Anti-PEG antibody binding material and method for detecting anti-PEG antibodies

Abstract

To provide an anti-PEG antibody binding material that makes it possible to detect a methoxy-terminated PEG-selective anti-PEG antibody.SOLUTION: An anti-PEG antibody binding material according to an embodiment contains cellulose oligomers represented by general formula (1), and can bind to a methoxy-terminated PEG-selective anti-PEG antibody. In formula (1), n is the average degree of polymerization and represents a number of 6-16, and m represents an integer of 1-16.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 blood circulation of the modified substance and reduces immunogenicity. For this reason, many PEG-modified pharmaceuticals 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, highly sensitive detection of anti-PEG antibodies present in the blood is required to evaluate the induction of anti-PEG antibodies and their effect on the therapeutic efficacy of PEG-modified drugs.

[0004] Anti-PEG antibodies include not only backbone-selective anti-PEG antibodies, which recognize the repeating oxyethylene unit that forms the backbone of the PEG structure and have high selectivity for the PEG chains of this backbone, but also methoxy-terminated PEG-selective anti-PEG antibodies. Methoxy-terminated PEG-selective anti-PEG antibodies have high selectivity for methoxy-terminated PEG, i.e., they specifically bind to PEG chains with methoxy groups at the terminus. Methoxy-terminated PEG-selective anti-PEG antibodies are also known to bind to PEG chains with more hydrophobic terminal ethoxy and butoxy groups.

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

[0006] Furthermore, Patent Documents 2 and 3 disclose biosensors containing hydrophilic particles, each of which includes a water-insoluble particle, a primer layer containing polysiloxane disposed on the surface of the particle, and a hydrophilic polymer layer disposed on the primer layer, the hydrophilic polymer layer being composed of a PEG chain bound to an antibody and a PEG chain bound to an inactive group. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-180921 [Patent Document 2] Japanese Patent Application Publication No. 2020-143964 [Patent Document 3] Patent Publication No. 2021-012089 [Non-patent literature]

[0008] [Non-Patent Document 1] T. Nohara et al., "Enzymatic Synthesis of Oligo(ethylene glycol)-Bearing Cellulose Oligomers for in Situ Formation of Hydrogels with Crystalline Nanoribbon Network Structures", Langmuir, 2016, 32, 47, 12520-12526 Summary of the Invention [Problem to be solved by the invention]

[0009] In the course of research aimed at highly sensitive detection of anti-PEG antibodies present in the blood, the inventors discovered that cellulose oligomers bearing specific oligoethylene glycol groups specifically bind to methoxy-terminated PEG-selective anti-PEG antibodies.

[0010] In view of the above, embodiments of the present invention aim to provide an anti-PEG antibody-binding material capable of binding to a methoxy-terminated PEG-selective anti-PEG antibody, and a method for detecting an anti-PEG antibody using the same. [Means for solving the problem]

[0011] The present invention includes the embodiments shown below. [1] An anti-PEG antibody binding material capable of binding to a methoxy-terminated PEG-selective anti-PEG antibody, comprising a cellulose oligomer represented by the following general formula (1): General formula (1): [ka] (In formula (1), n ​​is the average degree of polymerization and represents a number from 6 to 16, and m represents an integer from 1 to 16.) [2] A biosensor comprising the anti-PEG antibody binding material described in [1] above. [3] An adsorbent comprising the anti-PEG antibody-binding material described in [1] above.

[0012] [4] The following general formula (1): [ka] (where n is an average degree of polymerization and represents a number from 6 to 16, and m is an integer from 1 to 16) is incubated with a test sample suspected of containing a methoxy-terminated PEG-selective anti-PEG antibody under conditions that allow binding of the cellulose oligomer with 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 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; A method for detecting an anti-PEG antibody, comprising: [Effects 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 a methoxy-terminated PEG-selective anti-PEG antibody, which can be used, for example, in a biosensor for detecting a methoxy-terminated PEG-selective anti-PEG antibody or in an adsorbent for adsorbing a methoxy-terminated PEG-selective anti-PEG antibody. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a conceptual diagram of a nanoribbon made of cellulose oligomer that constitutes an anti-PEG antibody binding material according to an embodiment. [Figure 2] FIG. 1 is an explanatory diagram showing a method for detecting an anti-PEG antibody according to an embodiment. [Figure 3] 1 is a graph showing the results of evaluating the binding of secondary antibodies to each cellulose oligomer in Test Example 1 (absorbance at 490 nm). [Figure 4] 1 is a graph showing the results of evaluating the binding of methoxy-terminated PEG-selective anti-PEG antibodies to each cellulose oligomer in Test Example 2 (absorbance at 490 nm). [Figure 5] 1 is a graph showing the results of evaluating the binding of backbone-selective anti-PEG antibodies to each cellulose oligomer in Test Example 3 (absorbance at 490 nm). [Figure 6] 1 is a graph showing the results of BSA concentration dependency (absorbance at 490 nm) in blank measurements in Test Example 4. [Figure 7] 1 is a graph showing the results (absorbance at 490 nm) of BSA concentration dependency in blank measurements (containing cellulose oligomer) in Test Example 5. [Figure 8] 1 is a graph showing the results of evaluating the adsorption of BSA to cellulose oligomers in Test Example 6 (absorbance at 280 nm). [Figure 9] 1 is a graph showing the results of measurement of the lower detection limit (absorbance at 490 nm) of a methoxy-terminated PEG-selective anti-PEG antibody in the presence of BSA in Test Example 7. [Figure 10]10 is a graph showing an enlarged view of the low concentration region of the graph in FIG. 9. [Figure 11] 1 is a graph showing the results of detection of methoxy-terminated PEG-selective anti-PEG antibodies in the presence of 10% FBS in Test Example 8 (absorbance at 490 nm). [Figure 12] 12 is a graph showing an enlarged view of the low concentration region of the graph in FIG. 11. [Figure 13] 1 is a graph showing the results of detection (absorbance at 490 nm) of methoxy-terminated PEG-selective anti-PEG antibodies in the presence of 10% FBS in Test Example 9. [Figure 14] 14 is a graph showing an enlarged view of the low concentration region of the graph in FIG. 13. DETAILED DESCRIPTION OF THE INVENTION

[0015] The anti-PEG antibody-binding material according to this embodiment contains a cellulose oligomer (hereinafter also referred to as an OEG-modified cellulose oligomer) represented by the following general formula (1): An OEG-modified cellulose oligomer is a cellulose oligomer having a structure in which glucose units are linked by β-1,4-glucosidic bonds, to which an oligoethylene glycol group having a methoxy group is bonded as a substituent to the anomeric carbon at the reducing end.

[0016] [ka]

[0017] In formula (1), n ​​represents a number from 6 to 16, and m represents an integer from 1 to 16. 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 or more and 16 or less. n may be 6.5 or more, or 7 or more. It may also be 14 or less, 13 or less, or 12 or less. The average degree of polymerization of the cellulose oligomer is a weighted average of the degrees of polymerization according to the mass ratio of the cellulose oligomer. The degree of polymerization of each OEG cellulose oligomer is not particularly limited, and may be, for example, 4 or more, 5 or more, 6 or more, or 20 or less, 16 or less, or 13 or less.

[0019] The above-mentioned m represents the number of moles of ethylene oxide units added in the oligoethylene glycol group and is 1 or more and 16 or less. m is preferably 3 or more, more preferably 4 or more. m is preferably 12 or less, more preferably 10 or less, and even more preferably 8 or less. The OEG cellulose oligomer may be one in which m has any one value from 1 to 16, or may be a mixture in which m has multiple values.

[0020] In one embodiment, the OEG-modified cellulose oligomer may be represented by the following general formula (2). In this case, the anomeric position at the reducing end has a β-configuration. Note that m and n in formula (2) are the same as m and n in formula (1). [ka]

[0021] The OEG-modified cellulose oligomer may be a cellulose oligomer assembly having a cellulose type II crystalline structure. That is, in one embodiment, the OEG-modified cellulose oligomer may be a cellulose oligomer assembly having a cellulose type II crystalline structure, and containing a compound represented by formula (1) as a constituent component. The cellulose oligomer assembly may have a sheet-like structure (cellulose nanosheet). Here, the sheet-like structure is a concept that encompasses a ribbon-like structure (cellulose nanoribbon) as shown in FIG. 1.

[0022] While naturally occurring cellulose chains have a parallel-arranged crystalline structure of cellulose type I, synthetic cellulose oligomers generally form the thermodynamically stable crystalline structure of cellulose type II. In this case, the substituents at the ends of the cellulose chains (OC2H4) m The -OCH3 group does not affect the crystal structure, and the cellulose oligomers bearing this group are aligned in the thickness direction of the cellulose nanosheet to form lamellar crystals, with the group's substituents exposed on the surface of the sheet.

[0023] In detail, when the OEG-modified cellulose oligomer is represented by arrows pointing from the reducing end to the non-reducing end of the cellulose chain and wavy lines as shown below the formula (1) above, the OEG-modified cellulose oligomer is arranged in the film thickness direction so that the arrows point alternately, as shown in the cross-sectional structure in Figure 1, and therefore the substituents are exposed alternately on the sheet surface.

[0024] The method for synthesizing the OEG-modified cellulose oligomer is not particularly limited, and may be, for example, an enzymatic synthesis reaction utilizing the reverse reaction of cellodextrin phosphorylase (CDP), as described in Non-Patent Document 1. That is, by reacting a primer having the above-mentioned substituent at the anomeric position of glucose or cellobiose via a β-bond, or a primer having the above-mentioned substituent at the anomeric position of cellobiose via an α-bond, with α-glucose-1-phosphate (αG1P), and CDP, αG1P is sequentially polymerized to the primer, and the OEG-modified cellulose oligomer represented by formula (1) can be synthesized.

[0025] The OEG-modified cellulose oligomer represented by formula (1) specifically binds to methoxy-terminated PEG-selective anti-PEG antibodies but does not bind to secondary antibodies used as probes or other proteins such as BSA (bovine serum albumin) or lysozyme. Therefore, the cellulose oligomer assembly has a bioinert surface that does not nonspecifically adsorb proteins and specifically binds to methoxy-terminated PEG-selective anti-PEG antibodies. OEG-modified cellulose oligomers also do not bind to backbone-selective anti-PEG antibodies. Therefore, OEG-modified cellulose oligomers can selectively bind to methoxy-terminated PEG-selective anti-PEG antibodies and can be used as an anti-PEG antibody-binding material that can selectively detect or adsorb methoxy-terminated PEG-selective anti-PEG antibodies.

[0026] A methoxy-terminated PEG-selective anti-PEG antibody to which an OEG-modified cellulose oligomer can be bound is, more specifically, a monoclonal antibody that specifically binds to PEG (polyethylene glycol) having a methoxy group at its terminal, and is also referred to as a methoxy-terminated PEG-selective monoclonal anti-PEG antibody. The methoxy-terminated PEG-selective anti-PEG antibody may also bind to PEG chains having a more hydrophobic terminal ethoxy or butoxy group. The class of the methoxy-terminated PEG-selective anti-PEG antibody is not particularly limited, and may be IgG or IgM.

[0027] The anti-PEG antibody-binding material according to this embodiment contains an OEG-modified cellulose oligomer and is therefore capable of recognizing and binding to methoxy-terminated PEG-selective anti-PEG antibodies, and can therefore be used, for example, as a biosensor for detecting methoxy-terminated PEG-selective anti-PEG antibodies or as an adsorbent for adsorbing methoxy-terminated PEG-selective anti-PEG antibodies.

[0028] The anti-PEG antibody binding material may consist solely of OEG-modified cellulose oligomers (e.g., the above-mentioned cellulose oligomer aggregates), or may contain other components, such as proteins, peptides, nucleic acids, lipids, sugars, cells, amino acids, metal ions, organic solvents, etc., to the extent that the effect is not impaired.

[0029] OEG-modified cellulose oligomers, specifically cellulose oligomer aggregates, are insoluble in water, and when synthesized by the enzymatic synthesis reaction described above, the OEG-modified cellulose oligomers are obtained as an aqueous dispersion. In one embodiment, the anti-PEG antibody-binding material may be a dry powder obtained by removing water from the aqueous dispersion. A powdered anti-PEG antibody-binding material may be prepared using the dry powder as is, or by powder-mixing with other components such as proteins. The powdered anti-PEG antibody-binding material can be easily dispersed in water by adding water prior to use, thereby preparing an aqueous dispersion.

[0030] In one embodiment, the anti-PEG antibody binding material may be an aqueous dispersion of an OEG-modified cellulose oligomer dispersed in water. When preparing an aqueous dispersion of an OEG-modified cellulose oligomer, a buffer may be added to the water to form a buffer solution. That is, the aqueous dispersion of an OEG-modified cellulose oligomer may be one in which the OEG-modified cellulose oligomer is dispersed in a buffer solution.

[0031] 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.

[0032] When preparing an aqueous dispersion of an OEG-modified cellulose oligomer, the content (concentration) of the OEG-modified 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).

[0033] In this specification, "% (w / v)" refers to a mass / volume percent concentration, which is the mass (g) of a substance of interest contained in a volume of 100 mL.

[0034] An anti-PEG antibody-binding material according to one embodiment may be an OEG-modified cellulose oligomer (for example, the above-mentioned cellulose oligomer assembly) immobilized on the surface of a substrate such as plastic or glass.

[0035] In one embodiment, a biosensor comprising the anti-PEG antibody binding material is used to perform a bioassay to detect and quantify methoxy-terminated PEG-selective anti-PEG antibodies, and the biosensor refers to each element or assembly or combination of elements used to perform such a bioassay.

[0036] Therefore, the biosensor may consist of, for example, only a powder of OEG-modified cellulose oligomer or only an aqueous dispersion of OEG-modified cellulose oligomer. Alternatively, the biosensor may be a kit (i.e., an anti-PEG antibody detection kit) that combines a secondary antibody as a probe with the powder or aqueous dispersion of OEG-modified cellulose oligomer. Furthermore, if the secondary antibody is an enzyme-labeled antibody, the biosensor may be an anti-PEG antibody detection kit that combines a substrate corresponding to the enzyme.

[0037] As described in the examples below, the detection sensitivity of methoxy-terminated PEG-selective anti-PEG antibodies using OEG-modified cellulose oligomers is improved by adding albumin such as BSA (bovine serum albumin) when incubating the OEG-modified cellulose oligomers with the test specimen.

[0038] Therefore, the biosensor may contain albumin such as BSA, and albumin is preferably added when incubating the OEG-modified cellulose oligomer with the test specimen. In this case, albumin may be included in the anti-PEG antibody-binding material or may be included in the anti-PEG antibody detection kit separately from the anti-PEG antibody-binding material. When albumin is included in the anti-PEG antibody-binding material, the albumin may be in the form of a powder mixed with the OEG-modified cellulose oligomer, or the albumin may be dissolved in an aqueous dispersion of the OEG-modified cellulose oligomer. The ratio of OEG-modified cellulose oligomer to albumin is not particularly limited, and may be, for example, 0.003 / 1 to 0.3 / 1 or 0.01 / 1 to 0.1 / 1 by mass ratio of OEG-modified cellulose oligomer / albumin. When the test specimen contains albumin, such as blood, additional albumin may or may not be added.

[0039] A method for detecting an anti-PEG antibody according to one embodiment comprises the following steps: (1) incubating an OEG-modified cellulose oligomer with an analyte under conditions that allow binding to a methoxy-terminated PEG-selective anti-PEG antibody; (2) incubating the OEG-modified cellulose oligomer after incubation with the specimen with a sample containing a secondary antibody under conditions that allow binding of the secondary antibody; and (3) Using the OEG-modified cellulose oligomer after incubation with the sample, a secondary antibody is used as a probe to detect methoxy-terminated PEG-selective anti-PEG antibodies contained in the specimen.

[0040] In step (1), a test sample is added to a container together with an aqueous dispersion of OEG-modified cellulose oligomer as the anti-PEG antibody binding material, and after mixing and dispersing, the mixture is incubated.

[0041] The test sample is a substance suspected of containing methoxy-terminated PEG-selective anti-PEG antibodies. For example, when detecting anti-PEG antibodies present in blood to evaluate their influence on the induction of anti-PEG antibodies or the therapeutic efficacy of PEG-modified pharmaceuticals, the test sample may be blood or a diluted version of the blood.

[0042] In step (1), as described above, albumin such as BSA may be added to improve the detection sensitivity of methoxy-terminated PEG-selective anti-PEG antibodies. In this case, the albumin concentration in the system (the albumin concentration excluding albumin originally contained in the test sample) is not particularly limited, but is preferably, for example, 0.05 to 10% (w / v), and more preferably 0.5 to 5% (w / v).

[0043] The incubation conditions in step (1) are not particularly limited as long as they allow binding of the OEG-modified cellulose oligomer with the methoxy-terminated PEG-selective anti-PEG antibody, and may be, for example, 4 to 60°C and 1 minute to 2 hours. The concentration of the OEG-modified cellulose oligomer in the system is not particularly limited, and may be, for example, 0.01 to 1% (w / v), or 0.01 to 0.1% (w / v).

[0044] In step (1), the methoxy-terminated PEG-selective anti-PEG antibody to be detected is bound to the OEG-modified cellulose oligomer (see Figure 2). The OEG-modified cellulose oligomer to which the methoxy-terminated PEG-selective anti-PEG antibody is bound is then separated from the analyte by centrifugation and washing.

[0045] In step (2), a sample containing a secondary antibody is added to the OEG-modified cellulose oligomer to which the anti-PEG antibody has been bound, and after mixing and dispersing, the mixture is incubated.

[0046] 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 methoxy-terminated PEG-selective anti-PEG antibody, and examples thereof include antibodies labeled with enzymes, radioisotopes, biotin, fluorescent dyes, etc.

[0047] The incubation conditions in step (2) are not particularly limited as long as they allow binding of the methoxy-terminated PEG-selective anti-PEG antibody to the secondary antibody, 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.

[0048] In step (2), a secondary antibody is bound as a probe to the anti-PEG antibody bound to the OEG-modified cellulose oligomer (see Figure 2).The OEG-modified cellulose oligomer to which the secondary antibody is bound is then separated from the sample containing the secondary antibody by centrifugation and washing.

[0049] In step (3), an OEG-modified cellulose oligomer bound to a secondary antibody is used as a probe to detect and quantify methoxy-terminated PEG-selective anti-PEG antibodies contained in the test sample.

[0050] 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 OEG-modified cellulose oligomer to which the secondary antibody has been bound, as shown in Figure 2. The resulting dye product is detected and quantified by absorbance (color development), fluorescence, or chemiluminescence.

[0051] In another embodiment, an adsorbent comprising the anti-PEG antibody binding material can be used to remove or purify methoxy-terminated PEG-selective anti-PEG antibodies by adsorbing the anti-PEG antibodies.

[0052] The adsorbent may be composed of a powder of OEG-modified cellulose oligomer, or may be composed of an aqueous dispersion of OEG-modified cellulose oligomer, and these may contain other optional components.

[0053] When an adsorbent is used to remove methoxy-terminated PEG-selective anti-PEG antibodies, for example, the adsorbent is added to a liquid containing the anti-PEG antibodies, and the anti-PEG antibodies are brought into contact with an OEG-modified cellulose oligomer to bind to each other. The anti-PEG antibodies can then be removed from the liquid by centrifugation or other methods, resulting in the removal of the adsorbent with the adsorbed anti-PEG antibodies.

[0054] When an adsorbent is used to purify a methoxy-terminated PEG-selective anti-PEG antibody, for example, the adsorbent is added to a liquid containing the anti-PEG antibody and impurities, and the anti-PEG antibody is contacted with an OEGylated cellulose oligomer to bind them. The OEGylated cellulose oligomer to which the anti-PEG antibody has been adsorbed is then separated and recovered from the liquid by centrifugation or other methods. The recovered OEGylated cellulose oligomer can be treated with, for example, an acid to release the bond between the anti-PEG antibody and the OEGylated cellulose oligomer, allowing the anti-PEG antibody to be separated and recovered from the OEGylated cellulose oligomer, yielding a highly pure methoxy-terminated PEG-selective anti-PEG antibody. [Example]

[0055] The present invention will be further explained below with reference to examples, but is not limited to these.

[0056] [Preparation of unmodified cellulose oligomer (Cell-Glc)] According to the method described in T. Serizawa et al., Polym. J., 2016, 48, 539-544, an unmodified cellulose oligomer (Cell-Glc) with an average degree of polymerization of 10 (n = 10) was synthesized using glucose as a primer, as shown in the following formula: [ka]

[0057] [Preparation of OEG-modified cellulose oligomer (Cell-OEGm)] OEG-modified cellulose oligomers represented by the formula (2) were synthesized using OEG-glucose as a primer according to the method described in Non-Patent Document 1 (T. Nohara et al., Langmuir, 2016, 32, 47, 12520-12526). The OEG-modified cellulose oligomers synthesized were: cellulose oligomer (Cell-OEG4) with n = 10, m = 4, cellulose oligomer (Cell-OEG6) with n = 10, m = 6, and cellulose oligomer (Cell-OEG8) with n = 11, m = 8 in formula (2).

[0058] The average degree of polymerization was calculated using a proton nuclear magnetic resonance (NMR) spectrometer (AVANCE III HD500 (Bruker Biospin, magnetic field strength: 500 MHz, number of accumulations: 16)) based on the integral values ​​of protons at the anomeric positions other than the reducing end (around δ4.2 ppm) and the anomeric position at the reducing end (around δ4.3 ppm) in the cellulose moiety of the OEG-modified cellulose oligomer.

[0059] [Preparation of cellulose oligomer (Cell-Cello-BiP)] According to the method described in K. Sugiura et al., Polym. J., 2021, 53.1133, a cellulose oligomer (Cell-Cello-BiP) with p = 5, q = 5, represented by the following formula, was synthesized using oligoethylene glycol with cellobiose at both ends as a primer. [ka]

[0060] [reagent] The reagents BSA and OPD used in the test examples are as follows: BSA: Bovine serum albumin, manufactured by Fujifilm Wako Pure Chemical Industries, protease-free OPD: o-phenylenediamine dihydrochloride, manufactured by Nacalai Tesque, for water quality analysis

[0061] [Test Example 1: Evaluation of secondary antibody binding] The adsorption of the secondary antibody used to evaluate the binding of anti-PEG antibodies was evaluated. Anti-rabbit IgG, HRP-labeled antibody (GeneTex) was used as the secondary antibody. It was diluted 1000-fold with 3.3% (w / v) BSA / PBS solution to prepare a secondary antibody solution with a secondary antibody content of 410 ng / mL. The 3.3% (w / v) BSA / PBS solution was prepared by dissolving 330 mg of BSA in 10 mL of PBS (pH 7.4).

[0062] Cellulose dispersions were prepared by dispersing cellulose oligomers at 5 mg / mL in PBS. The cellulose oligomers used were unmodified cellulose oligomer (Cell-Glc), OEG-modified cellulose oligomers (Cell-OEG4, Cell-OEG6, Cell-OEG8), and cellulose oligomer (Cell-Cello-BiP).

[0063] 1. Secondary Antibody Adsorption 10 μL of the cellulose dispersion was added to a 0.2 mL PCR tube, followed by 100 μL of secondary antibody solution, followed by incubation for 1 hour at 25°C. The tube was then centrifuged (15,000 rpm, 25°C, 5 minutes), the supernatant was removed, and the cellulose was redispersed in 100 μL of PBS. This centrifugation and redispersion procedure was repeated three times for washing.

[0064] 2. Enzyme reaction After centrifugation and removal of the supernatant, 100 μL of substrate solution was added to the PCR tube. The substrate solution was a citrate phosphate buffer (pH 4.0) containing 2 mg / mL OPD and 0.11% (v / v) hydrogen peroxide. After the substrate solution was added and dispersed, the mixture was incubated at 25°C for 30 minutes. Next, 100 μL of 3N sulfuric acid was added, and the mixture was centrifuged (15,000 rpm, 25°C, 10 minutes). Then, 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 "Synergy H1").

[0065] The results are shown in Figure 3, and it was found that the HRP-labeled antibody (secondary antibody) did not adsorb to any of the unmodified cellulose oligomers (Cell-Glc), OEG-modified cellulose oligomers (Cell-OEG4, Cell-OEG6, Cell-OEG8), and cellulose oligomer (Cell-Cello-BiP).

[0066] [Test Example 2: Evaluation of binding of methoxy-terminated PEG-selective anti-PEG antibodies] The primary antibody used was PEG-B-47 (Abcam). PEG-B-47 is a methoxy-terminated PEG-selective monoclonal anti-PEG antibody (IgG) derived from rabbits and immunogen PEG-KLH (Merry R. Sherman et al., Molecular Immunology, 2008, 57, 236-246). PEG-B-47 was diluted 68,500-fold with 3.3% (w / v) BSA / PBS solution to prepare a primary antibody solution containing 11 ng / mL of PEG-B-47.

[0067] The cellulose dispersion, secondary antibody solution, and substrate solution were the same as those used in Test Example 1.

[0068] 1. Antigen-antibody reaction of primary antibody To a 0.2 mL PCR tube, 10 μL of cellulose dispersion (5 mg / mL, PBS) was added, followed by 100 μL of primary antibody solution. After dispersion, the mixture was incubated at 25°C for 1 hour to carry out an antigen-antibody reaction. The antigen-antibody reaction system had a cellulose oligomer concentration of 0.45 mg / mL, a primary antibody concentration of 10 ng / mL, and a BSA concentration of 3% (w / v).

[0069] After incubation, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes), the supernatant was removed, and the mixture was redispersed in 100 µL of PBS. This centrifugation and redispersion procedure was repeated three times for washing.

[0070] 2. Antigen-antibody reaction of secondary antibodies Next, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed, after which 100 μL of secondary antibody solution (secondary antibody content: 410 ng / mL in 3.3% BSA / PBS solution) was added to the PCR tube and dispersed, followed by incubation for 1 hour at 25°C. After that, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed, after which the mixture was redispersed in 100 μL of PBS, and this centrifugation and redispersion procedure was repeated three times for washing.

[0071] 3. Enzyme reaction The mixture was then centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant removed. 100 μL of substrate solution (OPD: 2 mg / mL, HO: 0.11 v / v%, pH 4.0 citrate phosphate buffer) was added to the PCR tube, dispersed, and incubated at 25°C for 30 minutes. 100 μL of 3N sulfuric acid was then added, and the mixture was centrifuged (15,000 rpm, 25°C, 10 minutes). The supernatant was then diluted 2-fold with ultrapure water, and 100 μL was added to a 96-well plate. The absorbance at 490 nm was measured using a plate reader (BioTek "Synergy H1").

[0072] The absorbance for evaluation was calculated by subtracting the absorbance measured for each cellulose oligomer from the absorbance measured for a similar experiment without the cellulose oligomer. The experiment was performed three times for each cellulose oligomer, and the average absorbance was calculated.

[0073] The results are shown in Figure 4. It can be seen that the methoxy-terminated PEG-selective anti-PEG antibody hardly bound to the unmodified cellulose oligomer (Cell-Glc) or cellulose oligomer (Cell-Cello-BiP).

[0074] In contrast, it is clear that methoxy-terminated PEG-selective anti-PEG antibodies bind to OEG-modified cellulose oligomers (Cell-OEG4, Cell-OEG6, and Cell-OEG8). In Test Example 1, the fact that HRP-labeled antibodies (secondary antibodies) do not adsorb to these cellulose oligomers also supports the specific binding of methoxy-terminated PEG-selective anti-PEG antibodies to OEG-modified cellulose oligomers.

[0075] Furthermore, the amount of anti-PEG antibody bound to the OEG-modified cellulose oligomer was greatest for Cell-OEG4, and the amount of binding decreased as the number of moles of ethylene oxide units added in the oligoethylene glycol group increased.

[0076] [Test Example 3: Evaluation of binding of backbone-selective anti-PEG antibodies] The primary antibody used was clone 6.3 (Merck, animal species: mouse, class: IgG1), a backbone-selective anti-PEG antibody (backbone-mAb). Clone 6.3 has previously been shown to bind to PEGs of 750 Da or greater (Samuel K. Lai, Commun. Chem. 2020, 3,124). Clone 6.3 was diluted 145,500-fold with 3.3% (w / v) BSA / PBS solution to prepare a primary antibody solution containing 11 ng / mL of clone 6.3.

[0077] The secondary antibody was anti-mouse IgG1, HRP-labeled antibody (Abcam Made) was diluted 2273-fold with 3.3% (w / v) BSA / PBS solution to prepare a secondary antibody solution with a secondary antibody content of 440 ng / mL. The cellulose dispersion and substrate solution used were the same as those used in Test Example 1.

[0078] 1. Antigen-antibody reaction of primary antibody 10 μL of the cellulose dispersion was added to a 0.2 mL PCR tube, followed by 100 μL of the primary antibody solution. After dispersion, the tube was incubated at 25°C for 1 hour to carry out an antigen-antibody reaction. The antigen-antibody reaction system consisted of a cellulose oligomer concentration of 0.45 mg / mL, a primary antibody concentration of 10 ng / mL, and a BSA concentration of 3% (w / v). After incubation, the tube was centrifuged (15,000 rpm, 25°C, 5 minutes), the supernatant was removed, and the tube was redispersed in 100 μL of PBS. This centrifugation and redispersion procedure was repeated three times for washing.

[0079] 2. Antigen-antibody reaction of secondary antibodies The mixture was then centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant removed, after which 100 μL of secondary antibody solution was added to the PCR tube and dispersed, followed by incubation for 1 hour at 25°C. The mixture was then centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant removed, after which the mixture was redispersed in 100 μL of PBS, and this centrifugation and redispersion procedure was repeated three times for washing.

[0080] 3. Enzyme reaction The mixture was then centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant removed. 100 μL of substrate solution (OPD: 2 mg / mL, HO: 0.11 v / v%, pH 4.0 citrate phosphate buffer) was added to the PCR tube, dispersed, and incubated at 25°C for 30 minutes. 100 μL of 3N sulfuric acid was then added, and the mixture was 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 "Synergy H1").

[0081] The absorbance to be evaluated was calculated by subtracting the absorbance obtained in a similar experiment without cellulose oligomer (control) from the absorbance measured for the cellulose oligomer. The experiment was carried out three times in three sets, and the average absorbance of the average values ​​of each set was calculated.

[0082] The results are shown in Figure 5. The absorbance calculated by subtracting the absorbance of the control was nearly 0, indicating that backbone-selective anti-PEG antibodies do not bind to the OEG-modified cellulose oligomers (Cell-OEG4, Cell-OEG6, Cell-OEG8). Therefore, it is believed that the cellulose oligomer according to this embodiment can selectively detect methoxy-terminated PEG-selective anti-PEG antibodies.

[0083] [Test Example 4: BSA concentration dependence in blank measurement] Non-specific adsorption of the secondary antibody to the container can cause a decrease in detection sensitivity. Therefore, to suppress non-specific adsorption of the secondary antibody to the container, we investigated the effect of adding BSA, a protein that is inactive in antigen-antibody reactions, to the reaction system.

[0084] 1. BSA adsorption To a 0.2 mL PCR tube, 10 μL of PBS was added, followed by 100 μL of BSA solution. The following BSA solutions were used: 0.11% (w / v) BSA / PBS solution (1.1 mg BSA in 1 mL PBS), 0.33% (w / v) BSA / PBS solution (3.3 mg BSA in 1 mL PBS), 1.1% (w / v) BSA / PBS solution (11 mg BSA in 1 mL PBS), 3.3% (w / v) BSA / PBS solution (33 mg BSA in 1 mL PBS), and 0% (w / v) PBS without BSA.

[0085] After adding and dispersing the BSA solution, the mixture was incubated at 25°C for 1 hour, and then the PCR tube was washed by replacing the contents with 100 µL of PBS three times.

[0086] 2. Secondary Antibody Adsorption Next, 100 μL of a secondary antibody solution (secondary antibody content: 410 ng / mL in 3.3% BSA / PBS solution) was added to the washed PCR tube, and the mixture was incubated for 1 hour at 25° C. The same secondary antibody solution as in Test Example 1 was used.

[0087] After incubation, the PCR tubes were washed by replacing the contents with 100 μL of PBS three times.

[0088] 3. Enzyme reaction Next, after removing the PBS, 100 μL of substrate solution (OPD: 2 mg / mL, H2O2: 0.11 v / v%, pH 4.0 citrate phosphate buffer) was added to the PCR tube and incubated at 25°C for 30 minutes. The substrate solution used was the same as in Test Example 1. Then, 100 μL of 3N sulfuric acid was added.

[0089] Next, 100 μL of the solution after the enzyme reaction was added to a 96-well plate, and the absorbance at 490 nm was measured using a plate reader (BioTek "Synergy H1"). The experiment was performed three times, and the average absorbance was calculated.

[0090] The results are shown in Figure 6. As the BSA concentration increased, the absorbance decreased, and the background decreased. This is thought to be because BSA adsorbed to the container (tube), suppressing nonspecific adsorption of the secondary antibody.

[0091] [Test Example 5: BSA concentration dependence in blank measurement (with cellulose oligomer)] The effect of BSA concentration on nonspecific adsorption of secondary antibodies when cellulose oligomers were added was examined.

[0092] 1. BSA adsorption 10 μL of cellulose dispersion was added to a 0.2 mL PCR tube, and then 100 μL of BSA solution was added and dispersed. The cellulose dispersion was prepared by dispersing OEG-modified cellulose oligomer (Cell-OEG4) in PBS at 5 mg / mL. The same five types of BSA solutions as in Test Example 4 were used.

[0093] After adding BSA solution and dispersing, the mixture was incubated at 25°C for 1 hour, then centrifuged (15,000 rpm, 25°C, 5 minutes), the supernatant was removed, and the mixture was redispersed in 100 μL of PBS. This centrifugation and redispersion procedure was repeated three times for washing.

[0094] 2. Secondary Antibody Adsorption Next, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed, after which 100 μL of a secondary antibody solution (secondary antibody content: 410 ng / mL in 3.3% BSA / PBS solution) was added to the PCR tube and dispersed, followed by incubation for 1 hour at 25°C. The same secondary antibody solution as in Test Example 4 was used.

[0095] After incubation, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes), the supernatant was removed, and the mixture was redispersed in 100 µL of PBS. This centrifugation and redispersion procedure was repeated three times for washing.

[0096] 3. Enzyme reaction The mixture was then centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed. 100 μL of substrate solution (OPD: 2 mg / mL, HO: 0.11 v / v%, pH 4.0 citrate phosphate buffer) was then added to the PCR tube, dispersed, and incubated at 25°C for 30 minutes. The substrate solution used was the same as in Test Example 4. 100 μL of 3N sulfuric acid was then added, and the mixture was centrifuged (15,000 rpm, 25°C, 10 minutes).

[0097] Next, 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 "Synergy H1"). The experiment was performed three times, and the average absorbance was calculated.

[0098] The results are shown in Figure 7. As the BSA concentration increased, the absorbance decreased, and the background decreased. We found that by adding a high concentration of BSA and incubating prior to treatment with the secondary antibody, nonspecific adsorption of the secondary antibody to the container, which is added next, can be suppressed, improving detection sensitivity (S / N ratio).

[0099] Test Example 6: Evaluation of adsorption of BSA to cellulose oligomers 48 μL of cellulose dispersion was added to a 0.2 mL PCR tube, followed by 72 μL of BSA solution. The cellulose dispersion was prepared by dispersing OEG-modified cellulose oligomer (Cell-OEG4) in PBS at a concentration of 5 mg / mL. The BSA solution was prepared by dissolving 50 mg of BSA in 1 mL of PBS to prepare a 5% (w / v) BSA / PBS solution. After mixing the two, the cellulose oligomer concentration was 2 mg / mL and the BSA concentration was 3% (w / v).

[0100] After mixing, the mixture was incubated at 25°C for 1 hour, then centrifuged (15,000 rpm, 25°C, 5 minutes), and 25 μL of the supernatant was taken and diluted 20-fold with ultrapure water, after which 50 μL was added to the microcell.

[0101] Then, UV-visible spectroscopy (UV-visible spectroscopy) analysis was performed at wavelengths of 200 to 800 nm to determine the absorbance at 280 nm. The measurement was carried out three times, and the average value was calculated.

[0102] As a blank, 48 μL of PBS containing no cellulose oligomer (Cell-OEG4) was added instead of the cellulose dispersion, and the average absorbance was calculated in the same manner.

[0103] The results are shown in Figure 8. In Figure 8, "Cellulose (+)" is an example in which a cellulose dispersion was used, and "Cellulose (-)" is an example in which PBS containing no cellulose oligomer (Cell-OEG4) was used as a blank.

[0104] As shown in Figure 8, the absorbance was almost the same whether or not cellulose oligomer (Cell-OEG4) was added, which indicates that BSA is hardly adsorbed to cellulose oligomer (Cell-OEG4).

[0105] [Test Example 7: Detection of methoxy-terminated PEG-selective anti-PEG antibodies in the presence of BSA]

[0106] The cellulose dispersion used was a 5 mg / mL dispersion of OEG-modified cellulose oligomer (Cell-OEG4) in PBS.

[0107] The primary antibody solution was prepared by diluting the above PEG-B-47 with 3.3% (w / v) BSA / PBS solution to give primary antibody concentrations of 1.1 ng / mL, 2.2 ng / mL, 5.5 ng / mL, 11 ng / mL, 22 ng / mL, 55 ng / mL, and 110 ng / mL (6850-fold dilution).

[0108] The secondary antibody solution and substrate solution used were the same as those used in Test Example 1.

[0109] 1. Antigen-antibody reaction of primary antibody 10 μL of cellulose dispersion (Cell-OEG 4, 5 mg / mL, PBS) was added to a 0.2 mL PCR tube, followed by 100 μL of primary antibody solution. After dispersion, the mixture was incubated at 25°C for 1 hour to carry out an antigen-antibody reaction. The antigen-antibody reaction system used consisted of a cellulose oligomer concentration of 0.45 mg / mL, a primary antibody concentration of 1–100 ng / mL, and a BSA concentration of 3% (w / v). A blank tube was prepared by adding 100 μL of a 3.3% (w / v) BSA / PBS solution without primary antibody and incubating for 1 hour.

[0110] After incubation, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes), the supernatant was removed, and the mixture was redispersed in 100 µL of PBS. This centrifugation and redispersion procedure was repeated three times for washing.

[0111] 2. Antigen-antibody reaction of secondary antibodies Next, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed, after which 100 μL of secondary antibody solution (secondary antibody content: 410 ng / mL in 3.3% BSA / PBS solution) was added to the PCR tube and dispersed, followed by incubation for 1 hour at 25°C. After that, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed, after which the mixture was redispersed in 100 μL of PBS, and this centrifugation and redispersion procedure was repeated three times for washing.

[0112] 3. Enzyme reaction The mixture was then centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant removed. 100 μL of substrate solution (OPD: 2 mg / mL, HO: 0.11 v / v%, pH 4.0 citrate phosphate buffer) was added to the PCR tube, dispersed, and incubated at 25°C for 30 minutes. 100 μL of 3N sulfuric acid was then added, and the mixture was centrifuged (15,000 rpm, 25°C, 10 minutes). The supernatant was then diluted 5-fold with ultrapure water, and 100 μL was added to a 96-well plate. The absorbance at 490 nm was measured using a plate reader (BioTek "Synergy H1").

[0113] The absorbance was measured for each concentration of the primary antibody. Three sets of three experiments were performed, and the average absorbance of each set was calculated.

[0114] The results are shown in Figures 9 and 10. The horizontal axis shows the concentration of the primary antibody (methoxy-mAb) in the reaction system in the antigen-antibody reaction of the primary antibody, and the vertical axis shows the absorbance at 490 nm. Figure 10 is a graph showing an enlarged view of the results in the low concentration range (0 to 10 ng / mL).

[0115] As shown in Figures 9 and 10, it was found that methoxy-terminated PEG-selective anti-PEG antibodies could be quantitatively detected in the presence of 3% (w / v) BSA. As shown in Figure 10, the slope in the low concentration range was 0.0612, the standard deviation σ was 0.00133, and the coefficient of determination R 2The lower detection limit (=3σ / slope) was 0.065 ng / mL, demonstrating extremely high sensitivity.

[0116] [Test Example 8: Detection limit in the presence of 10% FBS] Although albumin is the most abundant protein in serum, serum also contains various other contaminants, such as globulins, sugars, amino acids, urea, and metal ions. In this study, we aimed to detect methoxy-terminated PEG-selective anti-PEG antibodies present in actual blood, and investigated the detection and quantification of methoxy-terminated PEG-selective anti-PEG antibodies in the presence of fetal bovine serum (FBS) as a model serum.

[0117] In Test Example 8, the cellulose dispersion used was a 5 mg / mL dispersion of OEG-modified cellulose oligomer (Cell-OEG4) in PBS. The FBS used was a 22 v / v% solution (22% FBS) prepared by diluting 100% FBS for cell culture (manufactured by BioWest) with ultrapure water. Note that the FBS does not contain γ-globulin (antibody).

[0118] The primary antibody solutions were prepared by diluting the PEG-B-47 with 6.6% (w / v) BSA / 2xPBS solution to yield seven different primary antibody concentrations of 2.2 ng / mL, 4.4 ng / mL, 11 ng / mL, 22 ng / mL, 44 ng / mL, 110 ng / mL, and 220 ng / mL. The 6.6% (w / v) BSA / 2xPBS solution was prepared by dissolving 660 mg of BSA in 10 mL of 2xPBS (a double-concentration solution of PBS; the same applies below).

[0119] The secondary antibody solution and substrate solution used were the same as those used in Test Example 1.

[0120] 1. Antigen-antibody reaction of primary antibody To a 0.2 mL PCR tube, 10 μL of cellulose dispersion (Cell-OEG 4, 5 mg / mL, PBS) was added, followed by 50 μL of 22% FBS. 50 μL of primary antibody solution was added and dispersed. The mixture was then incubated at 25°C for 1 hour to carry out an antigen-antibody reaction. The antigen-antibody reaction system used consisted of a cellulose oligomer concentration of 0.45 mg / mL, a primary antibody concentration of 1–100 ng / mL, an FBS concentration of 10% (v / v), and a BSA concentration of 3% (w / v). A blank tube was prepared by adding 50 μL of a 6.6% (w / v) BSA / 2x PBS solution without primary antibody and incubating for 1 hour.

[0121] After incubation, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes), the supernatant was removed, and the mixture was redispersed in 100 µL of PBS. This centrifugation and redispersion procedure was repeated three times for washing.

[0122] 2. Antigen-antibody reaction of secondary antibodies Next, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed, after which 100 μL of secondary antibody solution (secondary antibody content: 410 ng / mL in 3.3% BSA / PBS solution) was added to the PCR tube and dispersed, followed by incubation for 1 hour at 25°C. After that, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed, after which the mixture was redispersed in 100 μL of PBS, and this centrifugation and redispersion procedure was repeated three times for washing.

[0123] 3. Enzyme reaction The mixture was then centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant removed. 100 μL of substrate solution (OPD: 2 mg / mL, HO: 0.11 v / v%, pH 4.0 citrate phosphate buffer) was added to the PCR tube, dispersed, and incubated at 25°C for 30 minutes. 100 μL of 3N sulfuric acid was then added, and the mixture was centrifuged (15,000 rpm, 25°C, 10 minutes). The supernatant was then aliquoted and diluted 5-fold with ultrapure water, and 100 μL was added to a 96-well plate. 100 μL of the centrifuged supernatant was then added to the 96-well plate, and the absorbance at 490 nm was measured using a plate reader (BioTek "Synergy H1").

[0124] The absorbance was measured for each concentration of the primary antibody. Three sets of three experiments were performed, and the average absorbance of each set was calculated.

[0125] The results are shown in Figures 11 and 12. The horizontal axis shows the concentration of the primary antibody (methoxy-mAb) in the reaction system in the antigen-antibody reaction of the primary antibody, and the vertical axis shows the absorbance at 490 nm. Figure 12 is a graph showing an enlarged view of the results in the low concentration range (0 to 10 ng / mL).

[0126] As shown in Figures 11 and 12, it was found that methoxy-terminated PEG-selective anti-PEG antibodies could be quantitatively detected in the presence of 10% (v / v) FBS and 3% (w / v) BSA. As shown in Figure 12, the slope in the low concentration range was 0.0554, the standard deviation σ was 0.00534, and the coefficient of determination R 2 The lower detection limit (=3σ / slope) was 0.29 ng / mL, which showed extremely high sensitivity compared to the detection limit of approximately 10 ng / mL of a previously reported sensing system in the presence of serum (20-fold dilution) (Shaoyi J. et al., Anal. Chem. 2017, 89, 16, 8217-8222).

[0127] [Test Example 9: Detection limit in the presence of 10% FBS] To confirm the effect of adding BSA, a similar test to Test Example 8 was carried out without adding BSA.

[0128] In Test Example 9, the primary antibody solutions were prepared by diluting the above-mentioned PEG-B-47 with 2xPBS to seven different concentrations of primary antibody: 2.2 ng / mL, 4.4 ng / mL, 11 ng / mL, 22 ng / mL, 44 ng / mL, 110 ng / mL, and 220 ng / mL. The cellulose dispersion, 22% FBS, secondary antibody solution, and substrate solution were the same as those in Test Example 8.

[0129] 1. Antigen-antibody reaction of primary antibody To a 0.2 mL PCR tube, 10 μL of cellulose dispersion (Cell-OEG4, 5 mg / mL, PBS) was added, followed by 50 μL of 22% FBS. 50 μL of primary antibody solution was then added and dispersed. The mixture was then incubated at 25°C for 1 hour to carry out an antigen-antibody reaction. The antigen-antibody reaction system used consisted of a cellulose oligomer concentration of 0.45 mg / mL, a primary antibody concentration of 1-100 ng / mL, and an FBS concentration of 10% (v / v). A blank was prepared by adding 50 μL of 2x PBS without primary antibody and incubating for 1 hour.

[0130] After incubation, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes), the supernatant was removed, and the mixture was redispersed in 100 µL of PBS. This centrifugation and redispersion procedure was repeated three times for washing.

[0131] 2. Antigen-antibody reaction of secondary antibodies Next, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed, after which 100 μL of secondary antibody solution (secondary antibody content: 410 ng / mL in 3.3% BSA / PBS solution) was added to the PCR tube and dispersed, followed by incubation for 1 hour at 25°C. After that, the mixture was centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant was removed, after which the mixture was redispersed in 100 μL of PBS, and this centrifugation and redispersion procedure was repeated three times for washing.

[0132] 3. Enzyme reaction The mixture was then centrifuged (15,000 rpm, 25°C, 5 minutes) and the supernatant removed. 100 μL of substrate solution (OPD: 2 mg / mL, HO: 0.11 v / v%, pH 4.0 citrate phosphate buffer) was added to the PCR tube, dispersed, and incubated at 25°C for 30 minutes. 100 μL of 3N sulfuric acid was then added, and the mixture was centrifuged (15,000 rpm, 25°C, 10 minutes). The supernatant was then collected and diluted 5-fold with ultrapure water. 100 μL of the solution was added to a 96-well plate, and the absorbance at 490 nm was measured using a plate reader (BioTek "Synergy H1").

[0133] The absorbance was measured for each concentration of the primary antibody. Three sets of three experiments were performed, and the average absorbance of each set was calculated.

[0134] The results are shown in Figures 13 and 14. The horizontal axis shows the concentration of the primary antibody (methoxy-mAb) in the reaction system in the antigen-antibody reaction of the primary antibody, and the vertical axis shows the absorbance at 490 nm. Figure 14 is a graph showing an enlarged view of the results in the low concentration range (0 to 10 ng / mL).

[0135] As shown in Figures 13 and 14, it was found that methoxy-terminated PEG-selective anti-PEG antibodies could be quantitatively detected in the presence of 10 v / v% FBS, even without the addition of BSA. As shown in Figure 14, the slope in the low concentration range was 0.0217, the standard deviation σ was 0.00219, and the coefficient of determination R 2 The mean value was 0.9979. The detection limit (=3σ / slope) was 0.30 ng / mL, which was equivalent to that in the presence of 3% (w / v) BSA. Therefore, no effect of adding BSA was observed in the presence of FBS. However, this demonstrated extremely high sensitivity compared to the previously reported detection limit of approximately 10 ng / mL of the sensing system in the presence of serum (20-fold dilution).

[0136] The various numerical ranges described in this specification can be arbitrarily combined with their upper and lower limits, and all such combinations are considered to be preferred numerical ranges described in this specification. Furthermore, a numerical range described as "X to Y" means from X to Y.

[0137] 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 spirit of the invention. These embodiments and their omissions, substitutions, modifications, etc. are included within the scope and spirit of the invention, as well as within the scope of the invention described in the claims and their equivalents.

Claims

1. The following general formula (1): 【Chemical 1】 In the formula (1), n ​​is an average degree of polymerization and represents a number of 6 to 16, and m represents an integer of 1 to 16. An anti-PEG antibody binding material capable of binding to a methoxy-terminated PEG-selective anti-PEG antibody.

2. A biosensor comprising the anti-PEG antibody binding material of claim 1.

3. An adsorbent comprising the anti-PEG antibody binding material of claim 1.

4. The following general formula (1): 【Chemistry 2】 Incubating a cellulose oligomer represented by formula (1), wherein n is an average degree of polymerization and is a number from 6 to 16, and m is an integer from 1 to 16, with a test sample suspected of containing a methoxy-terminated PEG-selective anti-PEG antibody under conditions that allow binding of 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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