Solid phase holding both Anti-human amyloid β42 antibody and polyethylene glycol

Abstract

The present invention provides a test reagent that has excellent detection sensitivity in measurement of amyloid β using an antibody. Specifically, the present invention provides, for example, (1) a solid phase holding both an anti-human Aβ42 antibody and polyethylene glycol, (2) a solid phase production method comprising bringing an anti-human Aβ42 antibody and polyethylene glycol into contact with a solid phase in a solution to obtain the solid phase, and (3) a human Aβ42 test method comprising using the solid phase to measure the amount of human Aβ42 in a sample.

Description

Solid phase carrying both anti-human amyloid beta 42 antibody and polyethylene glycol

[0001] The present invention relates to a solid phase that retains both an anti-human amyloid β42 antibody and polyethylene glycol (PEG), and the like.

[0002] Alzheimer's disease (AD) is one of the most common forms of dementia and is a neurodegenerative disease characterized histologically by the accumulation of intraneuronal neurofibrillary tangles and extracellular amyloid plaques throughout the cerebral cortex and limbic system. Amyloid-β, particularly the ratio of amyloid-β1-42 to amyloid-β1-40, in cerebrospinal fluid (CSF) is useful as a diagnostic marker for AD.

[0003] Many commercially available amyloid beta (Aβ) measurement kits use cerebrospinal fluid (CSF) as a sample, but collecting CSF has the problem of being highly invasive. On the other hand, it is known that amyloid beta is also present in the blood of Alzheimer's disease patients. Measuring amyloid beta in blood is advantageous because it is less invasive than measuring amyloid beta in CSF.

[0004] However, since the concentration of Aβ1-42 in blood is approximately 10-50 pg / mL, which is lower than the concentration in CSF (300-2000 pg / mL), high detection sensitivity is required for the test. Furthermore, since the difference in Aβ1-42 concentration between blood samples negative and positive for amyloid accumulation in the brain is not large, the development of a test reagent with excellent reproducibility is required. Therefore, it is necessary to improve the detection sensitivity of Aβ1-42 test reagents and suppress fluctuations in measurement values.

[0005] Regarding the measurement of amyloid β in a blood sample using an antibody, Patent Document 1 reports a method in which the measurement is carried out in the presence of an anionic polymer.

[0006] Furthermore, methods for measuring target antigens in samples other than blood samples using particles to which both an antibody and PEG are immobilized have been reported. For example, Patent Document 2 describes (1) a method for producing modified particles, including (a) a step of binding an anti-exosome antibody and PEG to particles, and (b) a step of binding a blocking agent to the particles to which the anti-exosome antibody and PEG are bound, and (2) a method for measuring exosomes in a culture supernatant of a cell line using the modified particles. Non-Patent Document 1 describes a method for measuring AFP in a cell lysate using particles to which both an anti-α-fetoprotein (AFP) antibody and PEG modified with pentaethylenehexamine are immobilized.

[0007] However, none of the above prior art techniques describes measuring amyloid β using a solid phase (eg, particles) that carries both an anti-amyloid β antibody and PEG.

[0008] International Publication No. 2021 / 200940 International Publication No. 2022 / 196630

[0009] Yukio Nagasaki et al., J Colloid Interface Sci. 2007 May 15;309(2):524-30

[0010] An object of the present invention is to develop a test reagent with excellent detection sensitivity in measuring amyloid β using an antibody.

[0011] After extensive research, the present inventors have found that the above-mentioned problems can be solved by measuring human Aβ42 using a solid phase carrying both an anti-human Aβ42 antibody and PEG. Interestingly and unexpectedly, the method of measuring human Aβ42 using a solid phase carrying both an anti-human Aβ42 antibody and PEG showed an improved S / N value (i.e., detection sensitivity) compared to the method of measuring human Aβ42 using a solid phase carrying an anti-human Aβ42 antibody but not PEG, whereas the method of measuring human Aβ40 using a solid phase carrying both an anti-human Aβ40 antibody and PEG did not show an improved S / N value compared to the method of measuring human Aβ40 using a solid phase carrying an anti-human Aβ40 antibody but not PEG (Test Example 2). Therefore, the improvement in the detection sensitivity of amyloid beta due to the use of PEG in measuring amyloid beta is thought to be specific to the case where human Aβ42 is selected as amyloid beta (i.e., measurement of human Aβ42 using an anti-human Aβ42 antibody). Based on these findings, the present inventors have completed the present invention.

[0012] That is, the present invention is as follows: [1] A solid phase holding both an anti-human amyloid β (Aβ)42 antibody and polyethylene glycol (PEG). [2] The solid phase of [1], wherein the solid phase is a particle. [3] The solid phase of [1] or [2], wherein both the anti-human Aβ42 antibody and PEG are held on the solid phase by a homogeneous bond. [4] Any of the solid phases of [1] to [3], wherein both the anti-human Aβ42 antibody and PEG are covalently bound to the solid phase. [5] The solid phase of [4], wherein the covalent bond is an amide bond. [6] The solid phase of [5], wherein the amide bond is formed by a reaction between the solid phase having a carboxy group, an anti-human Aβ42 antibody having an amino group, and a modified PEG having an amino group. [7] Any of the solid phases of [1] to [6], wherein the molecular weight of the PEG is 8,000 or less. [8] The solid phase of any one of [1] to [7], wherein the anti-human Aβ42 antibody is an antibody that recognizes an epitope comprising GGVVIA (SEQ ID NO: 5). [9] A method for producing a solid phase, comprising contacting an anti-human Aβ42 antibody and PEG with the solid phase in a solution to obtain the solid phase of any one of [1] to [8].

[10] The method of [9], comprising: (1) contacting an anti-human Aβ42 antibody with the solid phase in a first solution to obtain a solid phase that retains the anti-human Aβ42 antibody; and (2) contacting the solid phase that retains the anti-human Aβ42 antibody with PEG in a second solution to obtain the solid phase of any one of [1] to [8].

[11] A method for testing human Aβ42, comprising measuring human Aβ42 in a sample using the solid phase of any one of [1] to [8].

[12] The method of

[11] , wherein the sample is a blood sample or cerebrospinal fluid.

[13] The method of

[11] , comprising: (1) immobilizing human Aβ42 in a sample on a solid phase using any of the solid phases of [1] to [8]; (2) labeling the human Aβ42 immobilized on the solid phase with a labeled antibody against human Aβ42 (wherein the labeled antibody against human Aβ42 is an antibody that recognizes an epitope different from that of the anti-human Aβ42 antibody retained on the solid phase); and (3) measuring the labeled human Aβ42 immobilized on the solid phase.

[14] A test reagent for human Aβ42, comprising a solid phase retaining both an anti-human Aβ42 antibody and PEG.

[15] A test kit for human Aβ42, comprising: (1) a solid phase carrying both an anti-human Aβ42 antibody and PEG; and (2) a labeled antibody against human Aβ42 (wherein the labeled antibody against human Aβ42 recognizes an epitope different from that of the anti-human Aβ42 antibody carried on the solid phase).

[0013] According to the present invention, human Aβ42, a type of amyloid β, can be detected with high detection sensitivity.

[0014] FIG. 1 is a diagram showing an outline of the relationship between standard amyloid β1-40 and amyloid β1-42 and the antibodies used in the examples.

[0015] The present invention provides a solid phase bearing both an anti-human amyloid beta (Aβ) 42 antibody and polyethylene glycol (PEG).

[0016] Human amyloid beta (Aβ) is a peptide consisting of approximately 40 amino acid residues that is produced by proteolysis (cleavage) of the amyloid precursor protein (APP), which consists of approximately 770 amino acid residues, by beta-secretase and gamma-secretase. There are multiple peptides of human Aβ due to differences in cleavage sites and modifications, but the main known Aβs are human Aβ1-42 and human Aβ1-40.

[0017] The amino acid sequence of standard human Aβ1-42 (SEQ ID NO: 2) is shown in FIG. 1. In the present invention, human Aβ42 is a peptide having, as the carboxy-terminal amino acid residues, an isoleucine residue and an alanine residue (IA) corresponding to the 41st and 42nd amino acid residues in the amino acid sequence of SEQ ID NO: 2. Human Aβ42 can also be expressed herein as human Aβx-42 (where x represents an integer from 1 to 41). Representative examples of human Aβ42 include human Aβ1-42 peptide and human Aβ3-42 peptide generated by cleavage between the second and third amino acid residues in the amino acid sequence of SEQ ID NO: 2.

[0018] The amino acid sequence (SEQ ID NO: 2) of human Aβ42 (or Aβx-42; hereafter abbreviated) may contain several amino acid residue mutations. Human Aβ42 may be (a) a standard Aβ42 having the standard amino acid sequence (SEQ ID NO: 2), or (b) a mutant Aβ42 having an amino acid sequence containing several (e.g., 1 to 5, preferably 1, 2, or 3) amino acid residue mutations (e.g., deletions, insertions, substitutions, additions) at amino acid residues 1 to 40 in the standard amino acid sequence (SEQ ID NO: 2). Note that if the amino acid residue mutations involve deletions, insertions, or additions of several amino acid residues, the total number of amino acid residues in mutant human Aβ1-42 may not be 42, resulting in a deviation of several amino acid residues. However, even in such cases, a polypeptide generated by cleavage of an amyloid precursor protein and having, as its carboxy-terminal constituent amino acid residues, I A corresponding to the 41st and 42nd amino acid residues in the amino acid sequence of SEQ ID NO: 2 is included within the scope of human Aβ42 in the present invention (Figure 1).

[0019] The amino acid sequence of standard human Aβ1-40 (SEQ ID NO: 1) is shown in FIG. 1. In the present invention, human Aβ40 is a peptide having, as the carboxy-terminal amino acid residues, valine residues and valine residues (VV) corresponding to the 39th and 40th amino acid residues in the amino acid sequence of SEQ ID NO: 1. Human Aβ40 can also be expressed herein as human Aβx-40 (where x represents an integer from 1 to 39). Representative examples of human Aβ40 include human Aβ1-40 peptide and human Aβ3-40 peptide generated by cleavage between the second and third amino acid residues in the amino acid sequence of SEQ ID NO: 1.

[0020] The amino acid sequence (SEQ ID NO: 1) of human Aβ40 (or Aβx-40, hereafter abbreviated) may contain several amino acid residue mutations. Human Aβ40 may be (a) a standard Aβ40 having the standard amino acid sequence (SEQ ID NO: 1), or (b) a mutant Aβ40 having an amino acid sequence containing several (e.g., 1 to 5, preferably 1, 2, or 3) amino acid residue mutations (e.g., deletions, insertions, substitutions, additions) at amino acid residues 1 to 38 of the standard amino acid sequence (SEQ ID NO: 1). Note that if the amino acid residue mutations involve deletions, insertions, or additions of several amino acid residues, the total number of amino acid residues in mutant human Aβ1-40 may not be 40, but may be a deviation of several amino acid residues. However, even in such cases, a polypeptide generated by cleavage of an amyloid precursor protein and having, as carboxy-terminal constituent amino acid residues, VV corresponding to the 39th and 40th amino acid residues in the amino acid sequence of SEQ ID NO: 1 is included within the scope of human Aβ40 in the present invention (Figure 1).

[0021] Human Aβ42 differs from human Aβ40, which has an amino acid sequence without IA at the carboxy terminus, in that it has an amino acid sequence with IA added to the carboxy terminus. Therefore, for testing human Aβ42, an antibody against an epitope containing IA at the carboxy terminus of human Aβ42 can be used as an anti-human Aβ42 antibody (anti-human Aβ42 antibody). Anti-human Aβ42 antibodies can be produced by using, as an antigen, a peptide or the like containing an epitope (e.g., VIA) that includes IA and other amino acid residues (e.g., V) present at the carboxy terminus. Alternatively, anti-human Aβ42 antibodies can be produced by binding IA (or an amino acid sequence further containing other amino acid residues such as VIA) present at the carboxy terminus of human Aβ42 to a carrier (e.g., a carrier protein such as BSA) as a hapten, and using this hapten-carrier conjugate as an antigen to produce antibodies against IA (or an amino acid sequence further containing other amino acid residues such as VIA).

[0022] Preferably, the anti-human Aβ42 antibody may be an antibody against an epitope comprising IA and other amino acid residues present at the carboxy terminus.Such anti-human Aβ42 antibody includes, for example, an antibody against an epitope comprising VIA, an antibody against an epitope comprising VVIA (SEQ ID NO: 6), an antibody against an epitope comprising GVVIA (SEQ ID NO: 7), an antibody against an epitope comprising GGVVIA (SEQ ID NO: 5), an antibody against an epitope comprising VGGVVIA (SEQ ID NO: 8), and an antibody against an epitope comprising MVGGVVIA (SEQ ID NO: 9).Among these, an antibody against an epitope comprising GGVVIA (SEQ ID NO: 5) is more preferred, and an antibody against an epitope consisting of GGVVIA (SEQ ID NO: 5) is even more preferred.

[0023] The anti-human Aβ42 antibody may be either a polyclonal antibody or a monoclonal antibody. The anti-human Aβ42 antibody may be any isotype of immunoglobulin (e.g., IgG, IgM, IgA, IgD, IgE, IgY). The anti-human Aβ42 antibody may also be a full-length antibody. A full-length antibody refers to an antibody comprising a heavy chain and a light chain, each of which comprises a variable region and a constant region (e.g., an antibody comprising two Fab portions and an Fc portion). The anti-human Aβ42 antibody may also be an antibody fragment derived from such a full-length antibody. An antibody fragment is a portion of a full-length antibody, for example, a constant region-deleted antibody (e.g., F(ab') ２ , Fab', Fab, Fv). The anti-human Aβ42 antibody may also be a modified antibody such as a single-chain antibody. Methods for producing polyclonal and monoclonal antibodies are well known, and therefore, anti-human Aβ42 antibodies can be produced using the above antigens according to well-known methods. Alternatively, commercially available antibodies may be used as the anti-human Aβ42 antibody.

[0024] Polyethylene glycol (PEG) is a ２ CH ２ O) ｎPEG is a compound having a repeating unit of the formula: PEG can be retained on a solid phase by physical adsorption, so unmodified PEG can be used as the PEG. However, in order to retain PEG strongly and stably on a solid phase, it is preferable to use modified PEG having a moiety that enables binding to the solid phase. Examples of the moiety that enables binding to the solid phase include a moiety that enables covalent bonding to the solid phase, a moiety that enables affinity bonding to the solid phase, a moiety that enables ionic bonding to the solid phase, and a moiety that can promote physical adsorption to the solid phase.

[0025] When a modified PEG having a moiety that enables covalent bonding with a solid phase is used, examples of the moiety that enables covalent bonding with a solid phase include a moiety that enables amide bonding with a solid phase (e.g., a carboxy group, an amino group), a moiety that enables tosylamide bonding with a solid phase (e.g., a tosyl group, an amino group), a moiety that enables epoxyamide bonding with a solid phase (e.g., an epoxy group, an amino group), a moiety that enables thioether bonding with a solid phase (e.g., a maleimide group, a sulfhydryl group (thiol group)), and a moiety that enables hydrazone bonding with a solid phase (e.g., a hydrazone group, a carbonyl group). The moiety that enables covalent bonding with a solid phase is preferably a moiety that enables amide bonding with a solid phase (e.g., a carboxy group, an amino group). The moiety that enables covalent bonding may or may not be activated with a crosslinker. The modified PEG is preferably a modified PEG having the functional group described above, more preferably a modified PEG having a functional group that enables amide bonding (e.g., an amino group), and even more preferably a modified PEG having an amino group.

[0026] When a modified PEG having a moiety that can bind with a solid phase affinity is used, the moiety that can bind with a solid phase affinity can be an affinity substance.Such affinity substances include, for example, streptavidin, biotin, digoxigenin, dinitrophenol, fluorescein, fluorescein isothiocyanate, a specific antigen (excluding β-amyloid) or an antibody against a specific antigen (excluding an antibody against β-amyloid), and a pair of complementary single-stranded nucleic acids that can form double-stranded nucleic acids (for example, a pair of single-stranded nucleic acids having a polyA sequence and a polyT sequence).The moiety that can bind with a solid phase affinity is preferably streptavidin, biotin, digoxigenin, dinitrophenol, fluorescein, or fluorescein isothiocyanate, and more preferably streptavidin or biotin.

[0027] When a modified PEG having a moiety that allows ionic bonding with a solid phase is used, the moiety that allows ionic bonding with a solid phase can include, for example, a positively charged moiety and a negatively charged moiety. Positively charged moieties include, for example, a positively charged phosphorus atom (e.g., phosphonium), a positively charged nitrogen atom (e.g., ammonium, a positively charged amino group), and a positively charged metal atom (e.g., a divalent metal ion such as a copper ion, an iron ion, or a calcium ion, and a monovalent metal ion such as a sodium ion). Negatively charged moieties include, for example, a halogen atom ion (e.g., chloride ion), a monovalent negatively charged moiety (e.g., hydroxide ion, carboxylate ion), and a divalent negatively charged moiety (e.g., sulfate ion).

[0028] When a modified PEG having a moiety capable of promoting physical adsorption to a solid phase is used, examples of the moiety capable of promoting physical adsorption to a solid phase include a moiety capable of promoting hydrophobic binding to a solid phase (e.g., a hydrocarbon chain with a long chain length) and a moiety capable of promoting hydrophilic binding to a solid phase (e.g., a hydrophilic modification capable of forming hydrogen bonds).

[0029] When a modified PEG having a moiety capable of promoting chemical adsorption to a solid phase is used, the moiety capable of promoting chemical adsorption to a solid phase can be, for example, a moiety capable of strong interaction with a metal surface (e.g., a thiol group).

[0030] The moiety that allows binding to a solid phase may preferably be a moiety that allows covalent binding to the solid phase, a moiety that allows affinity binding to the solid phase, or a moiety that allows ionic binding to the solid phase, more preferably a moiety that allows covalent binding to the solid phase, or a moiety that allows affinity binding to the solid phase, even more preferably a moiety that allows covalent binding to the solid phase.

[0031] When a modified PEG having a moiety that allows binding to a solid phase is used as the PEG, the modified PEG may have a moiety that allows binding to a solid phase at one or both ends, and preferably has a moiety at one end. In the modified PEG, the moiety that allows binding to a solid phase can be (CH ２ CH ２ O) ｎ Any divalent group can be used as such a linker. Examples of such a divalent group include a divalent hydrocarbon group, -C(=O)-, and -NR １ - (R １ represents a hydrogen atom or a divalent hydrocarbon group), —O—, —S—, and —C(═S)—, as well as combinations of two or more of these (for example, 2, 3, or 4).

[0032] The divalent hydrocarbon group is a straight-chain, branched-chain, or cyclic divalent hydrocarbon group, preferably a straight-chain or branched-chain divalent hydrocarbon group. As the divalent hydrocarbon group, a hydrocarbon group having 1 to 12 carbon atoms is preferred, and a hydrocarbon group having 1 to 6 carbon atoms is more preferred. Examples of the divalent hydrocarbon group include alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, and arylene.

[0033] Preferably, the divalent hydrocarbon group may be alkylene. As the alkylene, alkylene having 1 to 12 carbon atoms is preferred, alkylene having 1 to 6 carbon atoms is more preferred, and alkylene having 1 to 4 carbon atoms is particularly preferred. The alkylene may be linear, branched, or cyclic, but linear alkylene is preferred. Examples of such alkylene include methylene, ethylene, propylene, butylene, pentylene, and hexylene.

[0034] When the modified PEG having a moiety that allows for ionic bonding with a solid phase has a moiety that allows for binding to a solid phase at one end, the other end can have any monovalent group, such as a monovalent hydrocarbon group.

[0035] The monovalent hydrocarbon group is a linear, branched, or cyclic monovalent hydrocarbon group, preferably a linear or branched monovalent hydrocarbon group. The monovalent hydrocarbon group is preferably a hydrocarbon group having 1 to 12 carbon atoms, more preferably a hydrocarbon group having 1 to 6 carbon atoms. Examples of the monovalent hydrocarbon group include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and aryl.

[0036] Preferably, the monovalent hydrocarbon group may be an alkyl. As the alkyl, an alkyl having 1 to 12 carbon atoms is preferred, an alkyl having 1 to 6 carbon atoms is more preferred, and an alkyl having 1 to 4 carbon atoms is particularly preferred. The alkyl may be linear, branched, or cyclic, but linear alkyl is preferred. Examples of such alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0037] PEGs having any weight-average molecular weight can be used. Such weight-average molecular weights may be, for example, in the range of 300 to 200,000. The weight-average molecular weight of PEG may be 500 or more, 1,000 or more, 1,500 or more, or 2,000 or more. The weight-average molecular weight of PEG may also be 100,000 or less, 70,000 or less, 50,000 or less, or 40,000 or less. More specifically, the weight-average molecular weight of PEG may be preferably 500 to 100,000, more preferably 1,000 to 70,000, even more preferably 1,000 to 50,000, and particularly preferably 2,000 to 40,000. When a modified PEG having a moiety that enables binding to a solid phase or a moiety that promotes adsorption is used as the PEG, the weight-average molecular weight of the entire modified PEG can be set to be within the above-mentioned range.

[0038] Preferably, PEG may have a weight-average molecular weight of 500 to 8,000 to achieve further improvement in detection sensitivity. The weight-average molecular weight of PEG may be 1,000 or more, 1,500 or more, or 2,000 or more. The weight-average molecular weight of PEG may also be 8,000 or less, 7,000 or less, 6,000 or less, or 5,000 or less. More specifically, the weight-average molecular weight of PEG may be preferably 1,000 to 7,000, more preferably 1,500 to 6,000, and even more preferably 2,000 to 5,000. When a modified PEG having a moiety that enables binding to a solid phase or a moiety that promotes adsorption is used as the PEG, the weight-average molecular weight of the entire modified PEG can be set to be within the above-mentioned range.

[0039] The solid phase can be one capable of retaining both the anti-human Aβ1-42 antibody and PEG. Examples of solid phases include solid phases that can be suspended or dispersed in a liquid phase (e.g., particles), and solid phases that can accommodate or carry a liquid phase (e.g., supports such as plates and membranes; and containers such as well plates, microtubes, test tubes, microchannels, glass capillaries, nanopillars, and monolith columns). Examples of solid phase materials include glass, silica, synthetic polymers (e.g., polystyrene, polyacrylamide, polyacrylic acid, polyhydroxyethyl methacrylate, polyvinyl alcohol), polysaccharides (e.g., glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, starch, glycogen, agarose, pectin, and cellulose), polypeptides (e.g., gelatin, proteoglycans, and fibronectin), metals (e.g., iron, copper, aluminum, calcium, and gold), metal compounds (e.g., iron oxide, cobalt oxide, and nickel ferrite), and carbon, as well as mixtures thereof. The solid phase material may also be a non-magnetic material or a magnetic material (eg, a metal such as iron).

[0040] Preferably, the solid phase may be a solid phase that can be suspended or dispersed in a liquid phase from the viewpoint of reactivity, and more preferably may be particles. Examples of particle materials include the solid phase materials described above. Preferred particles include, for example, gelatin particles (e.g., magnetic gelatin particles) and ferrite particles (e.g., magnetic ferrite particles). As such a solid phase, conventionally known ones can be used as appropriate, and commercially available ones can also be used as appropriate.

[0041] The solid phase can hold both the anti-human Aβ42 antibody and PEG by homogeneous or heterogeneous bonds. For example, when the solid phase holds both the anti-human Aβ42 antibody and PEG by homogeneous bonds, the solid phase can hold both the anti-human Aβ42 antibody and PEG by covalent bonds, affinity bonds, or ionic bonds, preferably by covalent bonds or affinity bonds, more preferably by covalent bonds, even more preferably by the specific covalent bonds detailed above, and particularly preferably by amide bonds. When the solid phase holds both the anti-human Aβ42 antibody and PEG by heterogeneous bonds, the solid phase can hold the anti-human Aβ42 antibody by covalent bonds, affinity bonds, or ionic bonds, and can hold the PEG by covalent bonds, affinity bonds, ionic bonds, or physical adsorption (wherein the type of bond for holding the PEG is different from the type of bond for holding the anti-human Aβ42 antibody). Preferably, the solid phase can hold both the anti-human Aβ42 antibody and PEG by homogeneous bonds.

[0042] In certain embodiments, the solid phase may hold the anti-human Aβ42 antibody by covalent or affinity binding, preferably by covalent binding.In such cases, the functional groups in the side chains of the amino acid residues constituting the anti-human Aβ42 antibody and the carboxyl group present at the carboxyl terminus of the anti-human Aβ42 antibody can be used for covalent binding to the solid phase.The functional groups in the side chains of the amino acid residues constituting the anti-human Aβ42 antibody include, for example, the amino group in the side chain of a lysine residue, the carboxyl group in the side chain of aspartic acid and glutamic acid, and the thiol group in a cysteine ​​residue.The thiol group in a cysteine ​​residue may be obtained by reducing the disulfide bond in the antibody.

[0043] In certain embodiments, the solid phase may hold PEG by covalent, affinity, ionic, or physical adsorption (e.g., hydrophobic bonds), preferably by covalent, affinity, or ionic bonds, more preferably by covalent or affinity bonds, and even more preferably by covalent bonds, with the specific covalent bonds detailed above being preferred, and with amide bonds being more preferred.

[0044] The solid phase of the present invention as described above can be prepared by contacting the solid phase with an anti-human Aβ42 antibody and PEG in a solution. The solid phase of the present invention can be prepared by covalent bonding (e.g., N-hydroxysuccinimide, periodate, glutaraldehyde, maleimide), affinity substance-based bonding, ionic bonding, or physical adsorption. The definitions, examples, and preferred examples of the solid phase, anti-human Aβ42 antibody, and PEG are the same as those described above.

[0045] The concentration of the anti-human Aβ42 antibody in the solution is not particularly limited, as long as it is a concentration that allows the solid phase to retain the anti-human Aβ42 antibody upon contact with the solid phase. Such a concentration may be, for example, in the range of 0.001 to 100 mg / ml. Such a concentration may be 0.01 mg / ml or more, 0.05 mg / ml or more, 0.1 mg / ml or more, or 0.2 mg / ml or more. Such a concentration may also be 50 mg / ml or less, 20 mg / ml or less, 10 mg / ml or less, or 5 mg / ml or less. More specifically, such a concentration may preferably be 0.01 to 50 mg / ml, 0.05 to 20 mg / ml, 0.1 to 10 mg / ml, or 0.2 to 5 mg / ml.

[0046] The concentration of PEG in the solution is not particularly limited, as long as it is a concentration that allows the solid phase to retain PEG upon contact with the solid phase. Such a concentration may be, for example, in the range of 0.1 to 1000 mg / ml. Such a concentration may be 0.5 mg / ml or more, 1 mg / ml or more, 5 mg / ml or more, or 10 mg / ml or more. Such a concentration may also be 700 mg / ml or less, 500 mg / ml or less, 200 mg / ml or less, or 100 mg / ml or less. More specifically, such a concentration may be preferably 0.5 to 700 mg / ml, more preferably 1 to 500 mg / ml, even more preferably 5 to 500 mg / ml, and particularly preferably 10 to 400 mg / ml.

[0047] The ratio of PEG to anti-human Aβ42 antibody in the solution can be appropriately set so that the solid phase can retain both PEG and anti-human Aβ42 antibody. This ratio can be determined, for example, based on the weight ratio of PEG to anti-human Aβ42 antibody (PEG / anti-human Aβ42 antibody). For example, this weight ratio (PEG / anti-human Aβ42 antibody) may be in the range of 1 to 1,000. This weight ratio may be 2 or more, 5 or more, 10 or more, or 20 or more. This weight ratio may also be 500 or less, 300 or less, 200 or less, or 100 or less. More specifically, this weight ratio may be preferably 2 to 500, more preferably 5 to 300, even more preferably 10 to 200, and particularly preferably 20 to 150.

[0048] The contact of the anti-human Aβ42 antibody and PEG with the solid phase in solution may be performed by simultaneously contacting the anti-human Aβ42 antibody and PEG with the solid phase. Alternatively, such contact may be performed by separately contacting the anti-human Aβ42 antibody and PEG with the solid phase. For example, the anti-human Aβ42 antibody may be first contacted with the solid phase, and then the PEG may be contacted, or the PEG may be first contacted with the solid phase, and then the anti-human Aβ42 antibody may be contacted. Preferably, the anti-human Aβ42 antibody may be first contacted with the solid phase, and then the PEG may be contacted.

[0049] An aqueous solution can be used as the solution. Examples of aqueous solutions include water (e.g., distilled water, sterilized water, sterilized distilled water, and pure water) and buffer solutions, with buffer solutions being preferred. Examples of buffer solutions include phosphate buffer solution, phosphate-buffered saline (PBS), tartrate buffer solution, citrate buffer solution, acetate buffer solution, glycine buffer solution, carbonate buffer solution, 2-morpholinoethanesulfonic acid (MES) buffer solution, trishydroxymethylaminomethane (Tris) buffer solution, borate buffer solution, 3-morpholinopropanesulfonic acid (MOPS) buffer solution, N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer solution, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer solution, 2-[4-(2-hydroxyethyl)1-piperazinylethanesulfonic acid (HEPES) buffer solution, and imidazole buffer solution. The pH of the solution may be, for example, 3.5 to 10.0, preferably 4.0 to 10.0, more preferably 4.5 to 9.5, even more preferably 5.0 to 9.0, and especially preferably 5.5 to 8.5.

[0050] Contact between the solid phase and the anti-human Aβ42 antibody and PEG can be achieved by mixing (e.g., stirring, inversion, pipetting) and / or incubation of the solid phase with the anti-human Aβ42 antibody and PEG. The contact time is not particularly limited, as long as the solid phase can retain both the anti-human Aβ42 antibody and PEG. Such contact time may be, for example, 1 minute to 10 hours, preferably 2 minutes to 5 hours, more preferably 3 minutes to 3 hours, even more preferably 5 minutes to 2 hours, and particularly preferably 10 minutes to 1 hour. The contact temperature may be, for example, 4 to 42°C, preferably 10 to 37°C, and more preferably 15 to 35°C.

[0051] In a specific embodiment, the solid phase of the present invention can be prepared by: (1) contacting an anti-human Aβ42 antibody with a solid phase in a first solution to obtain a solid phase carrying the anti-human Aβ42 antibody (antibody sensitization step); and (2) contacting PEG with the solid phase carrying the anti-human Aβ42 antibody in a second solution to obtain a solid phase carrying both the anti-human Aβ42 antibody and PEG (PEG sensitization step).

[0052] The first solution used in (1) above can be the solution described above. The concentration of the anti-human Aβ42 antibody in the first solution is the same as the concentration of the anti-human Aβ42 antibody in the solution described above. Details of the contact, such as the contact time and contact temperature, are also the same as those described above. In addition, the definitions, examples, and preferred examples of the solid phase, anti-human Aβ42 antibody, and retention are also the same as those described above.

[0053] The second solution used in (2) above can be the same as the solution described above. The concentration of PEG in the second solution is the same as the concentration of PEG in the solution described above. The second solution may be the same as or different from the first solution. Details of the contact, such as the contact time and contact temperature, are also the same as those described above. In addition, the definitions, examples, and preferred examples of the solid phase, anti-human Aβ42 antibody, PEG, and retention are also the same as those described above.

[0054] The method for preparing a solid phase of the present invention may further comprise washing the solid phase after the above steps (1) and / or (2). The solid phase can be washed using an aqueous solution (e.g., a buffer solution). The number of washes is usually 1 to 3 times. The aqueous solution used for washing may contain a component such as a surfactant.

[0055] The method for preparing a solid phase of the present invention may also further comprise one or more steps for quenching and / or masking.

[0056] The term "quenching the reaction" refers to stabilizing the solid phase by reacting unreacted groups present on the solid phase with a quenching agent. For example, when the solid phase holds an anti-human Aβ42 antibody and / or PEG by covalent bonding, a substance having a group (e.g., an amino group) capable of reacting with the unreacted group (e.g., a carboxy group) not subjected to the covalent bond (e.g., an amino group-containing substance such as Tris) can be used as the quenching agent. When the solid phase holds an anti-human Aβ42 antibody and / or PEG by affinity bonding, an affinity substance or a compound having the same capable of reacting with the unreacted affinity substance not subjected to affinity bonding can be used as the quenching agent. When the solid phase holds an anti-human Aβ42 antibody and / or PEG by ionic bonding, a positively or negatively charged substance can be used as the quenching agent. When the solid phase holds an anti-human Aβ42 antibody and / or PEG by physical adsorption (e.g., hydrophobic bonding), a hydrophobic substance can be used as the quenching agent.

[0057] Masking refers to treating a solid phase with a masking agent to suppress nonspecific adsorption of proteins in a sample to the solid phase. Examples of masking agents include proteins such as albumin (e.g., bovine serum albumin (BSA)), casein, collagen, and gelatin.

[0058] In one or more steps for reaction quenching and / or masking, the solid phase of the present invention can be treated with one or more aqueous solutions (e.g., buffer solutions) containing a reaction quenching agent and / or a masking agent. The reaction quenching agent and the masking agent may coexist in one aqueous solution, or the reaction quenching agent and the masking agent may exist separately in separate aqueous solutions. Furthermore, when two or more steps for reaction quenching and / or masking are set, the reaction quenching step may be performed first and then the masking step, or the masking step may be performed first and then the reaction quenching step. Note that the reaction quenching step and the masking step are relative steps, and the aqueous solution used in the reaction quenching step may contain a relatively large amount of reaction quenching agent and a relatively small amount of masking agent, or the aqueous solution used in the masking step may contain a relatively small amount of reaction quenching agent and a relatively large amount of masking agent. The aqueous solution used for reaction quenching and / or masking may contain a chelating agent (e.g., EDTA), a preservative (e.g., NaN ３ ) may further comprise additional ingredients such as

[0059] In a specific embodiment, the solid phase of the present invention can be prepared by the following steps: (1) contacting an anti-human Aβ42 antibody with a solid phase (e.g., a particle) in a first solution to obtain a solid phase that covalently holds the anti-human Aβ42 antibody (antibody sensitization step); and (2) contacting a modified PEG having a moiety that enables covalent bonding to the solid phase with the solid phase in a second solution to obtain a solid phase that covalently holds both the anti-human Aβ42 antibody and PEG (PEG sensitization step).

[0060] (1) and (2) of this embodiment can be carried out in the same manner as (1) and (2) of the above-described embodiment. The covalent bond can be a covalent bond as described above, with an amide bond being preferred. When the covalent bond is an amide bond, the solid phase may have a carboxy group, and the anti-human Aβ42 antibody and the modified PEG may have an amino group, or the solid phase may have an amino group, and the anti-human Aβ42 antibody and the modified PEG may have a carboxy group. The formation of an amide bond by the reaction of a carboxy group with an amino group can be carried out by any reaction. For example, this can be achieved by activating a carboxyl group with carbodiimide using EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiiimide) and then reacting this with an amino group. More preferably, this can be achieved by activating a carboxyl group with carbodiimide using EDC, then forming an NHS ester with NHS (N-hydroxysuccinimide) or an analog thereof (e.g., sulfo-NHS), and then reacting this with an amino group. The reaction of a carboxyl group with a primary amine using EDC and NHS is widely known and can be carried out according to standard methods. EDC and NHS are commercially available, and the reaction of a carboxyl group with a primary amine can be carried out according to the protocols attached to the commercially available products.

[0061] In a preferred embodiment, the solid phase of the present invention can be prepared by the following steps: (1) contacting an anti-human Aβ42 antibody having an amino group with a solid phase (e.g., a particle) having a carboxy group in a first solution to obtain a solid phase holding the anti-human Aβ42 antibody via an amide bond (antibody sensitization step); and (2) contacting a modified PEG having an amino group with the solid phase holding the anti-human Aβ42 antibody via an amide bond in a second solution to obtain a solid phase holding both the anti-human Aβ42 antibody and PEG via an amide bond (PEG sensitization step).

[0062] The present invention also provides a method for testing human Aβ42. The testing method of the present invention comprises measuring human Aβ42 in a sample using a solid phase that supports both an anti-human Aβ42 antibody and PEG. Measuring Aβ42 includes measuring the amount of human Aβ42 (quantitative) and determining the presence or absence of human Aβ42 (qualitative). The testing method of the present invention preferably comprises measuring the amount of human Aβ42 (preferably human Aβ1-42) in a sample using a solid phase that supports both an anti-human Aβ42 antibody and PEG.

[0063] The sample is not particularly limited as long as it may contain human Aβ42, and may be, for example, a human-derived sample. Examples of human-derived samples include blood samples (e.g., whole blood, serum, and plasma), urine, saliva, lymph, tissue fluid, cerebrospinal fluid, ascites, sweat, semen, tears, mucus, milk, pleural fluid, bronchoalveolar lavage fluid, and amniotic fluid. Preferably, the human-derived liquid sample is a blood sample or cerebrospinal fluid, and more preferably a blood sample. When plasma is used as the blood sample, the plasma may be, for example, EDTA plasma, heparin plasma, citrate plasma, sodium fluoride plasma, or plasma containing ACD (acid-citrate-dextrose) or CPD (citrate phosphate dextrose).

[0064] Measurement of human Aβ42 in a sample can be performed by immunoassay. Examples of such immunoassays include direct competitive assays, indirect competitive assays, and sandwich assays. Such immunoassays may preferably be sandwich assays. Examples of such immunoassays include chemiluminescent immunoassays (CLIA) (e.g., chemiluminescent enzyme immunoassays (CLEIA)), turbidimetric immunoassays (TIA), enzyme immunoassays (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassays (RIA), latex agglutination assays, fluorescent immunoassays (FIA), and immunochromatography. In the sandwich assay, the solid phase of the present invention can be used as a solid-phase antibody.

[0065] In a specific embodiment, the testing method of the present invention can be carried out by the following steps: (1) immobilizing human Aβ42 in a sample on a solid phase using a solid phase that supports both an anti-human Aβ42 antibody and PEG; (2) labeling the human Aβ42 immobilized on the solid phase using a labeled antibody against human Aβ42 (wherein the labeled antibody against human Aβ42 is an antibody that recognizes an epitope different from that of the anti-human Aβ42 antibody immobilized on the solid phase); and (3) measuring the labeled human Aβ42 immobilized on the solid phase.

[0066] (1) can be carried out in the same manner as in immunoassays using solid-phase antibodies.

[0067] (2) can be carried out in the same manner as in immunoassays using labeled antibodies. Examples of labeling substances for preparing labeled antibodies include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., one of streptavidin and biotin, one of the complementary sense and antisense strand nucleic acids), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein, rhodamine B isothiocyanate (RBITC), tetramethylrhodamine isothiocyanate, dansyl chloride, phycoerythrin, sulfonated cyanines (e.g., Alexa Fluor 532, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 600, Alexa Fluor 610, Alexa Fluor 620, Alexa Fluor 630, Alexa Fluor 640, Alexa Fluor 650, Alexa Fluor 651, Alexa Fluor 652, Alexa Fluor 653, Alexa Fluor 654, Alexa Fluor 655, Alexa Fluor 655, Alexa Fluor 655, Alexa Fluor 656, Alexa Fluor 657, Alexa Fluor 658, Alexa Fluor 659, Alexa Fluor 660, Alexa Fluor 661, Alexa Fluor 662, Alexa Fluor 663, Alexa Fluor 664, Alexa Fluor 665, Alexa Fluor 665, Alexa Fluor 666), and the like. 633, Alexa Fluor 647), 6-carboxyfluorescein (6-FAM), tetrachloro-6-carboxyfluorescein (TET), hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (6-JOE), carboxy-6-rhodamine (ROX), ATTO compounds (e.g., ATTO488, ATTO532, ATTO550, ATTORho6G, ATTO647N), 6-tetramethylrhodamine-5(6)-carboxamide)hexanoate (TAMRA), cyanine dyes), luminescent substances (e.g., luciferin, aequorin, acridinium esters, tris(2,2'-bipyridyl)ruthenium, luminol), radioactive substances (e.g., ３ H. １４ C. ３２ P. ３５ S. １２５ The labeled antibody can be prepared by any method.

[0068] The labeled antibody against human Aβ42 is an antibody that recognizes an epitope different from that of the anti-human Aβ42 antibody retained on a solid phase. Such a labeled antibody is not particularly limited, as long as it is capable of labeling human Aβ42 retained on a solid phase (i.e., as long as a sandwich immunoassay is possible in combination with an anti-human Aβ42 antibody retained on a solid phase). For example, the epitope of the labeled antibody against human Aβ42, based on the standard Aβ1-42 having the standard amino acid sequence (SEQ ID NO: 2), includes an epitope in consecutive amino acid residues 1 to 36 (preferably 1 to 30, more preferably 1 to 25, even more preferably 1 to 20, and particularly preferably 1 to 15) in the amino acid sequence of SEQ ID NO: 2 (Figure 1). More specifically, the epitope of the labeled antibody against human Aβ42 may be, for example, any consecutive 5 to 10 amino acid residues from the 1 to 36 amino acid residues in the amino acid sequence of SEQ ID NO: 2. For example, when detecting human Aβ1-42 of human Aβ42, an antibody recognizing an epitope containing the first amino acid residue of standard Aβ1-42 having the standard amino acid sequence (SEQ ID NO: 2) can be used as a labeled antibody. Specifically, the epitope may be an epitope containing (preferably consisting of) DAEFR (SEQ ID NO: 3). Furthermore, when detecting human Aβ3-42 of human Aβ42, an antibody recognizing an epitope containing the third amino acid residue of standard Aβ1-42 having the standard amino acid sequence (SEQ ID NO: 2) can be used as a labeled antibody. Specifically, the epitope may be an epitope containing (preferably consisting of) EFRHD (SEQ ID NO: 10). Furthermore, the epitope of the labeled antibody against human Aβ may be RHDSGY (SEQ ID NO: 11) or HDSGYE (SEQ ID NO: 12). More preferably, the epitope may be an epitope containing (preferably consisting of) DAEFR (SEQ ID NO: 3).

[0069] The labeled antibody against human Aβ42 may be either a polyclonal antibody or a monoclonal antibody. Such a labeled antibody may be any isotype of immunoglobulin (e.g., IgG, IgM, IgA, IgD, IgE, IgY). Such a labeled antibody may also be a full-length antibody as described above, an antibody fragment derived from a full-length antibody, or a modified antibody such as a single-chain antibody. Methods for producing polyclonal and monoclonal antibodies are well known, and labeled antibodies against human Aβ42 can be produced using the above antigens according to well-known methods. Alternatively, commercially available antibodies may be used as labeled antibodies against human Aβ42.

[0070] In (3), measurement of human Aβ42 (preferably, the amount, such as concentration) can be performed by detecting the label. Label detection can be performed based on a method appropriately selected from known methods depending on the type of label. When the label is an enzyme, the label can be detected by detecting enzymatic activity using a signal-generating substrate (e.g., a fluorescent substrate, a luminescent substrate, or a chromogenic substrate). When the label is an affinity substance, the label can be detected by using an enzyme or signal-generating substance capable of binding to the affinity substance to detect the enzyme or signal-generating substance bound to the affinity substance. Such an enzyme or signal-generating substance capable of binding to an affinity substance may be an enzyme or signal-generating substance bound to a substance capable of binding to the affinity substance. When the label is a fluorescent substance, a luminescent substance, or a radioactive substance, the label can be detected by detecting the signal generated from the label.

[0071] The present invention also provides a test reagent for human Aβ42, comprising a solid phase supporting both an anti-human Aβ42 antibody and PEG. The definitions, examples, and preferred examples of human Aβ42, anti-human Aβ42 antibody, PEG, solid phase, and support are as described above.

[0072] The present invention also provides a test kit for human Aβ42, comprising: (1) a solid phase carrying both an anti-human Aβ42 antibody and PEG; and (2) a labeled antibody against human Aβ42 (wherein the labeled antibody against human Aβ42 is an antibody that recognizes an epitope different from that of the anti-human Aβ42 antibody carried on the solid phase).

[0073] The definitions, examples, and preferred examples of human Aβ42, anti-human Aβ42 antibody, PEG, solid phase, retention, and labeled antibody against human Aβ42 are as described above. The kit of the present invention preferably includes the above (1) and (2) in a form separated from each other. Specifically, the labeled antibody may be contained in a container (e.g., a tube or plate) that is a solid phase different from (1). The kit of the present invention may also be configured according to the type of immunoassay to be employed. For example, when the sandwich method is employed, the kit of the present invention may include, as optional components, a label, an aqueous solution (e.g., a buffer solution), a substrate that reacts with the labeled substance, and a human Aβ42 preparation.

[0074] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0075] Reference Example 1: Preparation of alkaline phosphatase-labeled anti-amyloid β antibody solution Desalted alkaline phosphatase (ALP) and N-(4-maleimidobutyryloxy)-succinimide (GMBS) were mixed and left to stand at 30°C for 1 hour to obtain maleimidated ALP. Next, in a coupling reaction solution (100 mM phosphate buffer, 1 mM EDTA 2Na, pH 6.0), a Fab'-labeled anti-amyloid β antibody recognizing amyloid β1-5 was mixed with maleimidated ALP at a molar ratio of 1:1 and reacted. The reacted antibody and ALP mixture was subjected to column chromatography using Superdex 200 10 / 300 (trade name, manufactured by GE) and purified with a label diluent (50 mM MES buffer, 150 mM NaCl, 0.3 mM ZnCl ２ , 1 mM MgCl ２ , 0.1% NaN ３The main peak was separated and purified using a 50 mM MES buffer (2.0% BSA, pH 6.8) at a flow rate of 0.5 mL / min to obtain an ALP-labeled anti-amyloid β antibody (ALP-labeled anti-Aβ antibody). During measurement, the ALP-labeled anti-Aβ antibody was diluted with a label diluent (50 mM MES buffer, 150 mM NaCl, 0.3 mM ZnCl ２ , 1 mM MgCl ２ , 0.1% NaN ３ , 2.0% BSA, pH 6.8) to prepare an ALP-labeled anti-Aβ antibody solution.

[0076] Test Example 1: Preparation of Particle Solution (1) Preparation of Anti-Amyloid β1-40 Antibody Immobilized Particle Solution After washing magnetic particles having carboxy groups with ion-exchanged water and 50 mM MES (pH 5.5), an EDC solution and an N-hydroxysuccinimide (NHS) solution were added, and the solution was subjected to rotary stirring at 25°C for 30 minutes. The supernatant after rotary stirring was removed, and the particles were washed twice with solid-phase buffer (50 mM MMES (pH 5.5)) by stirring. Next, a mouse anti-amyloid β1-40 antibody (anti-Aβ40 antibody) that specifically binds to amyloid β1-40, solubilized in the solid-phase buffer at 1 mg / ml, was added to the washed particles to a final concentration of 0.5 mg / ml, and the solution was subjected to rotary stirring at 25°C for 60 minutes (antibody sensitization step). Aminoethyl PEG (EVERBRITE® series) having the molecular weight shown in Table 1, dissolved at 375 mg / ml in the solid-phase buffer, was added (final concentration: 62.5 mg / ml) and stirred at 25°C for 30 minutes (PEG sensitization step). As a control, particles were prepared using the same procedure except that PEG was not added. Without removing the supernatant after stirring, a 1 M Tris, 10% BSA (pH 7.0) solution was added to each sample and stirred at 25°C for 30 minutes (reaction termination step). The supernatant after stirring was removed, and a masking solution (0.1 M Tris, 2% BSA, 1 mM EDTA, 0.1% NaN ３, pH 9.0) was added and stirred at 37°C for 16-24 hours (masking step). After stirring, the supernatant was removed, and buffer exchange was performed with a storage solution (50 mM Tris, 150 mM NaCl, 1 mM EDTA 2Na). Anti-Aβ1-40 antibody-immobilized particles (conditions 1 to 7) were prepared using PEG without PEG and PEG with molecular weights of 2,000, 5,000, 10,000, 20,000, 30,000, and 40,000, respectively. After stirring, the supernatant was removed, and the prepared magnetic particles were suspended in particle diluent (50 mM MES buffer, 150 mM NaCl, 1 mM EDTA 2Na, 0.5% BSA, pH 6.0), yielding seven conditions of anti-Aβ1-40 antibody-immobilized particle solutions.

[0077] (2) Preparation of Anti-Amyloid β1-42 Antibody Immobilized Particle Solution After washing magnetic gelatin particles with carboxyl groups with ion-exchanged water and 50 mM MES (pH 5.5), EDC solution and N-hydroxysuccinimide (NHS) solution were added and the mixture was stirred at 25°C for 30 minutes. The supernatant after the stirring was removed, and the particles were washed twice with solid-phase buffer (50 mM MES (pH 5.5)) by stirring. Next, a mouse anti-amyloid β1-42 antibody (anti-Aβ42 antibody) that specifically binds to amyloid β1-42, solubilized in the solid-phase buffer at 1 mg / ml, was added to the washed particles to a final concentration of 0.5 mg / ml, and the mixture was stirred at 25°C for 60 minutes (antibody sensitization step). Aminoethyl PEG (EVERBRITE® series) having the molecular weight shown in Table 1, dissolved at 375 mg / ml in the solid-phase buffer, was added (final concentration: 62.5 mg / ml) and stirred at 25°C for 30 minutes (PEG sensitization step). As a control, particles were prepared using the same procedure except that PEG was not added. Without removing the supernatant after stirring, a 1 M Tris, 10% BSA (pH 7.0) solution was added to each sample and stirred at 25°C for 30 minutes (reaction termination step). The supernatant after stirring was removed, and a masking solution (0.1 M Tris, 2% BSA, 1 mM EDTA, 0.1% NaN ３, pH 9.0) was added and stirred at 37°C for 16-24 hours (masking step). After stirring, the supernatant was removed, and buffer exchange was performed with a storage solution (50 mM Tris, 150 mM NaCl, 1 mM EDTA 2Na). Anti-Aβ1-42 antibody-immobilized particles (conditions 1 to 7) were prepared using PEG without PEG and PEG with molecular weights of 2,000, 5,000, 10,000, 20,000, 30,000, and 40,000, respectively. After stirring, the supernatant was removed, and the prepared magnetic particles were suspended in particle diluent (50 mM MES buffer, 150 mM NaCl, 1 mM EDTA 2Na, 0.1% Proclin, 0.5% BSA, pH 6.0), yielding seven conditions of anti-Aβ1-42 antibody-immobilized particle solutions.

[0078]

[0079] Test Example 2: Measurement of amyloid β (1) Measurement of amyloid β1-40 Amyloid β1-40 antigen solution (manufactured by Fujirebio Inc., concentrations: 0, 100, 1,000 pg / mL) was used as the measurement subject.

[0080] 20 μL of sample and 150 μL of anti-Aβ1-40 antibody solid-phase particle solution were dispensed into a reaction vessel and stirred. After that, the mixture was incubated at 37 ° C for 10 minutes, and the particles in the reaction vessel were collected with a magnet. The reaction vessel was then washed with a washing solution (0.05% Tween (registered trademark) 20 / PBS), whereby B / F separation and washing were performed. 150 μL of ALP-labeled anti-Aβ antibody solution was further dispensed into the reaction vessel, and after stirring, the mixture was incubated at 37 ° C for 10 minutes. The particles in the reaction vessel were collected with a magnet, and the reaction vessel was then washed with a washing solution, whereby B / F separation and washing were performed. Then, 200 μL of Lumipulse (registered trademark) substrate solution (Fujirebio Inc.) containing the chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) was dispensed into the reaction vessel, stirred, and incubated at 37°C for 5 minutes, after which the amount of luminescence (counts) was measured using a luminometer. Actual measurements were performed using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse G1200 (Fujirebio Inc.)).

[0081] (2) Measurement of Amyloid β1-42 Amyloid β1-42 antigen solution (manufactured by Fujirebio Inc., concentrations: 0, 30, 1,000 pg / mL) was used as the measurement subject.

[0082] 80 μL of sample was taken and dispensed into a reaction vessel containing 150 μL of anti-Aβ1-42 antibody solid-phase particle solution and stirred. This was then incubated at 37°C for 10 minutes, followed by B / F separation and washing using a magnetic field. 150 μL of ALP-labeled anti-Aβ antibody solution was further dispensed into this reaction vessel, stirred, and incubated at 37°C for 10 minutes. The particles in the reaction vessel were collected with a magnet, and the reaction vessel was washed with a washing solution, thereby performing B / F separation and washing. Subsequently, 200 μL of Lumipulse (registered trademark) substrate solution (manufactured by Fujirebio Inc.) containing the chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) was dispensed into the reaction vessel, stirred, and incubated at 37°C for 5 minutes, after which the luminescence (counts) was measured using a luminometer. Actual measurements were carried out using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse G1200 (Fujirebio)).

[0083] Table 2 shows the results for amyloid β1-40, including count values, average count values, and the values ​​(S / N ratios) of "average count values ​​for 100 pg / mL samples" / "average count values ​​for 0 pg / mL samples," "S / N ratios for each of conditions 2 to 7" / "S / N ratio for condition 1" x 100(%). Table 3 shows the results for amyloid β1-42, including count values, average count values, and the values ​​(S / N ratios) of "average count values ​​for 100 pg / mL samples" / "average count values ​​for 0 pg / mL samples," "S / N ratios for each of conditions 9 to 14" / "S / N ratio for condition 8" x 100(%).

[0084]

[0085]

[0086] As shown in Table 2, for amyloid β1-40, the S / N value was highest under the condition without PEG addition (condition 1), and the S / N value was lower under the conditions with PEG addition (conditions 2 to 7) compared to condition 1. In particular, when PEG with a molecular weight of 2,000 and PEG with a molecular weight of 5,000 were used, the S / N values ​​were 59% and 63%, respectively, of the S / N value under condition 1.

[0087] On the other hand, as shown in Table 3, for amyloid β1-42, under the conditions where PEG with a molecular weight of 2,000 to 40,000 was added (conditions 9 to 14), the S / N value was improved to 106 to 142% of the S / N value under condition 1. In particular, under the conditions where PEG with a molecular weight of 2,000 or 5,000 was used (conditions 9 and 10), the S / N value was improved by about 40% compared to condition 1, and under the conditions where PEG with a molecular weight of 10,000 or 20,000 was used (conditions 11 and 12), the S / N value was improved by about 20% compared to condition 1.

[0088] Test Example 3: Study of PEG Concentration Using solutions of immobilized anti-Aβ1-42 antibody particles containing different concentrations of aminoethyl PEG, a study was conducted to determine whether the concentration of PEG added after antibody sensitization affected reactivity. Specifically, aminoethyl PEG (EVERBRITE (registered trademark)) with a molecular weight of 5,000 dissolved in a solid-phase buffer was added to the particles to give final concentrations of aminoethyl PEG of 0 mg / ml, 15 mg / ml, 30 mg / ml, 62.5 mg / ml, and 83.3 mg / ml, respectively. The remaining steps were performed in the same manner as in the case of the immobilized anti-Aβ1-42 antibody particle solution described in Test Example 1, to obtain five conditions of immobilized anti-Aβ1-42 antibody particle solutions (conditions 15 to 19).

[0089] Thereafter, amyloid β1-42 was measured for amyloid β1-42 antigen solutions (manufactured by Fujirebio Inc., concentrations: 0, 30, 1,000 pg / mL) and one plasma sample using the measurement method described for amyloid β1-42 measurement in Test Example 2. Table 4 shows the count values, average count values, the value of "average count value for 30 pg / mL sample" / "average count value for 0 pg / mL sample" (S / N value), and the value of "S / N value for each of conditions 16 to 19" / "S / N value for condition 15" × 100 (%).

[0090]

[0091] As shown in Table 4, when PEG was added to a final concentration of 15 mg / ml to 83.3 mg / ml during particle preparation, an improvement in the S / N value was confirmed at all concentrations, and a tendency for the S / N value to increase depending on the PEG concentration added was observed.

[0092] Test Example 4: Investigation of particle preparation process The influence of the presence or absence of a PEG sensitization step, a reaction termination step, and a masking step in the preparation process of anti-amyloid β1-42 antibody-immobilized particles on the amyloid β1-42 measurement system was examined. Amyloid β1-42 antigen solutions (manufactured by Fujirebio Inc., concentrations: 0, 30, 1,000 pg / mL) and one plasma sample were measured for amyloid β1-42 by the method described in Test Example 2, using anti-Aβ1-42 antibody-immobilized particles prepared without the PEG sensitization step of the preparation process for anti-Aβ1-42 antibody-immobilized particles described in Test Example 1 (Condition 20), anti-Aβ1-42 antibody-immobilized particles prepared by the same method as the method described in Test Example 1 (Condition 21), and anti-Aβ1-42 antibody-immobilized particles prepared without the reaction termination step and masking step of the preparation process for anti-Aβ1-42 antibody-immobilized particles described in Test Example 1 (Condition 22).

[0093]

[0094] As a result, when conditions 20 and 21 were compared, the addition of PEG improved the S / N ratio by 137%. Furthermore, under condition 22, which omitted the masking step with a BSA-containing reagent, there was no effect on back counts, and the count increase from the second concentration onwards improved, resulting in a 145% improvement in the S / N ratio. This demonstrates that the sensitivity improvement effect is achieved not by the effect of stopping the reaction, but by the binding of PEG to the magnetic particles.

[0095] From the above, it was shown that the particles prepared by adding PEG after antibody sensitization did not have an improved S / N ratio for amyloid β1-40, but the S / N value (i.e., detection sensitivity) was specifically improved only for amyloid β1-42, and that the detection sensitivity improved as the molecular weight of PEG decreased.

[0096] Test Example 5: Study using anti-amyloid β1-42 antibodies of different clones In order to confirm that the improvement in S / N value due to the addition of PEG, as confirmed in Test Example 2, is not an event specific to the anti-amyloid β1-42 antibody but an event caused by the amyloid β1-42 being measured, an anti-amyloid β1-42 antibody of a clone different from the anti-amyloid β1-42 antibody used in Test Examples 1 to 4 was used to confirm the reactivity to amyloid β1-42. Using PEG-free anti-Aβ1-42 antibody-immobilized particles (Condition 23) and anti-Aβ1-42 antibody-immobilized particles (Condition 24) prepared via a PEG sensitization step, which were prepared in the same manner as Conditions 1 and 3 described in Test Example 1, except that different clones of anti-Aβ1-42 antibodies were used, amyloid β1-42 antigen solutions (manufactured by Fujirebio Inc., concentrations: 0, 30, 100, and 1,000 pg / mL) and two plasma samples were measured using the measurement method described for amyloid β1-42 measurement in Test Example 2. Table 6 shows the count values, average count values, and the value (S / N ratio) of "average count values ​​for 30 pg / mL samples" / "average count values ​​for 0 pg / mL samples," and the value (relative to Condition 23) of "S / N ratio under Condition 24" / "S / N ratio under Condition 23" × 100 (%).

[0097]

[0098] As a result, even when anti-Aβ1-42 antibody immobilized particles were used that used an anti-Aβ1-42 antibody of a clone different from the anti-Aβ1-42 antibody used in Test Examples 1 to 4, the S / N value was improved by 43% under the PEG-sensitized condition (Condition 24) compared to the condition without PEG sensitization (Condition 23).

[0099] From the above, it was shown that the improvement in S / N value due to the use of anti-Aβ1-42 antibody immobilized particles prepared by adding PEG is not an event specific to the anti-amyloid β1-42 antibody, but an event caused by the amyloid β1-42 being measured.

Claims

1. A solid phase bearing both an anti-human amyloid beta (Aβ) 42 antibody and polyethylene glycol (PEG).

2. The solid phase of claim 1, wherein the solid phase is a particle.

3. The solid phase of claim 1, wherein both the anti-human Aβ42 antibody and the PEG are attached to the solid phase by a covalent bond.

4. The solid phase of claim 1, wherein both the anti-human Aβ42 antibody and PEG are covalently attached to the solid phase.

5. The solid phase of claim 4, wherein the covalent bond is an amide bond.

6. The solid phase according to claim 5, wherein the amide bond is formed by reaction between the solid phase having a carboxy group, the anti-human Aβ42 antibody having an amino group, and the modified PEG having an amino group.

7. The solid phase according to claim 1, wherein the molecular weight of PEG is 8,000 or less.

8. The solid phase according to claim 1, wherein the anti-human Aβ42 antibody is an antibody that recognizes an epitope containing GGVVIA (SEQ ID NO: 5).

9. A method for producing a solid phase, comprising contacting a solid phase in a solution with an anti-human Aβ42 antibody and PEG to obtain the solid phase according to any one of claims 1 to 8.

10. The method of claim 9, comprising: (1) contacting an anti-human Aβ42 antibody with a solid phase in a first solution to obtain a solid phase carrying the anti-human Aβ42 antibody; and (2) contacting PEG with the solid phase carrying the anti-human Aβ42 antibody in a second solution to obtain a solid phase of any one of claims 1 to 8.

11. A method for testing human Aβ42, which comprises measuring human Aβ42 in a sample using the solid phase according to any one of claims 1 to 8.

12. The method of claim 11, wherein the sample is a blood sample or cerebrospinal fluid.

13. The method according to claim 11, comprising: (1) immobilizing human Aβ42 in a sample on a solid phase using the solid phase according to any one of claims 1 to 8; (2) labeling the human Aβ42 immobilized on the solid phase with a labeled antibody against human Aβ42 (wherein the labeled antibody against human Aβ42 is an antibody that recognizes an epitope different from that of the anti-human Aβ42 antibody retained on the solid phase); and (3) measuring the labeled human Aβ42 immobilized on the solid phase.

14. A test reagent for human Aβ42, comprising a solid phase bearing both an anti-human Aβ42 antibody and PEG.

15. A test kit for human Aβ42, comprising: (1) a solid phase carrying both an anti-human Aβ42 antibody and PEG; and (2) a labeled antibody against human Aβ42 (wherein the labeled antibody against human Aβ42 is an antibody that recognizes an epitope different from that of the anti-human Aβ42 antibody carried on the solid phase).

Images