Anti-peg antibody-binding material and detection method for Anti-peg antibody

Abstract

To provide an anti-PEG antibody-binding material that makes it possible to detect an anti-PEG antibody.SOLUTION: An anti-PEG antibody-binding material according to embodiments includes a cellulose oligomer represented by general formula (1). In formula (1), n represents a number from 6 to 16, m represents a number that is at least 3, and A represents a C1-24 divalent organic group which links the polyethylene glycol chain to the oxygen atom bonded to the anomeric carbon at the reducing end of the cellulose oligomer and includes at least one type of heteroatom selected from the group consisting of nitrogen atoms, oxygen atoms and sulfur atoms.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to an anti-PEG antibody-binding material and a method for detecting an anti-PEG antibody using the same. [Background technology]

[0002] Modification of the surface of proteins, liposomes, micelles, etc. with PEG (polyethylene glycol) improves the retention in the blood of the modified substance and reduces immunogenicity. For this reason, many PEG-modified drugs are currently in clinical use.

[0003] However, it has been revealed that PEG modification induces the secretion of antibodies against PEG (anti-PEG antibodies). If a PEG-modified drug is administered while anti-PEG antibodies are present in the blood, the PEG-modified drug may be removed from the blood (ABC phenomenon), resulting in reduced blood retention and side effects. Therefore, there is a need to detect anti-PEG antibodies present in the blood with high sensitivity in order to evaluate the induction of anti-PEG antibodies and their effect on the therapeutic efficacy of PEG-modified drugs.

[0004] Anti-PEG antibodies include backbone-selective anti-PEG antibodies and methoxy-terminated PEG-selective anti-PEG antibodies. Backbone-selective anti-PEG antibodies are antibodies that recognize the repeating oxyethylene structure that is the backbone of the PEG structure and specifically bind to the PEG chain. Methoxy-terminated PEG-selective anti-PEG antibodies are antibodies that have high selectivity for methoxy-terminated PEG, that is, they specifically bind to PEG chains that have a methoxy group at the end.

[0005] As an example of technology relating to biosensors containing PEG chains, Patent Document 1 discloses a biosensor containing PEG-modified nanoparticles in which functional groups or functional moieties are attached to metal (oxide) particles or semiconductor particles via two or more PEG chains.

[0006] Furthermore, Patent Documents 2 and 3 disclose biosensors that include hydrophilic particles, which include water-insoluble particles, a primer layer containing polysiloxane disposed on the surface of the particles, and a hydrophilic polymer layer disposed on the primer layer, the hydrophilic polymer layer being composed of PEG chains to which antibodies are bound and PEG chains to which inactive groups are bound. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2005-180921 A [Patent Document 2] JP 2020-143964 A [Patent Document 3] Patent Publication No. 2021-012089 Summary of the Invention [Problem to be solved by the invention]

[0008] In the course of their research aimed at detecting anti-PEG antibodies present in the blood with high sensitivity, the inventors discovered that certain cellulose oligomers bearing PEG chains specifically bind to anti-PEG antibodies.

[0009] The embodiments of the present invention have been made in consideration of the above points, and an object of the present invention is to provide an anti-PEG antibody-binding material capable of binding to an anti-PEG antibody, and a method for detecting an anti-PEG antibody using the same. [Means for solving the problem]

[0010] The present invention includes the embodiments set forth below. [1] An anti-PEG antibody binding material comprising a cellulose oligomer represented by general formula (1): [ka] (In formula (1), n ​​is an average degree of polymerization and is a number from 6 to 16, m is an average degree of polymerization and is a number equal to or greater than 3, and A is a divalent organic group that links an oxygen atom bonded to the anomeric carbon at the reducing end of a cellulose oligomer to a polyethylene glycol chain, and is a divalent organic group having 1 to 24 carbon atoms and containing at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom.) [2] The anti-PEG antibody-binding material according to [1], wherein A in the formula (1) represents a divalent organic group having 2 to 10 carbon atoms containing at least one group selected from the group consisting of a triazole group, an amide group, and a thioether group. [3] In the formula (1), A is [ka] where R 1 represents a hydrocarbon group having 1 to 10 carbon atoms, R 2 represents a single bond or a hydrocarbon group having 1 to 10 carbon atoms. [4] The anti-PEG antibody-binding material according to any one of [1] to [3], wherein m in formula (1) is a number of 10 or more.

[0011] [5] A biosensor comprising the anti-PEG antibody binding material according to any one of [1] to [4]. [6] An adsorbent comprising the anti-PEG antibody-binding material described in any one of [1] to [4].

[0012] [7] A method for detecting an anti-PEG antibody, comprising: incubating a cellulose oligomer represented by the above general formula (1) with a specimen suspected of containing an anti-PEG antibody under conditions that allow binding to the anti-PEG antibody; incubating the cellulose oligomer after incubation with the specimen with a sample containing the secondary antibody under conditions that allow binding of a secondary antibody to the anti-PEG antibody; and using the cellulose oligomer after incubation with the sample to detect the anti-PEG antibody contained in the specimen with the secondary antibody as a probe. Effect of the Invention

[0013] According to an embodiment of the present invention, there is provided an anti-PEG antibody-binding material capable of binding to an anti-PEG antibody, which can be used, for example, in a biosensor for detecting an anti-PEG antibody or in an adsorbent for adsorbing an anti-PEG antibody. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a conceptual diagram of a cellulose nanosheet (cellulose oligomer assembly) made of cellulose oligomers constituting an anti-PEG antibody binding material according to one embodiment. [Diagram 2] 1 is a reaction scheme of a PEG-introduced cellulose oligomer according to one embodiment. [Diagram 3] FIG. 1 is an explanatory diagram showing a method for detecting an anti-PEG antibody according to one embodiment. [Figure 4] FIG. 2 shows mass spectra of the cellulose oligomer assemblies obtained in Synthesis Examples 1 to 4. [Diagram 5] FIG. 2 shows ATR / FT-IR spectra of the cellulose oligomer assemblies obtained in Synthesis Examples 1 to 4. [Figure 6] 1 is a graph showing the results of evaluation of the binding of an anti-PEG antibody (PEG-B-47) to each cellulose oligomer assembly in Test Example 1 (absorbance at 490 nm). [Figure 7] 1 is a graph showing the results of evaluation of the binding of anti-PEG antibody (15-2b) to each cellulose oligomer assembly in Test Example 2 (absorbance at 490 nm). [Figure 8] 1 is a graph showing the results of evaluation of the binding of anti-PEG antibody (6.3) to each cellulose oligomer assembly in Test Example 3 (absorbance at 490 nm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The anti-PEG antibody-binding material according to this embodiment contains a cellulose oligomer represented by general formula (1) (hereinafter referred to as a PEG-introduced cellulose oligomer). The PEG-introduced cellulose oligomer is a cellulose oligomer having a structure in which glucose is linked by a β-1,4-glucoside bond, and a substituent containing a polyethylene glycol chain (PEG chain) is introduced to the anomeric carbon at the reducing end. In detail, a PEG chain having a methoxy group at its end is bound to the oxygen atom bonded to the anomeric carbon via a linking group A. By interposing the linking group A in this way, PEG chains of various chain lengths can be introduced into the cellulose oligomer.

[0016] [ka]

[0017] In formula (1), n ​​is a number from 6 to 16, m is a number of 3 or more, and A is a divalent organic group. The wavy line in formula (1) indicates that the configuration of the anomeric position at the reducing end is an α-form, a β-form, or a mixture of an α-form and a β-form.

[0018] The above n represents the average degree of polymerization (DP) of the cellulose oligomer and is 6 to 16. n may be 6 to 14, 6.5 to 13, or 7 to 12. The average degree of polymerization of the cellulose oligomer is a weighted average (number average degree of polymerization) of the degrees of polymerization according to the ratio of the number of molecules of cellulose oligomers having different degrees of polymerization. The degree of polymerization of each PEG-introduced cellulose oligomer is not particularly limited, and may be, for example, 4 to 20, 5 to 16, or 6 to 13.

[0019] The above m represents the average degree of polymerization (DP) of the PEG chain, and is 3 or more. m can be set according to the selectivity of the anti-PEG antibody to be bound, and by setting m to, for example, 10 or more, backbone-selective anti-PEG antibody is easily bound. The upper limit of m is not particularly limited, but is preferably, for example, 500 or less. In one embodiment, m may be 3 to 500, 10 to 250, 16 to 150, 18 to 100, or 20 to 50. The average degree of polymerization of the PEG chain is a weighted average value of the degrees of polymerization according to the molecular ratio of PEG chains having different degrees of polymerization. Note that, when there is no distribution in the molecular weight of the PEG chain (i.e., when the polydispersity is 1), m takes an integer value as a simple degree of polymerization.

[0020] The above A is an oxygen atom bonded to the anomeric carbon at the reducing end of the cellulose oligomer and a PEG chain (i.e., (CH2CH2O) m ) and represents a divalent organic group having 1 to 24 carbon atoms and containing at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom.

[0021] The number of carbon atoms in the linking group A is preferably 1 to 20, more preferably 2 to 15, and even more preferably 2 to 10. Examples of the heteroatom-containing group contained in the linking group A include a triazole group, an amide group, a thioether group, a maleimide group, an ether group, and an ester group, and the cellulose oligomer and the PEG chain are linked via these heteroatom-containing groups. Among these, from the viewpoints of ease of synthesis, bond stability, and the like, it is preferable that the linking group A contains a triazole group, an amide group, or a thioether group.

[0022] In one embodiment, A in formula (1) preferably represents a divalent organic group having 2 to 10 carbon atoms containing at least one type selected from the group consisting of a triazole group, an amide group, and a thioether group.

[0023] More specifically, A in formula (1) is [ka] It is preferred that

[0024] Here, R 1 R represents a hydrocarbon group (divalent hydrocarbon group) having 1 to 10 carbon atoms and is bonded to the oxygen atom bonded to the anomeric carbon at the reducing end. 1 R is preferably a linear or branched alkanediyl group, and more preferably a linear alkanediyl group. 1 The hydrocarbon group preferably has 1 to 6 carbon atoms, and more preferably 1 to 3 carbon atoms.

[0025] R 2 R represents a single bond or a hydrocarbon group having 1 to 10 carbon atoms and is bonded to the PEG chain. 2 R is preferably a single bond or a linear or branched alkanediyl group, and more preferably a single bond (i.e., a direct bond). 2 The hydrocarbon group preferably has 1 to 6 carbon atoms, and more preferably 1 to 3 carbon atoms.

[0026] In one embodiment, the PEG-introduced cellulose oligomer is preferably represented by the following general formula (2). In this case, the steric configuration of the anomeric position at the reducing end is a β-form. 1 and R 2 is as stated above. [ka]

[0027] The PEG-introduced cellulose oligomer may be included in a cellulose oligomer assembly having a cellulose type II crystal structure. The cellulose oligomer assembly may have a sheet-like structure (cellulose nanosheet). Here, the sheet-like structure is a concept that includes a ribbon-like structure (cellulose nanoribbon) as shown in FIG. 1.

[0028] While naturally occurring cellulose chains have a cellulose type I crystal structure in which they are arranged in parallel, artificially synthesized cellulose oligomers generally form the thermodynamically stable cellulose type II crystal structure. In this case, the substituents at the ends of the cellulose chains do not affect the crystal shape, and cellulose oligomers with substituents are aligned in the thickness direction of the cellulose nanosheet to form lamellar crystals, with the substituents exposed on the surface of the sheet.

[0029] In detail, as one embodiment, a structure in which a substituent containing a PEG chain is introduced into some of the cellulose oligomers constituting the cellulose nanosheet is shown in FIG. 1. In FIG. 1, the PEG-introduced cellulose oligomer is shown with an arrow pointing from the reducing end to the non-reducing end, as shown below the above formula (1), and the substituent containing a PEG chain is shown with a wavy line. In addition, for a cellulose oligomer in which a PEG chain is not introduced, the cellulose chain is shown with an arrow pointing from the reducing end to the non-reducing end. As shown in the cross-sectional structure in FIG. 1, the cellulose oligomer containing the PEG-introduced cellulose oligomer is arranged in the film thickness direction so that the directions of the arrows are alternated, and the substituent containing a PEG chain is exposed on the sheet surface.

[0030] The synthesis method of the PEG-introduced cellulose oligomer is not particularly limited, but may be as follows. That is, for example, a cellulose oligomer having a functional group introduced at the anomeric position of the reducing end is used to prepare a cellulose nanosheet from the functional group-introduced cellulose oligomer. The PEG chain can be introduced into the cellulose oligomer via the linking group A by reacting a compound having a PEG chain (PEG compound) with the functional group exposed on the sheet surface of the cellulose nanosheet. In this way, a PEG chain with a long chain length can be introduced by using a cellulose nanosheet having a functional group exposed on the sheet surface and introducing the PEG chain by reacting with the functional group. Therefore, it becomes possible to bind with an anti-PEG antibody that specifically binds to the long PEG chain.

[0031] Examples of the reaction between the functional group and a compound having a PEG chain include a click reaction between an azide group and an alkynyl group, an amidation reaction between a carboxy group and an amino group, an esterification reaction between a carboxy group and a hydroxy group, a thiol-ene reaction between a thiol group and a carbon-carbon double bond, and a reaction between a thiol group and an organic halide.

[0032] FIG. 2 is a reaction scheme of a PEG-introduced cellulose oligomer according to one embodiment. A cellulose nanosheet having an ethynyl group exposed on the surface as a functional group is used, and a metal catalyst such as a copper catalyst is used to cause a click reaction with a PEG compound having an azide group. This introduces a PEG chain into the cellulose oligomer via a linking group A containing a triazole group. This is expressed by the chemical formula shown in reaction formula (3) below. In reaction formula (3), a click reaction is caused between a cellulose oligomer having a propargyl group (CEL-C≡CH) and methoxypolyethylene glycol azide (mPEG azide). [ka]

[0033] In reaction formula (3), the functional group of the cellulose oligomer is an alkynyl group, and the functional group of the PEG compound is an azide group, but this may be reversed. That is, as shown in reaction formula (4) below, a cellulose oligomer having an azide group as a functional group and a PEG compound having an ethynyl group may be subjected to a click reaction. [ka]

[0034] In another embodiment, as shown in the following reaction scheme (5), a PEG chain may be introduced into the cellulose oligomer via the linking group A containing an amide group by subjecting a cellulose oligomer having an amino group as a functional group to an amidation reaction with a PEG compound having a carboxy group. [ka]

[0035] In reaction formula (5), the functional group of the cellulose oligomer is an amino group, and the functional group of the PEG compound is a carboxy group, but this may be reversed. That is, as shown in the following reaction formula (6), a cellulose oligomer having a carboxy group as a functional group and a PEG compound having an amino group may be subjected to an amidation reaction. [ka]

[0036] In another embodiment, as shown in the following reaction formula (7), a PEG chain may be introduced into the cellulose oligomer via the linking group A containing a thioether group by subjecting a cellulose oligomer having a thiol group as a functional group to a PEG compound having a maleimide group through a thiol-ene reaction. [ka]

[0037] In reaction formula (7), the functional group of the cellulose oligomer is a thiol group, and the functional group of the PEG compound is a maleimide group, but this may be reversed. That is, as shown in reaction formula (8) below, a cellulose oligomer having a maleimide group as a functional group and a PEG compound having a thiol group may be subjected to a thiol-ene reaction. [ka]

[0038] In another embodiment, as shown in the following reaction scheme (9), a PEG chain may be introduced into a cellulose oligomer via a linking group A containing a thioether group by reacting a cellulose oligomer having a thiol group as a functional group with a PEG compound having a halogen atom. [ka]

[0039] The anti-PEG antibody binding material according to this embodiment contains a PEG-introduced cellulose oligomer, and is preferably contained as a cellulose oligomer assembly such as a cellulose nanosheet. That is, in one embodiment, the anti-PEG antibody binding material preferably contains a cellulose oligomer assembly containing a PEG-introduced cellulose oligomer. The cellulose oligomer constituting the cellulose oligomer assembly may be only a PEG-introduced cellulose oligomer, or may contain a cellulose oligomer without a PEG chain introduced therein together with the PEG-introduced cellulose oligomer.

[0040] When a cellulose oligomer assembly consisting of functionalized cellulose oligomers is used as described above and reacted with a PEG compound, the anti-PEG antibody binding material may contain a cellulose oligomer assembly containing a PEG-introduced cellulose oligomer and a functionalized cellulose oligomer. In the cellulose oligomer assembly, the ratio of the PEG-introduced cellulose oligomers to the total cellulose oligomers constituting the assembly (PEG introduction rate) is not particularly limited and may be, for example, 1 mol% or more, preferably 5 to 80 mol%, more preferably 7 to 60 mol%, and even more preferably 10 to 50 mol%.

[0041] The PEG-introduced cellulose oligomer binds to anti-PEG antibodies such as backbone-selective anti-PEG antibodies and methoxy-terminated PEG-selective anti-PEG antibodies, but does not bind to secondary antibodies as probes or other proteins such as BSA (bovine serum albumin) and lysozyme. Therefore, it can be used as an anti-PEG antibody binding material that can detect or adsorb anti-PEG antibodies.

[0042] Examples of anti-PEG antibodies to which PEG-introduced cellulose oligomers can be bound include backbone-selective anti-PEG antibodies and / or methoxy-terminated PEG-selective anti-PEG antibodies. The class of anti-PEG antibodies is not particularly limited, and examples include IgG, IgG1, IgG2b, and IgM.

[0043] A backbone-selective anti-PEG antibody is a monoclonal antibody that recognizes the repeating oxyethylene structure that is the backbone of the PEG structure and specifically binds to the PEG chain. The molecular weight of the PEG chain that can specifically bind varies depending on the type of anti-PEG antibody. Therefore, the average degree of polymerization m of the PEG chain in the above formula (1) can be set according to the type of anti-PEG antibody to be detected or adsorbed.

[0044] For example, since the anti-PEG antibody "6.3" specifically binds to the backbone of a PEG chain of 750 Da (DP=16) or more, when attempting to detect or adsorb this, the average degree of polymerization m of the PEG chain in formula (1) should be set to 16 or more. For example, the anti-PEG antibody "AGP4" specifically binds to a PEG chain of 2000 Da (DP=45) or more, when attempting to detect or adsorb this, the average degree of polymerization m of the PEG chain in formula (1) should be set to 45 or more.

[0045] The methoxy-terminated PEG-selective anti-PEG antibody is a monoclonal antibody that recognizes PEG chains having a methoxy group at the end and specifically binds to the PEG chain. The methoxy-terminated PEG-selective anti-PEG antibody may also bind to PEG chains having an ethoxy group or a butoxy group at the end, which are more hydrophobic. The molecular weight of the PEG chain that can be specifically bound varies depending on the type of anti-PEG antibody. Therefore, the average degree of polymerization m of the PEG chains in the above formula (1) may be set according to the type of anti-PEG antibody to be detected or adsorbed.

[0046] For example, anti-PEG antibody "15-2b" specifically binds to PEG chains having a methoxy group at the end of 560 Da (DP=12) or more, so when attempting to detect or adsorb this, the average degree of polymerization m of the PEG chains in formula (1) should be set to 12 or more. For example, anti-PEG antibody "PEG-B-47" specifically binds to PEG chains having a methoxy group at the end and a DP of 3 or more, so when attempting to detect or adsorb this, the average degree of polymerization m of the PEG chains in formula (1) should be set to 3 or more.

[0047] The anti-PEG antibody-binding material according to the embodiment contains a PEG-introduced cellulose oligomer and is therefore capable of recognizing and binding to an anti-PEG antibody, and can therefore be used, for example, as a biosensor for detecting an anti-PEG antibody or an adsorbent for adsorbing an anti-PEG antibody.

[0048] The anti-PEG antibody binding material may consist solely of a PEG-introduced cellulose oligomer or a cellulose oligomer assembly that is an assembly containing the same, or may contain other components such as proteins, peptides, nucleic acids, lipids, sugars, cells, amino acids, metal ions, organic solvents, etc., within the scope that does not impair the effect of the material.

[0049] The PEG-introduced cellulose oligomer or cellulose oligomer aggregate is a substance that does not dissolve in water, and when prepared by the above synthesis method, it is obtained as an aqueous dispersion of cellulose nanosheets containing the PEG-introduced cellulose oligomer. The anti-PEG antibody-binding material according to one embodiment may be a dry powder obtained by removing water from the aqueous dispersion, and the powdered anti-PEG antibody-binding material may be prepared by using the dry powder as is or by powder-mixing it with other components such as proteins. The powdered anti-PEG antibody-binding material can be easily dispersed in water by adding water when used, and an aqueous dispersion can be prepared.

[0050] The anti-PEG antibody binding material according to one embodiment may be an aqueous dispersion in which a PEG-introduced cellulose oligomer or a cellulose oligomer assembly is dispersed in water. In this case, a buffer solution in which a buffering agent is added to water may be used as the dispersion medium, that is, the aqueous dispersion may be one in which a PEG-introduced cellulose oligomer or a cellulose oligomer assembly is dispersed in a buffer solution. In the aqueous dispersion, the content (concentration) of the PEG-introduced cellulose oligomer is not particularly limited and may be, for example, 0.01 to 5% (w / v) or 0.03 to 0.3% (w / v).

[0051] In this specification, "% (w / v)" refers to a mass volume percent concentration, which is the mass (g) of the subject substance contained in a volume of 100 mL.

[0052] The buffer is not particularly limited, and examples thereof include buffers such as phosphate buffered saline (PBS), 2-morpholinoethanesulfonic acid (MES) buffer, trishydroxymethylaminomethane (Tris) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer.

[0053] An anti-PEG antibody-binding material according to one embodiment may be a PEG-introduced cellulose oligomer or cellulose oligomer assembly immobilized on the surface of a substrate such as plastic or glass.

[0054] In one embodiment, a biosensor comprising the anti-PEG antibody binding material is used to perform a bioassay to detect and quantify anti-PEG antibodies, the biosensor referring to each element or assembly or combination of elements used to perform such a bioassay.

[0055] Therefore, the biosensor may be composed of, for example, only a powder of PEG-introduced cellulose oligomer or cellulose oligomer assembly, or only an aqueous dispersion of PEG-introduced cellulose oligomer or cellulose oligomer assembly. Alternatively, the biosensor may be a kit combining a powder or aqueous dispersion of PEG-introduced cellulose oligomer or cellulose oligomer assembly with a secondary antibody as a probe (i.e., an anti-PEG antibody detection kit), and further, for example, when the secondary antibody is an enzyme-labeled antibody, it may be an anti-PEG antibody detection kit combining a substrate corresponding to the enzyme.

[0056] The biosensor may contain albumin such as BSA, and it is preferable that albumin is added when incubating the PEG-introduced cellulose oligomer or cellulose oligomer assembly with the test specimen. In this case, albumin may be contained in the anti-PEG antibody binding material, or may be contained in the anti-PEG antibody detection kit separately from the anti-PEG antibody binding material. When albumin is contained in the anti-PEG antibody binding material, the albumin may be in a powder form mixed with the PEG-introduced cellulose oligomer or cellulose oligomer assembly, or the albumin may be dissolved in an aqueous dispersion. The ratio of the PEG-introduced cellulose oligomer to albumin is not particularly limited, and may be, for example, 0.002 / 1 to 0.3 / 1, or 0.01 / 1 to 0.1 / 1 in terms of the mass ratio of the PEG-introduced cellulose oligomer / albumin. When the test specimen contains albumin, such as blood, albumin may or may not be added additionally.

[0057] A method for detecting an anti-PEG antibody according to one embodiment includes the following steps. (1) incubating a PEG-incorporated cellulose oligomer with a test specimen under conditions that allow binding to an anti-PEG antibody; (2) incubating the PEG-introduced cellulose oligomer after incubation with the analyte with a sample containing a secondary antibody under conditions that allow binding of the secondary antibody; and (3) detecting anti-PEG antibodies contained in a specimen using the PEG-introduced cellulose oligomer after incubation with the specimen as a probe with a secondary antibody;

[0058] In step (1), a test specimen is added to a container together with an aqueous dispersion of a PEG-introduced cellulose oligomer as the anti-PEG antibody-binding material, mixed and dispersed, and then incubated. As the aqueous dispersion, an aqueous dispersion of a cellulose oligomer aggregate, which is an aggregate containing a PEG-introduced cellulose oligomer, is preferably used.

[0059] The specimen is a test substance suspected of containing anti-PEG antibodies. For example, when detecting anti-PEG antibodies present in blood to evaluate the effect of the induction of anti-PEG antibodies or the therapeutic effect of a PEG-modified drug, blood or a diluted version of the blood may be used as the specimen.

[0060] In step (1), as described above, albumin such as BSA may be added to improve the detection sensitivity of the anti-PEG antibody. In this case, the concentration of albumin in the system (the albumin concentration excluding the albumin originally contained in the test sample) is not particularly limited, but is preferably, for example, 0.05 to 10% by mass, and more preferably 0.5 to 5% by mass.

[0061] The incubation conditions in step (1) are not particularly limited as long as they allow binding of the PEG-introduced cellulose oligomer to the anti-PEG antibody, and may be, for example, 4 to 60° C. and 1 minute to 2 hours. The concentration of the PEG-introduced cellulose oligomer in the system is not particularly limited, and may be, for example, 0.001 to 1% (w / v), or 0.01 to 0.1% (w / v).

[0062] In step (1), the anti-PEG antibody to be detected is bound to the PEG-introduced cellulose oligomer (see FIG. 3). The PEG-introduced cellulose oligomer to which the anti-PEG antibody is bound is then separated from the specimen by centrifugation and washing.

[0063] In step (2), a sample containing a secondary antibody is added to the PEG-introduced cellulose oligomer (preferably a cellulose oligomer assembly containing it) to which an anti-PEG antibody has been bound, and the mixture is mixed and dispersed, followed by incubation.

[0064] The sample containing the secondary antibody may be an aqueous solution of the secondary antibody. The secondary antibody is not particularly limited as long as it can be used as a probe by specifically binding to the anti-PEG antibody, and may be, for example, an antibody labeled with an enzyme, a radioisotope, a fluorescent dye, or the like.

[0065] The incubation conditions in step (2) are not particularly limited as long as the anti-PEG antibody and the secondary antibody can bind to each other, and may be, for example, 4 to 60° C. and 1 minute to 2 hours. The concentration of the secondary antibody in the system is not particularly limited, and may be, for example, 100 to 1000 ng / mL.

[0066] In step (2), a secondary antibody is bound as a probe to the anti-PEG antibody bound to the PEG-introduced cellulose oligomer (see FIG. 3). Then, the PEG-introduced cellulose oligomer bound to the secondary antibody (preferably a cellulose oligomer assembly containing the PEG-introduced cellulose oligomer) is separated from the sample containing the secondary antibody by centrifugation and washing.

[0067] In step (3), a PEG-introduced cellulose oligomer having a secondary antibody bound thereto is used to detect and quantify anti-PEG antibodies contained in a specimen using the secondary antibody as a probe.

[0068] For example, when an enzyme-labeled antibody is used as the secondary antibody, an enzyme reaction is carried out by adding a substrate corresponding to the enzyme to the cellulose oligomer assembly to which the secondary antibody is bound, as shown in Figure 3. The pigment of the product thus generated is detected and quantified by absorbance (color development), fluorescence, chemiluminescence, or the like.

[0069] In another embodiment, an adsorbent comprising the anti-PEG antibody-binding material can be used to remove or purify the anti-PEG antibody by adsorbing the anti-PEG antibody.

[0070] The adsorbent may be composed of a powder or an aqueous dispersion of a PEG-introduced cellulose oligomer or a cellulose oligomer assembly containing the same, and these may contain other optional components.

[0071] When an adsorbent is used to remove anti-PEG antibodies, for example, the adsorbent is added to a liquid containing anti-PEG antibodies, and the anti-PEG antibodies are brought into contact with the PEG-introduced cellulose oligomer to bind to each other. The adsorbent to which the anti-PEG antibodies are adsorbed is then removed from the liquid by centrifugation or the like, thereby removing the anti-PEG antibodies from the liquid.

[0072] When the adsorbent is used for purifying an anti-PEG antibody, for example, the adsorbent is added to a liquid containing impurities together with the anti-PEG antibody, and the anti-PEG antibody and the PEG-introduced cellulose oligomer are brought into contact with each other to bind to each other. The adsorbent to which the anti-PEG antibody is adsorbed is then separated and recovered from the liquid by centrifugation or the like. The recovered adsorbent is, for example, treated with an acid to release the bond between the anti-PEG antibody and the PEG-introduced cellulose oligomer, so that the anti-PEG antibody can be separated and recovered from the adsorbent, and a highly pure anti-PEG antibody can be obtained. EXAMPLES

[0073] The present invention will be further explained below with reference to examples, but is not limited to these.

[0074] [Enzymatic synthesis of cellulose oligomers bearing propargyl groups] A 1M aqueous solution of α-glucose-1-phosphate (αG1P), a 1M 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (pH 7.5), a 100μM EDTA solution, and a 1M aqueous solution of 2-propynyl-β-D-glucoside (Glc-C≡CH) were mixed in a predetermined amount, and ultrapure water was used to prepare a solution of αG1P at 200mM, HEPES at 500mM, EDTA at 50μM, and Glc-C≡CH at 50mM. Here, "M" stands for "mol / L". Cellodextrin phosphorylase (CDP) was added to the obtained solution at 1U / mL, and then incubated at 60°C for 3 days. The liquid containing the precipitated product was centrifuged (20,400 × g, 5 min, 4 °C), the supernatant was removed, and then ultrapure water was added to redisperse the product. This centrifugal separation (under the same conditions) was repeated five times to obtain a cellulose oligomer having propargyl groups (CEL-C≡CH).

[0075] Here, the above 2-propynyl-β-D-glucoside was synthesized as follows. Tetra-O-acetyl-2-propynyl-β-D-glucoside was synthesized according to a previous report (HB Mereyala et al., Carbohydr. Polym. 1998, 307, 351-354). Then, 0.97 g of tetra-O-acetyl-propargyl-β-D-glucoside and 0.034 g of sodium methoxide were reacted in the presence of 25 mL of methanol and 25 mL of tetrahydrofuran at 25°C for 12 hours or more, and the reaction mixture was purified by silica gel column chromatography to obtain 2-propynyl-β-D-glucoside.

[0076] The fact that the product of the enzymatic synthesis was a cellulose oligomer having a propargyl group at one end was confirmed by using a proton nuclear magnetic resonance (NMR) spectrometer (AVANCE III HD500 (Bruker Biospin, magnetic field strength: 500 MHz, accumulation number 16)) using a sample prepared by dissolving the product in a 4% aqueous solution of sodium deuterium hydroxide at a concentration of 2% by mass or more.

[0077] The mass spectrum of the product was obtained by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and the average degree of polymerization of the cellulose oligomer was 7.5, and the polydispersity index (PDI) was 1.02. The measurement conditions of MALDI-TOF-MS and the calculation method of the average degree of polymerization and the polydispersity index (PDI) are as follows.

[0078] Measurements were performed using a MALDI-TOF MS instrument (AXIMA-performance, Shimadzu Corporation) under the following conditions: accelerating voltage 20 kV, linear (positive) ion mode, mass range 1-4000, max leaser rap rate 10, power 100, profiles 100, shots 2, ion gate blank 500 Da, pulsed extraction optimized at 1500 Da. The average degree of polymerization and polydispersity index (PDI) were calculated based on the integral value of the peak of sodium or potassium adduct of cellulose oligomer (CEL-C≡CH) with a propargyl group at one end with a degree of polymerization of 6 to 11.

[0079] [Introduction of PEG chains into cellulose oligomers] A click reaction was carried out between a cellulose oligomer having a propargyl group (CEL-C≡CH) and methoxypolyethylene glycol azide (mPEG azide) according to the reaction scheme shown in Figure 2 and the reaction shown in the above reaction formula (3). Four types of PEG-introduced cellulose oligomers with different PEG incorporation rates were prepared according to the following synthesis examples 1 to 4.

[0080] [Synthesis Example 1] CEL-C≡CH aqueous dispersion, 40 mM methyl-PEG24-azide (mPEG azide with m=24 in reaction formula (3)), DMSO (SP grade), 20 mM CuSO4 aqueous solution, 8 mM sodium ascorbate aqueous solution, and ultrapure water were added to a 5 mL tube so that CEL-C≡CH was 2 mg / mL, methyl-PEG24-azide was 2 mM, CuSO4 was 0.5 mM, and ascorbic acid was 0.2 mM, and the mixture was reacted for 1 hour by stirring at 25°C. At that time, the water / DMSO (volume ratio) was 1 / 1, and the volume of the reaction solution was 5000 μL. The liquid after the reaction was centrifuged (20,400 × g, 5 minutes, 4 ° C.), the supernatant was removed, and then ultrapure water was added to redisperse the product. The same centrifugation (under the same conditions) was repeated five times to obtain the cellulose oligomer aggregate (mEG 24 _1) was obtained.

[0081] [Synthesis Example 2] The amounts of methyl-PEG24-azide, CuSO4, and ascorbic acid were set to 4 mM, 1.0 mM, and 0.4 mM, respectively, and the rest of the conditions were the same as in Synthesis Example 1. The cellulose oligomer aggregate (mEG 24 _2) was obtained.

[0082] [Synthesis Example 3] The amounts of methyl-PEG24-azide, CuSO4, and ascorbic acid were set to 6 mM, 1.5 mM, and 0.6 mM, respectively, and the rest of the conditions were the same as in Synthesis Example 1. The cellulose oligomer aggregate (mEG 24 _3) was obtained.

[0083] [Synthesis Example 4] CEL-C≡CH aqueous dispersion, 40 mM methyl-PEG12-azide (mPEG azide with m=12 in reaction formula (3)), DMSO (SP grade), 20 mM CuSO4 aqueous solution, 8 mM sodium ascorbate aqueous solution, and ultrapure water were added to a 5 mL tube so that CEL-C≡CH was 2 mg / mL, methyl-PEG12-azide was 2 mM, CuSO4 was 0.5 mM, and ascorbic acid was 0.2 mM, and the mixture was reacted for 1 hour by stirring at 25°C. At that time, the water / DMSO (volume ratio) was 1 / 1, and the volume of the reaction solution was 2500 μL. The liquid after the reaction was centrifuged (20,400 × g, 5 minutes, 4 ° C.), the supernatant was removed, and then ultrapure water was added to redisperse the product. The same centrifugation (under the same conditions) was repeated five times to obtain the cellulose oligomer aggregate (mEG 12 _1) was obtained.

[0084] Cellulose oligomer aggregates (mEG 24 _1~3 and mEG 12 For _1), mass spectrum was obtained by MALDI-TOF-MS method. The results are shown in Figure 4. In Figure 4, the peaks surrounded by dashed lines with DP = 6 to 11 are peaks of CEL-C≡CH to which no PEG chain has been introduced, and the numbers 6 to 11 represent the degree of polymerization of cellulose in CEL-C≡CH. The peaks surrounded by dashed lines with DP = 6' to 10' are peaks of PEG-introduced cellulose oligomers to which a PEG chain of m = 24 has been introduced, and the numbers 6' to 10' represent the degree of polymerization of cellulose in the PEG-introduced cellulose oligomers. The peaks surrounded by dashed lines with DP = 7" to 9" are peaks of PEG-introduced cellulose oligomers to which a PEG chain of m = 12 has been introduced, and the numbers 7" to 9" represent the degree of polymerization of cellulose in the PEG-introduced cellulose oligomers.

[0085] From the integral values ​​of the peaks of the PEG-introduced cellulose oligomers and the CEL-C≡CH peaks, each cellulose oligomer assembly (mEG 24 _1~3 and mEG 12The PEG introduction rate of _1) was determined. The results are shown in Table 1 below. The PEG introduction rate is the proportion (mol %) of the PEG-introduced cellulose oligomers in all cellulose oligomers constituting the cellulose oligomer assembly.

[0086] [Table 1]

[0087] Cellulose oligomer aggregates (mEG 24 _1~3 and mEG 12 ATR / FT-IR measurements were performed on _1), and the spectrum shown in Figure 5 was obtained. The spectrum obtained showed peaks derived from the hydroxyl groups of cellulose forming the cellulose type II crystal structure, peaks derived from the acetylene groups, and peaks derived from the amorphous PEG chains. This shows that the click reaction does not affect the cellulose crystal structure, and the PEG chains exposed on the surface of the cellulose nanosheet are in an amorphous state.

[0088] [reagent] The reagents BSA and OPD used in the following test examples are as follows. BSA: Bovine serum albumin, Fujifilm Wako Pure Chemical Industries, protease-free OPD: o-phenylenediamine dihydrochloride, manufactured by Nacalai Tesque, for water quality analysis

[0089] [Test Example 1: Evaluation of binding of anti-PEG antibody (PEG-B-47)] PEG-B-47 (Abcam) was used as the primary antibody, an anti-PEG antibody. PEG-B-47 is a methoxy-terminated PEG-selective monoclonal anti-PEG antibody of rabbit species and IgG class, which specifically binds to PEG chains with methoxy groups at the ends of 140 Da (DP=3) or more. PEG-B-47 was diluted with a 3.3% by mass BSA / PBS (pH 7.4) solution to prepare a primary antibody solution with a PEG-B-47 content of 110 ng / mL.

[0090] The cellulose oligomer assemblies (mEG 24 _1 and mEG 12 _1) was dispersed in PBS at 5 mg / mL to prepare two types of cellulose dispersions according to the examples. Also, the cellulose oligomer assembly having a propargyl group (CEL-C≡CH) synthesized above was dispersed in PBS at 5 mg / mL to prepare a cellulose dispersion according to the comparative example.

[0091] The secondary antibody used to evaluate the binding of the anti-PEG antibody was an anti-rabbit IgG, HRP-labeled antibody (GeneTex), which was diluted with a 3.3% by mass BSA / PBS solution to prepare a secondary antibody solution with a secondary antibody content of 440 ng / mL.

[0092] 1. Antigen-antibody reaction of primary antibody 10 μL of the cellulose dispersion was added to a 0.2 mL PCR tube, and then 100 μL of the primary antibody solution was added and dispersed, followed by incubation at 25° C. for 1 hour to carry out an antigen-antibody reaction. The system for this primary antibody antigen-antibody reaction had a cellulose oligomer assembly concentration of 0.45 mg / mL, a primary antibody concentration of 100 ng / mL, and a BSA concentration of 3 mass%. After incubation, the tube was centrifuged (15,000 rpm, 25° C., 5 minutes) and the supernatant was removed, after which the tube was redispersed in 100 μL of PBS, and this operation was repeated three times for washing.

[0093] 2. Antigen-antibody reaction of secondary antibodies After washing, i.e. after centrifugation and removal of the supernatant, 100 μL of secondary antibody solution was added to the PCR tube for dispersion, and the tube was incubated at 25° C. for 1 hour to carry out an antigen-antibody reaction. The secondary antibody antigen-antibody reaction system had a cellulose oligomer assembly concentration of 0.45 mg / mL and a secondary antibody concentration of 400 ng / mL. After incubation, the tube was centrifuged (15,000 rpm, 25° C., 5 minutes) and the supernatant was removed, after which the tube was redispersed in 100 μL of PBS, and this operation was repeated three times for washing.

[0094] 3. Enzyme reaction Next, after washing, i.e., after centrifuging and removing the supernatant, 100 μL of substrate solution was added to the PCR tube, dispersed, and incubated at 25° C. for 30 minutes. As the substrate solution, citrate phosphate buffer (pH 4.0) containing 2 mg / mL OPD and 0.11 v / v% hydrogen peroxide was used. After incubation, 100 μL of 3N sulfuric acid was added and centrifuged (15,000 rpm, 25° C., 10 minutes). 100 μL of the supernatant was added to a 96-well plate, and the absorbance at 490 nm was measured using a plate reader (BioTek's "Synergy H1").

[0095] The results are shown in Figure 6. In Figure 6, the graph on the left, "PEG-B-47(+)" shows "mEG 24 _1" and "mEG 12 "_1" is the result of the above test. The test was carried out in three sets of three experiments, and the average absorbance of the average value of each set was calculated.

[0096] As a control, reactions 1 to 3 were carried out in the same manner as above except that 100 μL of 3.3% by mass BSA / PBS (pH 7.4) solution not containing PEG-B-47 was added instead of 100 μL of the primary antibody solution in the antigen-antibody reaction of the primary antibody. The results are shown as the graph "PEG-B-47(-)" on the right side of Figure 6.

[0097] Furthermore, in the antigen-antibody reaction of the primary antibody, 10 μL of PBS not containing cellulose oligomer aggregates was added instead of 10 μL of the cellulose dispersion liquid, and the other conditions were the same as above, and the reactions 1 to 3 were carried out. The results are shown as "(-)" in the graphs of "PEG-B-47(+)" and "PEG-B-47(-)" in Figure 6.

[0098] As shown in FIG. 6, the cellulose oligomer assembly CEL-C≡CH according to the comparative example had the same absorbance as the cellulose oligomer-free "(-)" and did not bind to the anti-PEG antibody PEG-B-47. 24 _1 or mEG 12In the case of _1), the absorbance is significantly higher than that of "(-)", indicating that the anti-PEG antibody PEG-B-47 is bound. Therefore, in the case of the cellulose oligomer assembly of the embodiment, the methoxy-terminated PEG-selective anti-PEG antibody can be detected.

[0099] [Test Example 2: Evaluation of binding of anti-PEG antibody (15-2b)] 15-2b (Merck) was used as the primary antibody, an anti-PEG antibody. 15-2b is a methoxy-terminated PEG-selective monoclonal anti-PEG antibody of animal species: mouse, class: IgG2b, which specifically binds to PEG chains with a methoxy group at the end of 560 Da (DP = 12) or more. 15-2b was diluted with a 3.3% by mass BSA / PBS (pH 7.4) solution to prepare a primary antibody solution with a 15-2b content of 110 ng / mL.

[0100] The cellulose oligomer aggregates (mEG 24 _1~3 and mEG 12 _1) was dispersed in PBS at 5 mg / mL to prepare four types of cellulose dispersions according to the examples. Also, the cellulose oligomer assembly having a propargyl group (CEL-C≡CH) synthesized above was dispersed in PBS at 5 mg / mL to prepare a cellulose dispersion according to the comparative examples.

[0101] The secondary antibody used to evaluate the binding of the anti-PEG antibody was an anti-mouse IgG, HRP-labeled antibody (Sigma-Aldrich), which was diluted with a 3.3% by mass BSA / PBS solution to prepare a secondary antibody solution with a secondary antibody content of 440 ng / mL.

[0102] 1. Antigen-antibody reaction of primary antibody 10 μL of the cellulose dispersion was added to a 0.2 mL PCR tube, and then 100 μL of the primary antibody solution was added and dispersed, followed by incubation at 25° C. for 1 hour to carry out an antigen-antibody reaction. The system for this primary antibody antigen-antibody reaction had a cellulose oligomer assembly concentration of 0.45 mg / mL, a primary antibody concentration of 100 ng / mL, and a BSA concentration of 3 mass%. After incubation, the tube was centrifuged (15,000 rpm, 25° C., 5 minutes) and the supernatant was removed, after which the tube was redispersed in 100 μL of PBS, and this operation was repeated three times for washing.

[0103] 2. Antigen-antibody reaction of secondary antibodies After washing, i.e. after centrifugation and removal of the supernatant, 100 μL of secondary antibody solution was added to the PCR tube for dispersion, and the tube was incubated at 25° C. for 1 hour to carry out an antigen-antibody reaction. The secondary antibody antigen-antibody reaction system had a cellulose oligomer assembly concentration of 0.45 mg / mL and a secondary antibody concentration of 400 ng / mL. After incubation, the tube was centrifuged (15,000 rpm, 25° C., 5 minutes) and the supernatant was removed, after which the tube was redispersed in 100 μL of PBS, and this operation was repeated three times for washing.

[0104] 3. Enzyme reaction Next, after washing, i.e., after centrifuging and removing the supernatant, 100 μL of substrate solution was added to the PCR tube, dispersed, and incubated at 25° C. for 30 minutes. As the substrate solution, citrate phosphate buffer (pH 4.0) containing 2 mg / mL OPD and 0.11 v / v% hydrogen peroxide was used. After incubation, 100 μL of 3N sulfuric acid was added and centrifuged (15,000 rpm, 25° C., 10 minutes). 100 μL of the supernatant was added to a 96-well plate, and the absorbance at 490 nm was measured using a plate reader (BioTek's "Synergy H1").

[0105] The results are shown in Figure 7. In Figure 7, the graph on the left, "15-2b(+)" shows "mEG 24 _1", "mEG 24 _2", "mEG 24 _3", "mEG 12"_1" and "CEL-C≡CH" are the results of the above test. The test was carried out in three sets of three experiments, and the further average absorbance of the average value of each set was calculated.

[0106] As a control, reactions 1 to 3 were carried out in the same manner as above except that 100 μL of 3.3% by mass BSA / PBS (pH 7.4) solution not containing 15-2b was added instead of 100 μL of the primary antibody solution in the antigen-antibody reaction of the primary antibody. The results are shown as the graph "15-2b(-)" on the right side of FIG. 7.

[0107] Furthermore, in the antigen-antibody reaction of the primary antibody, 10 μL of PBS not containing cellulose oligomer assemblies was added instead of 10 μL of the cellulose dispersion liquid, and the other conditions were the same as above, and the reactions 1 to 3 were carried out. The results are shown as "(-)" in the graphs "15-2b(+)" and "15-2b(-)" in Figure 7.

[0108] As shown in FIG. 7, the cellulose oligomer assembly CEL-C≡CH according to the comparative example had the same absorbance as the cellulose oligomer-free "(-)" and did not bind to the anti-PEG antibody 15-2b. 24 _1~3 or mEG 12 In the case of _1), the absorbance is significantly higher than that of "(-)", indicating that anti-PEG antibody 15-2b is bound. Therefore, in the case of the cellulose oligomer assembly according to the embodiment, the methoxy-terminated PEG-selective anti-PEG antibody can be detected.

[0109] [Test Example 3: Evaluation of binding of anti-PEG antibody (6.3)] 6.3 (Merck) was used as the primary antibody, an anti-PEG antibody. 6.3 is a backbone-selective anti-PEG antibody with animal species: mouse, class: IgG1, and specifically binds to PEG chains of 750 Da (DP = 16) or more (Samuel K. Lai, Commun. Chem. 2020, 3, 124). 6.3 was diluted with a 3.3 mass% BSA / PBS (pH 7.4) solution to prepare a primary antibody solution with a 6.3 content of 110 ng / mL. The cellulose dispersion was prepared in the same manner as in Test Example 1 using the cellulose oligomer of Synthesis Example 1. The same secondary antibody solution as in Test Example 1 was used.

[0110] 1. Antigen-antibody reaction of primary antibody 10 μL of the cellulose dispersion was added to a 0.2 mL PCR tube, and then 100 μL of the primary antibody solution was added and dispersed, followed by incubation at 25° C. for 1 hour to carry out an antigen-antibody reaction. The system for this primary antibody antigen-antibody reaction had a cellulose oligomer assembly concentration of 0.45 mg / mL, a primary antibody concentration of 100 ng / mL, and a BSA concentration of 3 mass%. After incubation, the tube was centrifuged (15,000 rpm, 25° C., 5 minutes) and the supernatant was removed, after which the tube was redispersed in 100 μL of PBS, and this operation was repeated three times for washing.

[0111] 2. Antigen-antibody reaction of secondary antibodies After washing, i.e. after centrifugation and removal of the supernatant, 100 μL of secondary antibody solution was added to the PCR tube for dispersion, and the tube was incubated at 25° C. for 1 hour to carry out an antigen-antibody reaction. The secondary antibody antigen-antibody reaction system had a cellulose oligomer assembly concentration of 0.45 mg / mL and a secondary antibody concentration of 400 ng / mL. After incubation, the tube was centrifuged (15,000 rpm, 25° C., 5 minutes) and the supernatant was removed, after which the tube was redispersed in 100 μL of PBS, and this operation was repeated three times for washing.

[0112] 3. Enzyme reaction Next, after washing, i.e., after centrifuging and removing the supernatant, 100 μL of substrate solution was added to the PCR tube, dispersed, and incubated at 25° C. for 30 minutes. As the substrate solution, citrate phosphate buffer (pH 4.0) containing 2 mg / mL OPD and 0.11 v / v% hydrogen peroxide was used. After incubation, 100 μL of 3N sulfuric acid was added and centrifuged (15,000 rpm, 25° C., 10 minutes). 100 μL of the supernatant was added to a 96-well plate, and the absorbance at 490 nm was measured using a plate reader (BioTek's "Synergy H1").

[0113] The results are shown in Figure 8. In Figure 8, the graph on the left, "6.3(+)" in "mEG 24 "_1" and "CEL-C≡CH" are the results of the above test. The test was carried out in three sets of three experiments, and the further average absorbance of the average value of each set was calculated.

[0114] As a control, reactions 1 to 3 were carried out in the same manner as above except that 100 μL of 3.3% by mass BSA / PBS (pH 7.4) solution not containing 6.3 was added instead of 100 μL of the primary antibody solution in the antigen-antibody reaction of the primary antibody. The results are shown as the graph "6.3(-)" on the right side of FIG. 8.

[0115] Furthermore, in the antigen-antibody reaction of the primary antibody, 10 μL of PBS not containing cellulose oligomer aggregates was added instead of 10 μL of the cellulose dispersion liquid, and the other conditions were the same as above, and the reactions 1 to 3 were carried out. The results are shown as "(-)" in the graphs "6.3(+)" and "6.3(-)" in Figure 8.

[0116] As shown in FIG. 8, the cellulose oligomer assembly CEL-C≡CH according to the comparative example had almost the same absorbance as the cellulose oligomer-free "(-)" and anti-PEG antibody 6.3 did not bind. In contrast, the cellulose oligomer assembly (mEG 24In the case of _1), the absorbance is high relative to "(-)", indicating that anti-PEG antibody 6.3 is bound. Therefore, in the case of the cellulose oligomer assembly of the embodiment, the backbone-selective anti-PEG antibody can be detected.

[0117] In addition, the various numerical ranges described in the specification can be arbitrarily combined with their upper and lower limits, and all such combinations are considered to be preferred numerical ranges described in the specification. In addition, a numerical range described as "X to Y" means from X to Y.

[0118] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the gist of the invention. These embodiments and their omissions, substitutions, modifications, etc. are included in the scope and gist of the invention as well as the invention and its equivalents described in the claims.

Claims

1. General formula (1): 【Chemistry 1】 The cellulose oligomer is represented by In formula (1), n ​​is an average degree of polymerization and is a number from 6 to 16; m is an average degree of polymerization and is a number equal to or greater than 3; A is a divalent organic group connecting an oxygen atom bonded to the anomeric carbon at the reducing end of a cellulose oligomer and a polyethylene glycol chain, the divalent organic group having 1 to 24 carbon atoms and containing at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom; Anti-PEG antibody binding material.

2. The anti-PEG antibody-binding material according to claim 1, wherein A in formula (1) represents a divalent organic group having 2 to 10 carbon atoms containing at least one type selected from the group consisting of a triazole group, an amide group, and a thioether group.

3. In the formula (1), A is 【Chemistry 2】 where R １ represents a hydrocarbon group having 1 to 10 carbon atoms, R ２ The anti-PEG antibody binding material of claim 1 , wherein represents a single bond or a hydrocarbon group having 1 to 10 carbon atoms.

4. The anti-PEG antibody binding material according to claim 1 , wherein m in formula (1) is a number of 10 or more.

5. A biosensor comprising the anti-PEG antibody binding material according to any one of claims 1 to 4.

6. An adsorbent comprising the anti-PEG antibody binding material according to any one of claims 1 to 4.

7. General formula (1): 【Chemistry 3】 in which n in formula (1) is an average degree of polymerization and is a number from 6 to 16, m is an average degree of polymerization and is a number not less than 3, and A is a divalent organic group connecting an oxygen atom bonded to the anomeric carbon at the reducing end of the cellulose oligomer to a polyethylene glycol chain, the divalent organic group having 1 to 24 carbon atoms and containing at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom, with a test subject suspected of containing an anti-PEG antibody under conditions that allow binding to the anti-PEG antibody; incubating the cellulose oligomer after incubation with the analyte with a sample containing a secondary antibody under conditions that allow binding of the secondary antibody to the anti-PEG antibody; and detecting the anti-PEG antibody contained in the specimen using the cellulose oligomer after incubation with the sample and the secondary antibody as a probe; A method for detecting an anti-PEG antibody, comprising:

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