kDa, 20 kDa, and 30 kDa PEGs containing succinimide groups at their ends.
• Table 7 shows the results. The effect of suppressing non-specific reaction was exhibited even when modified with PEG of any molecular weight. Moreover, it was confirmed that the effect increases depending on the molecular weight of PEG.
• TAT or non-specific reaction substances in the non-specimen were quantified.
• a non-specific reaction inhibitor was added to R1 so that the concentration after addition of the sample was 0.05 mg / mL or 0.1 mg / mL, and the non-specific reaction substance was absorbed during the first operation. It was decided to.
• the same volume of PBS(-) was added to R1 to which the non-specific reaction inhibitor was not added to avoid the influence of dilution due to the additive.
• the mixing ratio of the measurement sample, R1 and R2 was 20 ⁇ L:90 ⁇ L:90 ⁇ L. Latex aggregation was detected at a wavelength of 700 nm. The measured value was calculated by comparing the absorbance from a calibration curve obtained by measuring TAT with a known concentration.
• the same sample A as in Examples 3-5 was used as the test sample.
• Table 8 shows the results.
• the non-specific inhibitory effect of the PEG-modified IgG-binding peptide could also be confirmed in the latex agglutination method.
• the reason why the measured value increased by adding peptides not modified with PEG is that the IgG-binding peptide binds to a non-specific reaction substance, but steric hindrance is enough to prevent the binding of the non-specific reaction substance and the anti-TAT antibody.
• Example 7 Confirmation of reaction suppression effect on anti-human IgG antibody and human IgG with or without PEG modification of IgG-binding nucleic acid aptamer>>
• the nucleic acid aptamer the reaction inhibitory effect depending on the presence or absence of PEG modification was confirmed using a model system for confirming the non-specific inhibitory effect in the same manner as in Example 2.
• nucleic acid aptamer is an approximately 7.5 kDa human IgG-binding nucleic acid aptamer (nucleic acid sequence: N(6)_ggAGGU(F)GcU(F)ccgaaaGGA(L)aC(F)U) that has been reported for the purpose of purifying human IgG.
• Test samples and measurements were performed under the same assay conditions as in Example 2.
• a test sample and a non-specific reaction inhibitor were allowed to coexist in a measurement sample, and human IgG was absorbed in the sample solution.
• the measurement was performed according to the operation manual attached to OctetR2.
• an antibody solution an anti-human IgG polyclonal rabbit antibody (manufactured by Dako) was diluted with PBS( ⁇ ) to 25 ⁇ g/mL to prepare an anti-human IgG antibody solution.
• Table 9 shows the results.
• the IgG-binding nucleic acid aptamer had a greater effect of suppressing the reaction between the anti-human IgG antibody and human IgG than when not modified.
• the IgG-binding nucleic acid aptamer has a molecular weight of about 7.5 kDa, and even when modified with 20 kDa PEG, the molecular weight is 27.5 kDa, which is smaller than that of the antibody fragment, but showed the effect of suppressing the reaction.
• the nonspecific reaction inhibitor of the present invention can be applied to immunoassays.
• amino acid sequences represented by SEQ ID NOs: 1 and 2 in the sequence listing are human IgG-binding peptide and human IgA-binding peptide, respectively.

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