Nonspecific reaction inhibitor

Abstract

Provided is a nonspecific reaction inhibitor that has high solubility, is capable of ensuring a sufficient addition amount, is inexpensive, and has small variation among different lots. This nonspecific reaction inhibitor comprises a conjugate of a polymer compound and a peptide or nucleic acid molecule specifically bonding to a nonspecific reaction substance.

Description

Non-specific reaction inhibitors

[0001] 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.

[0002] Japanese Patent Application Laid-Open No. 11-287801 discloses a method for suppressing nonspecific reactions, in which an antibody against a nonspecific reaction substance is coexisted in a test reagent to reduce nonspecific reactions caused by the nonspecific reaction substance in the sample. However, this technique has the problem that when IgG or IgM is used as the antibody against the nonspecific reaction substance, an immune nephelometric reaction occurs due to the formation of an immune complex with the nonspecific reaction substance, which can lead to inaccurate measurement values ​​in latex immunoturbidimetry. To address this problem, the amount of IgG or IgM added to the nonspecific reaction substance was reduced, but this sometimes resulted in insufficient nonspecific suppression effect.

[0003] To eliminate the immunological nephelometric reaction, IgG was converted to F(ab'). ２ By fragmenting the antibody into F(ab') and reducing its hydrophobicity, it becomes possible to add a large amount to the reagent and to make it less likely to cause an immunological nephelometric reaction. However, there is a problem with the durability of the nonspecific inhibitory effect in the reagent, and the cause is the F(ab') ２ It was predicted that this was due to the weakening of the nonspecific inhibitory effect caused by degradation of Fab' to Fab'. Japanese Patent No. 5189098 suggests that the low nonspecific inhibitory effect of Fab' may be due to an insufficient molecular size, and to solve this problem, it discloses a method of modifying antibody fragments such as Fab' with a polymer compound to enlarge them, thereby enhancing the nonspecific inhibitory effect.

[0004] Japanese Patent Application Laid-Open No. 11-287801 Patent No. 5189098

[0005] However, in recent years, there has been a demand for more sensitive immunoassays. As the amount of sample used increases in an effort to increase the sensitivity of reagents, the amount of nonspecific reaction substances present in the reaction system increases significantly, necessitating the coexistence of large amounts of nonspecific reaction inhibitors in the reagent. Similarly, when a large amount of nonspecific substances is present in the sample, it is also necessary to coexist large amounts of nonspecific reaction inhibitors in the reagent. Under these circumstances, the technology of Patent Publication No. 5189098 uses relatively expensive biological raw materials such as antibodies and antibody fragments, which poses challenges in terms of cost and raw material lot variations. Furthermore, since the technology relies on the large molecular size, the effect of improving solubility in the reagent is insufficient. Therefore, when the sample amount is increased in an effort to achieve high sensitivity, the amount of inhibitor added is not sufficient to obtain a sufficient nonspecific reaction inhibitor effect.

[0006] In view of these circumstances, an object of the present invention is to provide a non-specific reaction inhibitor that is highly soluble, can be added in a sufficient amount, is inexpensive, and has little lot-to-lot variation.

[0007] As a result of extensive research aimed at achieving the above object, it was found that an efficient nonspecific reaction inhibitory effect can be achieved by modifying a peptide that has binding ability to nonspecific reaction substances and is even smaller in molecular weight than Fab' with a polymer such as polyethylene glycol (PEG). Fab' is a molecule with a molecular weight of approximately 55 kDa, but according to the disclosure of Japanese Patent No. 5189098, even a peptide with such a molecular weight loses its nonspecific reaction inhibitory effect. However, the inventors unexpectedly discovered that even a very small peptide of approximately 1 kDa can be efficiently inhibited by binding a PEG of approximately 5 kDa, despite its molecular weight being smaller than Fab'.

[0008] The above-mentioned problems of the present invention can be solved by the following inventions: [1] A non-specific reaction inhibitor for immunoassay, comprising a conjugate of a polymer compound and a peptide or nucleic acid molecule that specifically binds to a non-specific reaction substance. [2] The non-specific reaction inhibitor of [1], wherein the substance that specifically binds to a non-specific reaction substance is a chemically synthesizable substance. [3] The non-specific reaction inhibitor of [1] or [2], wherein the conjugate is 50 kDa or less. [4] The non-specific reaction inhibitor of any of [1] to [3], wherein the polymer compound is polyethylene glycol. [5] The non-specific reaction inhibitor of any of [1] to [4], wherein the non-specific reaction substance is immunoglobulin. [6] An immunoassay method, characterized by using a conjugate of a polymer compound and a peptide or nucleic acid molecule that specifically binds to a non-specific reaction substance. [7] The immunoassay method of [6], wherein the immunoassay method is latex agglutination optical assay, immunonephelometry, immunoturbidimetry, chemiluminescence immunoassay, enzyme immunoassay, fluorescence immunoassay, or radioimmunoassay. [8] An immunoassay reagent comprising a complex of a peptide or nucleic acid molecule that specifically binds to a non-specific reactant and a polymer compound. [9] The immunoassay method of [8], characterized in that the complex of a peptide or nucleic acid molecule that specifically binds to a non-specific reactant and a polymer compound is contained in the stabilizing reagent.

[0009] The present invention is cheaper than biological raw materials, and since chemical synthesis is possible, lot-to-lot variations can be reduced. Furthermore, since the present invention has a lower molecular weight, it has high solubility and can be added in sufficient amounts.

[0010] FIG. 1 is an explanatory diagram illustrating each step of measurement using Octet (trademark) R2.

[0011] The non-specific reaction inhibitor of the present invention comprises a complex of a polymer compound and a peptide or nucleic acid molecule (hereinafter sometimes referred to as a binding partner) that specifically binds to a non-specific reaction substance. As used herein, a "non-specific reaction substance" refers to a substance that induces a non-specific reaction in an immunological assay utilizing an antigen-antibody reaction. Specific factors include, when a human body fluid is used as a sample, human IgM, human IgG, human IgA, human IgE, human IgD, and factors that bind to these antibodies, such as complement, rheumatoid factor, and Fc receptor. When a body fluid of an animal other than a human is used as a sample, specific factors include the animal's IgM, IgG, IgA, IgE, and factors that bind to these antibodies.

[0012] Examples of the immunoassay include latex agglutination optical assay, immunonephelometry, immunoturbidimetry, chemiluminescence immunoassay, enzyme immunoassay, fluorescence immunoassay, and radioimmunoassay. Assays without a B / F separation step, such as latex agglutination optical assay, immunonephelometry, and immunoturbidimetry, are particularly susceptible to the influence of nonspecific reaction substances. In such assays, the concentration of the test sample during the antigen-antibody reaction is often only a few percent. The higher the test sample concentration, the greater the amount of nonspecific reaction inhibitor required to be added. Even when the test sample concentration in the reaction solution is higher than conventional concentrations (4% or more, 5% or more, or 8% or more), the nonspecific reaction inhibitor of the present invention can be added in sufficient amounts. Both assays utilize antigen-antibody reactions, and the antibodies used to detect the target antigens include polyclonal and monoclonal antibodies. In both assays, 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.

[0013] The binding partner used in the present invention that specifically binds to a non-specific reaction substance can be a peptide or a nucleic acid molecule (e.g., an aptamer), but the present invention is characterized in that it does not use an antibody or an antibody fragment of Fab' or larger. The molecular weight of the binding partner is typically 0.5 to 47 kDa, preferably 0.8 to 15 kDa, and more preferably 1 to 8 kDa. In the present invention, by using a binding partner with a molecular weight smaller than Fab', high solubility can be achieved and a sufficient amount can be added. According to the present invention, the amount of non-specific reaction inhibitor added in the reaction system in which immune complex formation occurs can be increased, thereby making it possible to accommodate test samples with high concentrations of non-specific reaction substances and to increase the concentration of the test sample in the reaction system.

[0014] 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 antibody fragment, it is possible to provide a nonspecific reaction inhibitor that is inexpensive and has little lot-to-lot variation. The binding partner can appropriately contain a functional group for modifying a polymer compound, biotin, a spacer, etc.

[0015] Examples of the non-specific reaction inhibitor of the present invention (i.e., a complex of a polymer compound and a binding partner of a non-specific reaction substance) include chemically modified peptides or nucleic acid molecules chemically modified with a polymer compound, and complexes of peptides or nucleic acid molecules and polymer compounds bound via a biotin-avidin bond.

[0016] The above-mentioned chemical modification can target, for example, thiol groups, amino groups, hydroxyl groups, or carboxyl groups and can be conjugated via a "reactive derivative." Examples of "reactive derivatives" used in modifications targeting thiol groups include thiol-selective reactive groups such as maleimides and vinyl sulfones. Alternatively, a polymer may be directly bonded to a reactive derivative, or a crosslinker containing a reactive derivative may be used. Examples of "reactive derivatives" used in modifications targeting amino groups include N-hydroxysuccinimide (NHS) esters and N-hydroxysulfosuccinimide (Sulfo-NHS) esters. Other examples include compounds containing aldehyde groups (e.g., glutaraldehyde) and polymers that already contain aldehyde groups. Examples of modifications targeting carboxyl groups include complexes obtained by reacting amino groups with carbodiimide (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride) as a catalyst. For example, a compound containing an isocyanate derivative can be used to prepare a polymer targeted at a hydroxyl group. Polymers incorporating reactive derivatives may be commercially available (e.g., from NOF Corporation) or may be prepared using conventional chemical techniques. The present invention also encompasses methods that utilize a high-affinity bond between a binding partner and a polymer, such as biotin and avidin, in which the bond between the binding partner and the polymer is not a covalent bond.

[0017] Examples of polymeric compounds that can be used in the present invention include polysaccharides, proteins, and organic polymers. Examples of polysaccharides include dextran, dextrin, agarose, carboxymethyl (CM)-cellulose, heparin, and soluble starch. Polysaccharides may be linear or branched. Modification with polysaccharides can be achieved by conventional methods such as the periodate oxidation method, cyanogen bromide method, carbodiimide method, cyanuric chloride method, epichlorohydrin method, SPDP (N-Succinimidyle 3-[2-pyridyldithio]propionate) reagent method, and active ester method. Polysaccharides with reactive derivatives may be commercially available or prepared using conventional chemical techniques.

[0018] The above-mentioned proteins are complexes in which multiple amino acids are linked by peptide bonds. They may be purified from animals, artificially prepared by genetic engineering, or prepared by chemical synthesis, such as synthetic peptides. Examples of proteins include casein, milk casein, gelatin, and recombinant albumin. Polyamino acids include homopolymers of arginine, lysine, and glutamic acid, as well as random polymers of lysine and glycine, and lysine and serine. Protein binding methods include binding crosslinkers targeting amino, carboxyl, and sulfide groups on proteins, and then binding the proteins to binding partners via the crosslinkers. Another method involves using carbodiimide as a catalyst to bind the proteins to binding partners. In the present invention, a preferred method involves reacting a crosslinker such as EMCS [N-(6-Maleimidocaproyloxy) succinimide; Dojin Co., Ltd.] or SMCC [succinimdyl 4-(N-maleimidomethyl)cyclohexane carbonate; Dojin Co., Ltd.] with the amino groups of a protein, and then binding the crosslinker to a binding partner having a terminal sulfide group. The protein having a reactive derivative introduced therein and used to prepare the product of the present invention may be obtained as a commercially available product, or may be prepared using conventional chemical techniques.

[0019] Examples of the organic high molecular weight polymer include polyethylene glycol, polyvinyl alcohol, polyacrylic alcohol, polyethyleneimine, polymethyl methacrylate, polyacrylic acid, polyallylamine, and polysaccharides. The organic high molecular weight polymer may be linear or branched, or may be a random copolymer of multiple types. It may also be a synthetic polymer with a spherical structure such as a dendrimer. The polymer may be synthetic or naturally derived.

[0020] Polyethylene glycol is a polymeric compound based on a polymerized structure of ethylene glycol. Other functional groups can be introduced into the hydroxyl groups of polyethylene glycol, and the functional groups can be used to conjugate the polyethylene glycol to an antibody. Activation methods for conjugating polyethylene glycol to an antibody include, for example, methods using cyanuric chloride, carbodiimidazole, N-hydroxysuccinimide, carbodiimide, etc. Commercially available polyethylene glycols with functional groups introduced can be used. Efficient preparation is possible using commercially available products in which maleimide groups, succinimide groups, amino groups, sulfide groups, etc., have been introduced into polyethylene glycol. Polyethylene glycols with maleimide groups or succinimide groups are more preferred, as they have good conjugation efficiency with binding partners having sulfide groups or amino groups at their termini. The polyethylene glycol may be linear or branched, and may include polyethylene glycols in which a portion of the polyethylene glycol is substituted with another chemical structure or polyethylene glycols modified with other polymers or compounds.

[0021] As a method for chemical modification with an organic polymer other than polyethylene glycol, there is a method similar to the above-mentioned method for chemical modification with polyethylene glycol, in which a functional group is introduced into an organic polymer and then bound to an antibody. Note that, in the case of an organic polymer that already contains a reactive derivative, it may not be necessary to introduce a new functional group into the organic polymer during the preparation stage. In the present invention, it is preferable to use a polymer into which a maleimide group, a succinimide group, an amino group, or a carboxyl group has been introduced. Note that the organic polymer into which a reactive derivative has been introduced may be obtained as a commercially available product or may be prepared using conventional chemical techniques.

[0022] The molecular size of the polymer compound is not particularly limited, but generally has an average molecular weight of about 200 Da to 1000 kDa, for example, 1 kDa to 1000 kDa, and preferably 10 kDa to 100 kDa. In the case of polyethylene glycol, the average molecular weight is preferably 3 kDa to 49.5 kDa. Depending on the type of polymer used, the molecular size can be appropriately selected taking into consideration hydrophilicity, three-dimensional structure, non-specific suppression effect, etc.

[0023] The non-specific reaction inhibitor of the present invention can be used by adding its active ingredient, a complex of a binding partner and a polymer compound, to an immunoassay system. Specifically, a solution of the binding partner modified with a polymer compound is prepared, and before reacting an antibody against the antigen to be measured with the antigen, the solution is added to the sample in advance to react the non-specific reaction substance with its corresponding binding partner, thereby suppressing the non-specific reaction caused by the non-specific reaction substance. Alternatively, the binding partner modified with a polymer compound may be contained in a solution of the antibody against the antigen to be measured, and this solution may be added to the sample to react the non-specific reaction substance with its corresponding binding partner, thereby suppressing the non-specific reaction caused by the non-specific reaction substance.

[0024] The immunoassay reagent of the present invention may be a conventional immunoassay reagent utilizing an antigen-antibody reaction, further comprising the nonspecific reaction inhibitor of the present invention, and may be composed of one or more liquids. When composed of one liquid, the reagent comprises a reaction reagent containing at least a support carrying an antigen or antibody for forming an immunological complex. A sample is reacted with the reagent according to a known method, and a signal is detected. The nonspecific reaction inhibitor of the present invention may be added to the reagent before the sample reacts with the reagent, but is preferably supplied in a state where it is added to the reagent. When composed of two or more liquids, the reagent comprises a stabilizing reagent and a reaction reagent containing at least a support carrying an antibody or antigen for forming an immunological complex. The stabilizing reagent is a reagent used to dilute or pretreat a sample to an appropriate concentration, and can be prepared according to a known method. According to a known method, a sample is reacted with the stabilizing reagent, and then with the reaction reagent, and a signal is detected. The non-specific reaction inhibitor of the present invention may be added to the stabilizing reagent and / or the reaction reagent before the reaction between the sample and the reaction reagent, and is preferably supplied in a state where it has been added to the stabilizing reagent and / or the reaction reagent, particularly preferably supplied in a state where it has been added to the stabilizing reagent.

[0025] Measurement items of immunoassay reagents include, for example, 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 (alpha-fetoprotein), β2m (beta-2-microglobulin), CEA (carcinoembryonic antigen), ferritin, and CA19-9 (carcinoembryonic antigen 19). -9), PAP (prostatic acid phosphatase), PSA (prostate-specific antigen), CRP (C-reactive protein), Mb (myoglobin), PCT (procalcitonin), presepsin, IL-2R (interleukin-2 receptor), RF (rheumatoid factor), ASO (antistreptolysin-O), FDP (fibrin degradation products), ATIII (antithrombin III), TAT (thrombin-antithrombin III complex), plasminogen, α2PI (α-2-plasmin inhibitor), D-dimer (fibrin degradation products D-f fragment dimer), IgG (immunoglobulin G), IgA (immunoglobulin A), IgM (immunoglobulin M), IgE (immunoglobulin E), C3 (third component of complement), C4 (fourth component of complement), urinary albumin, hCG (human chorionic gonadotropin), hPL (human placental lactogen), insulin, HBs antigen (hepatitis B surface antigen), HBs antibody (anti-hepatitis B surface antigen antibody), HBc antibody (anti-hepatitis B core antigen antibody), HCV antibody (anti-hepatitis C virus antibody), treponeme (anti-Treponema pallidum antibody), TSH (thyroid-stimulating hormone), L H (luteinizing hormone), FSH (follicle-stimulating hormone), prolactin, testosterone, estradiol, progesterone, digoxin, digitoxin, quinidine, procainamide, NAPA (N-acetylprocainamide), theophylline, phenytoin, phenobarbital, carbamazepine, valproic acid, ethosuximide, gentamicin, tobramycin, amikacin, zincomycin, cyclosporin A, B12 (vitamin B12), folic acid, T3 (triiodothyronine), T4 (thyroxine), and estrogen.

[0026] Chemically synthesized peptides or nucleic acid aptamers were used as small molecules that bind to non-specific reaction substances, and polyethylene glycol (hereinafter referred to as PEG) was used as the polymer compound to modify them. The present invention will be specifically explained below using examples, but these are not intended to limit the scope of the present invention.

[0027] Example 1 Preparation of PEG-Modified Peptides One molecule of PEG was conjugated to one molecule of peptide via the N-terminal amino group. The peptides used were a publicly known approximately 1.5 kDa human IgG-binding peptide (amino acid sequence: DCAWHLGELVWCT; SEQ ID NO: 1) used for the purification of human IgG, and a reported approximately 1.8 kDa human IgA-binding peptide (amino acid sequence: HMVCLSYRGRPVCFSL; SEQ ID NO: 2) used for the purification of human IgA. PEG used had a succinimide group at one end (molecular weight 20 kDa; manufactured by NOF Corporation).

[0028] [Preparation of PEG-modified IgG-binding peptide] Human IgG-binding peptide was dissolved in 0.2 mol / L NaHCO 3 to a concentration of 1.0 mg / mL. ３ After dissolving the peptide in PBS (pH 8.5), equimolar amounts of PEG bearing a succinimide group at one end were added to the peptide, and the mixture was allowed to react overnight at 4°C while stirring on a rotator. After the reaction, the mixture was passed through a human IgG affinity column using phosphate-buffered saline (PBS) as the running buffer to remove unreacted PEG. The mixture was eluted with 0.2 mol / L citrate buffer (pH 3.0), concentrated by ultrafiltration, and unreacted peptide was removed. The buffer was then replaced with PBS. The concentration was measured using a Quantitative Colorimetric Peptide Assay (Thermo Fisher) and the mixture was stored at 4°C.

[0029] [Preparation of PEG-modified IgA-binding peptide] Human IgA-binding peptide was dissolved in PBS (pH 7.4) containing 40% dimethyl sulfoxide (DMSO) to a concentration of 1.0 mg / mL. PEG bearing a succinimide group at one end was added in an equimolar amount to the peptide, and the mixture was allowed to react overnight at 4°C while stirring on a rotator. After the reaction, the mixture was passed through a human IgA affinity column using PBS as the running buffer to remove unreacted PEG. The mixture was eluted with 0.2 mol / L citrate buffer (pH 3.0), concentrated by ultrafiltration, and unreacted peptide was removed. The buffer was then replaced with PBS. Concentration was measured using a Quantitative Colorimetric Peptide Assay (Thermo Fisher) and the mixture was stored at 4°C.

[0030] Example 2 Confirmation of the Reaction Inhibitory Effect on Human IgG or Human IgA Depending on the Presence or Absence of PEG Modification As a model system for confirming the non-specific inhibitory effect, an anti-human IgG antibody and human IgG, or an anti-human IgA antibody and human IgA, was used to confirm the reaction inhibitory effect, depending on the presence or absence of PEG modification, for an IgG-binding peptide or an IgA-binding peptide with a smaller molecular weight than the antibody fragment.

[0031] [Method] Measurements were performed using Bio-Layer Interferometry (BLI) using the Octet™ R2 (SARTORIUS) biomolecular interaction analysis system. The basic principles of Bio-Layer Interferometry are briefly described below. When light of a specific wavelength is projected onto a layer of biomolecules immobilized on the sensor chip surface, light is reflected from both the biomolecular layer and an internal reference layer, generating an optical interference wave. When molecules in the measurement sample bind to the biomolecules on the sensor chip surface, the thickness of the layer at the sensor tip increases, causing a wavelength shift in the interference wave. By measuring this change in wavelength shift, quantification and kinetic analysis of the number of molecules binding to the biomolecules immobilized on the sensor chip surface can be performed in real time. In this example, the test sample and a nonspecific reaction inhibitor were coexisted in the measurement sample, and human IgG or human IgA was absorbed in the sample solution. Measurements were performed according to the operating manual provided with the Octet R2.

[0032] [Confirmation of the effect of suppressing the reaction to human IgG] An anti-human IgG polyclonal rabbit antibody (manufactured by Dako) was diluted with PBS(-) to 25 μg / mL to prepare an anti-human IgG antibody solution. The sample solution used was human IgG diluted to 1 mg / mL in PBS(-). To 20 μL of this test sample, an IgG-binding peptide or an IgG-binding peptide modified with 20 kDa PEG was added to a final concentration of 25 μg / mL or 50 μg / mL, and the resulting solution was adjusted to a total volume of 200 μL with the first reagent (hereinafter referred to as R1) of LPIA Genesis TAT ​​(manufactured by LSI Medience Corporation), a reagent for measuring thrombin-antithrombin III complex (hereinafter referred to as TAT).

[0033] A 96-well black plate (Greiner Bio-One) was filled with the sample solution and anti-human IgG antibody solution at 200 μL / well. PBS(-) was added to the baseline, dissociation, and washing wells at 200 μL / well. This plate and a biosensor (Biosensor / ProL: SARTRIUS) were placed in the designated positions of the OctetR2. The OctetR2 was operated under the conditions shown in Table 1 below to acquire data, and the wavelength shift change (Response) was calculated using the analysis software provided with the OctetR2. Measurements were performed at 30°C.

[0034]

[0035] [Confirmation of the inhibitory effect on human IgA] Anti-human IgA polyclonal rabbit antibody (manufactured by Dako) was diluted with PBS(-) to 25 μg / mL to prepare an anti-human IgA antibody solution. Human IgA at 250 μg / mL in PBS(-) was used as the test sample. To 20 μL of this test sample, an IgA-binding peptide or a 20 kDa PEG-modified IgA-binding peptide was added to a final concentration of 25 μg / mL or 50 μg / mL, and the total volume was adjusted to 200 μL using R1. Measurements using OctetR2 were performed in the same manner as for confirming the inhibitory effect on human IgG.

[0036] [Results] The inhibitory effect on human IgG is shown in Table 2, and the inhibitory effect on human IgA is shown in Table 3. When the IgG-binding peptide or IgA-binding peptide was modified with PEG, the inhibitory effect on the reaction between anti-human IgG antibody and human IgG, or between anti-human IgA antibody and human IgA, was greater than when it was not modified. Furthermore, the IgG-binding peptide or IgA-binding peptide was 1 to 2 kDa, and even when modified with 20 kDa PEG, the molecular weight was 21 to 22 kDa, which is smaller than that of the antibody fragment, but it was found that it still exhibited an inhibitory effect.

[0037] The reason why the addition of non-PEG-modified peptides increased the measured values ​​is that, although the peptides bound to human IgG or human IgA, no steric hindrance was generated that was sufficient to prevent the binding of anti-human IgG antibodies to human IgG or anti-human IgA antibodies to human IgA, and therefore human IgG bound to the IgG-binding peptide bound to the anti-human IgG antibody, and human IgA bound to the IgA-binding peptide bound to the anti-human IgA antibody. This is thought to have resulted in an increase in the thickness of the layer at the tip of the sensor, resulting in a larger change in wavelength shift than when only human IgG or human IgA binds to anti-human IgG or anti-human IgA antibodies when no non-specific reaction inhibitor is added.

[0038]

[0039]

[0040] Example 3 Confirmation of the effect of suppressing non-specific reactions with and without PEG modification Using human plasma that exhibits non-specific reactions, the effect of suppressing non-specific reactions with and without PEG modification was confirmed for an IgG-binding peptide with a smaller molecular weight than an antibody fragment.

[0041] [Method] Sample A, which is human plasma exhibiting a nonspecific reaction in LPIA Genesis TAT ​​(manufactured by LSI Medience Corporation), and which is characterized by IgG as a specific reaction substance, was used as the test sample. Measurement was performed using the same method as in Example 2. In this example, Sample A and a nonspecific reaction inhibitor were present in the measurement sample, and the nonspecific reaction substance was absorbed in the sample solution. The antibody solution used was an anti-TAT antibody solution prepared by diluting the anti-TAT antibody used in the second reagent (hereinafter referred to as R2) of LPIA Genesis TAT, which is characterized by containing latex particles to which a monoclonal antibody having specific binding ability to TAT is immobilized, with PBS(-) to a concentration of 25 μg / mL. The sample solution used was prepared by adding an IgG-binding peptide or a 20 kDa PEG-modified IgG-binding peptide as a non-specific reaction inhibitor to 20 μL of the test sample diluted 10-fold with PBS(-) to a final concentration of 25 μg / mL or 50 μg / mL, and adjusting the total volume to 200 μL using R1.

[0042] [Results] The results are shown in Table 4. Similar to the results of Example 2, the IgG-binding peptide was more effective in inhibiting non-specific reactions when modified with PEG than when not modified. Furthermore, although the PEG-modified IgG-binding peptide had a smaller molecular weight than the antibody fragment, it still exhibited the effect of inhibiting non-specific reactions.

[0043] The reason why the measured value increased upon addition of the non-PEG-modified peptide is that although the IgG-binding peptide bound to the non-specific reaction substance, no steric hindrance was generated that was sufficient to prevent the non-specific reaction substance from binding to the anti-TAT antibody, and the non-specific reaction substance bound to the IgG-binding peptide bound to the anti-TAT antibody, which is thought to have increased the thickness of the layer at the tip of the sensor and resulted in a larger change in wavelength shift than when only the non-specific reaction substance binds to the anti-TAT antibody without the addition of a non-specific reaction inhibitor.

[0044]

[0045] Example 4: Comparison of the effects of PEG-modified peptides and antibody fragments The results of Examples 2 and 3 revealed that PEG-modified peptides exhibit a non-specific reaction inhibitory effect even though they have a smaller molecular weight than antibody fragments. This is a new discovery that overturns the report in Japanese Patent No. 5189098 that antibody fragments have a small non-specific reaction inhibitory effect due to their small molecular weight, and that the non-specific reaction inhibitory effect can be exerted by modifying them with PEG to increase their size. Therefore, the non-specific reaction inhibitory effects were compared when a peptide that binds to a non-specific reaction substance was PEG-modified and when an antibody fragment that binds to a non-specific reaction substance was PEG-modified.

[0046] [Method] PEG-modified antibody fragments were prepared by chemically modifying Fab' reactive with human IgG with 20 kDa PEG containing a maleimide group at the terminus. Goat anti-human IgG polyclonal IgG (purified from antiserum manufactured by International Immunology Corporation) was digested with pepsin to prepare F(ab'), which was then reduced with 0.1 mol / L TCEP-HCl at 37°C for 210 minutes to prepare Fab'. 20 kDa PEG conjugated with a maleimide group (manufactured by NOF Corporation) was added to a 5 mg / mL Fab' solution and allowed to react at 37°C for 30 minutes. The reaction solution was concentrated to approximately 5 mg / mL by ultrafiltration, and the buffer was replaced with 0.2 mol / L TBS to obtain PEG-modified anti-human IgG Fab'.

[0047] The non-specific reaction inhibitory effects of the PEG-modified peptides described in Examples 2 and 3 and the prepared PEG-modified antibody fragments were compared using the same assay conditions as those described in Example 3. The amount of non-specific reaction inhibitor added was 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, or 400 μg / mL, and the same sample A as in Example 3 was used as the test sample.

[0048] [Results] The results are shown in Tables 5 and 6. When the added amounts were equal, the PEG-modified peptide had a higher effect of inhibiting nonspecific reactions than the PEG-modified antibody fragment. This demonstrates that even a small amount of the PEG-modified peptide has a sufficient effect of inhibiting nonspecific reactions.

[0049]

[0050]

[0051] Example 5: Confirmation of the effect of suppressing non-specific reactions when the molecular weight of the modifying PEG is increased. Japanese Patent No. 5189098 confirms that the effect of suppressing non-specific reactions increases as the molecular weight of the PEG used to modify an antibody fragment increases. Therefore, the effect of suppressing non-specific reactions of PEG-modified peptides was confirmed when the molecular weight of the modifying PEG was changed.

[0052] [Method] PEG-modified IgG-binding peptides chemically modified with PEGs of 5 kDa, 10 kDa, 20 kDa, and 30 kDa containing a succinimide group at the end were prepared by the method described in Example 1.

[0053] [Assay conditions for evaluating the effect of non-specific reaction inhibitors] The assay was carried out under the same conditions as those described in Example 3. Sample solutions containing PEG-modified peptides chemically modified with 5 kDa, 10 kDa, 20 kDa, and 30 kDa PEG were prepared as non-specific reaction inhibitors, and the non-specific reaction inhibitory effect of each was confirmed. The amount of non-specific reaction inhibitor added was 5 μg / mL, 10 μg / mL, or 20 μg / mL, and the test sample used was the same sample A as in Examples 3 and 4.

[0054] [Results] The results are shown in Table 7. Regardless of the molecular weight of the PEG used for modification, the effect of inhibiting nonspecific reactions was observed. It was also confirmed that the effect increased depending on the molecular weight of the PEG.

[0055]

[0056] Example 6: Confirmation of non-specific suppression effect when using an automatic analyzer The non-specific suppression effect when measuring by latex agglutination was confirmed using an automatic analyzer.

[0057] [Method] The nonspecific reaction inhibitory effect of the IgG-binding peptide and PEG-modified IgG-binding peptide used in Example 2 was confirmed by latex agglutination. Measurements were performed using an automated STACIA analyzer (manufactured by LSI Medience Corporation). Measurements using the STACIA involved two main steps. In the first step, the sample to be measured was diluted with R1 and heated at 37°C for 3.5 minutes. In the second step, R2 was added to the reaction solution, and the solution was heated at 37°C for 6 minutes to induce latex agglutination. The agglutination reaction was optically monitored to quantify TAT or nonspecific reaction substances in the non-analyte. In this example, a nonspecific reaction inhibitor was added to R1 so that the concentration after sample addition was 0.05 mg / mL or 0.1 mg / mL, and nonspecific reaction substances were absorbed during the first step. To R1, which did not contain a nonspecific reaction inhibitor, the same volume of PBS(-) was added to avoid the influence of dilution by additives. The mixture ratio of the measurement sample, R1, and R2 was 20 μL:90 μL:90 μL. Latex aggregation was detected at a wavelength of 700 nm. Measurement values ​​were calculated by comparing absorbance with a calibration curve obtained by measuring TAT with known concentrations. The test sample used was the same sample A as in Examples 3 to 5.

[0058] [Results] The results are shown in Table 8. The nonspecific reaction inhibitory effect of the PEG-modified IgG-binding peptide was also confirmed in the latex agglutination assay. The reason for the increase in measured values ​​due to the addition of unPEG-modified peptide is that, although the IgG-binding peptide bound to nonspecific reaction substances, no steric hindrance was generated to the extent that it prevented the binding of the nonspecific reaction substances to the anti-TAT antibody, and the nonspecific reaction substances bound to the IgG-binding peptide bound to the anti-TAT antibody. This is thought to have resulted in a change in absorbance due to the increase in the size or complexity of the aggregates compared to when only nonspecific reaction substances bind to the anti-TAT antibody without the addition of a nonspecific reaction inhibitor.

[0059]

[0060] Example 7: Confirmation of the reaction inhibitory effect of IgG-binding nucleic acid aptamer on anti-human IgG antibody and human IgG depending on whether it is PEG-modified or not As in Example 2, the reaction inhibitory effect of the nucleic acid aptamer depending on whether it is PEG-modified or not was confirmed using a model system for confirming the non-specific inhibitory effect.

[0061] [PEG-modified IgG-binding nucleic acid aptamer] The nucleic acid aptamer was a human IgG-binding nucleic acid aptamer of approximately 7.5 kDa (nucleic acid sequence: N(6)_ggAGGU(F)GcU(F)ccgaaaGGA(L)aC(F)U(F)cc (uppercase letters = RNA, lowercase letters = DNA, N(F) = 2'-Fluoro RNA, N(L) = LNA, N(6) = Amino C6) that has been reported for the purpose of purifying human IgG. The PEG-modified nucleic acid aptamer used was one molecule of nucleic acid aptamer to which one molecule of PEG was bound via the N-terminal amino group (nucleic acid sequence: P_N(6)_ggAGGU(F)GcU(F)ccgaaaGGA(L)aC(F)U(F)cc (uppercase letters = RNA, lowercase letters = DNA, N(F) = 2'-Fluoro RNA, N(L) = LNA, N(6) = Amino C6 linker, P = PEG (Jenkem ME 20kDa)).

[0062] [Method] The test sample and measurement were performed under the same assay conditions as in Example 2. In this example, the test sample and a nonspecific reaction inhibitor were allowed to coexist in the measurement sample, and human IgG was absorbed in the sample solution. Measurements were performed according to the operating manual provided with the OctetR2. Anti-human IgG polyclonal rabbit antibody (manufactured by Dako) was diluted with PBS(-) to 25 μg / mL to prepare an anti-human IgG antibody solution. Human IgG at 1 mg / mL in PBS(-) was used as the test sample. 20 μL of this test sample was added to an IgG-binding nucleic acid aptamer or a 20 kDa PEG-modified IgG-binding nucleic acid aptamer to a final concentration of 0.3 mg / mL, and the reaction buffer (145 mmol / L NaCl, 5.4 mmol / L KCl, 0.8 mmol / L MgCl) was added. ２ , 1.8 mmol / L CaCl ２, 20 mmol / L Tris (pH 7.6), 0.05% Tween 20) to a total volume of 200 μL.

[0063] [Results] The results are shown in Table 9. When the IgG-binding nucleic acid aptamer was modified with PEG, the inhibitory effect on the reaction between anti-human IgG antibody and human IgG was greater than when it was not modified. Furthermore, the IgG-binding nucleic acid aptamer was approximately 7.5 kDa, and even when modified with 20 kDa PEG, the molecular weight was 27.5 kDa, which is smaller than the molecular weight of the antibody fragment, but still showed the inhibitory effect.

[0064]

[0065] The non-specific reaction inhibitor of the present invention can be used in immunological measurements.

[0066] The amino acid sequences represented by SEQ ID NOs: 1 and 2 in the sequence listing are human IgG-binding peptides and human IgA-binding peptides, respectively.

Claims

1. A non-specific reaction inhibitor for immunoassay, comprising a complex of a peptide or nucleic acid molecule that specifically binds to a non-specific reaction substance and a polymer compound.

2. 2. The non-specific reaction inhibitor according to claim 1, wherein the peptide or nucleic acid molecule that specifically binds to the non-specific reaction substance is a chemically synthesizable substance.

3. 3. The non-specific reaction inhibitor according to claim 1, wherein the complex has a molecular weight of 50 kDa or less.

4. 2. The non-specific reaction inhibitor according to claim 1, wherein the polymer compound is polyethylene glycol.

5. 2. The non-specific reaction inhibitor according to claim 1, wherein the non-specific reaction substance is immunoglobulin.

6. An immunoassay method characterized by using a complex of a peptide or nucleic acid molecule that specifically binds to a non-specific reactive substance and a polymer compound.

7. 7. The immunoassay method according to claim 6, wherein the immunoassay method is a latex agglutination optical assay, an immunonephelometry, an immunoturbidimetry, a chemiluminescence immunoassay, an enzyme immunoassay, a fluorescence immunoassay, or a radioimmunoassay.

8. An immunoassay reagent comprising a complex of a peptide or nucleic acid molecule that specifically binds to a non-specific reactive substance and a polymer compound.

9. 9. The immunoassay reagent according to claim 8, wherein the stabilized reagent contains a complex of a peptide or nucleic acid molecule that specifically binds to a non-specific reaction substance and a polymer compound.