Method for evaluating denatured state of antibody

Abstract

The present invention provides, as a method for evaluating a denaturation state of an antibody with an easy operation, a method for evaluating a denaturation state of an antibody, comprising: a step for bringing a compound represented by general formula (1) or a salt or solvate thereof into contact with a test sample including an antibody; and a step for measuring a maximum emission wavelength based on the contact. R1 and R2 in the formula are defined in the specification.

Description

Method for evaluating the denatured state of an antibody

[0001] The present invention relates to a method for evaluating the denatured state of an antibody.

[0002] Antibodies are widely used in antibody drugs, immunochromatographic in vitro diagnostics, and the like. In these applications, detection methods for denatured antibodies play an extremely important role from the perspective of quality control. Conventionally, UV-visible spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy, size exclusion chromatography (SEC), and dynamic light scattering (DLS) have been used for these measurements, but these analytical techniques require specialized knowledge and lack simplicity. In particular, SEC and DLS are methods that detect antibody aggregates based on protein size and are not capable of evaluating antibody structure and properties. While methods using X-ray crystallography and NMR are commonly used for structural analysis, they are not suitable for evaluating antibody quality control due to their lack of simplicity.

[0003] Patent Document 1 discloses a method for measuring the amount of a denatured antibody using an oligopeptide that recognizes the denatured antibody.

[0004] Japanese Patent Application Laid-Open No. 2017-187476

[0005] Patent Document 1 discloses that the above method is capable of quickly and easily measuring the amount of antibody denatured due to stress during production, etc. However, there is room for improvement in terms of ease of operation. In particular, when detecting oligopeptides that recognize denatured antibodies using ELISA (Enzyme-Linked Immunosorbent Assay), a washing step for B / F (Bound / Free) separation is required, making the operation complicated. An object of the present invention is to provide a method for evaluating the denatured state of an antibody that is easy to operate.

[0006] The present inventors conducted extensive research in light of the above-mentioned problems and discovered that a specific compound immediately emits light when brought into contact with a test sample containing an antibody, and that a spectral shift occurs in the case of a denatured antibody, thereby completing the present invention. The present invention encompasses the following aspects [1] to [4]. [1] A method for evaluating the denatured state of an antibody, comprising the steps of: contacting a compound represented by the following general formula (1) or a salt or solvate thereof with a test sample containing an antibody; and measuring the maximum emission wavelength upon said contact:

[0007]

[0008] [In the formula, R １ represents a methyl group or one of groups represented by the following formula (i-1) or (i-2):

[0009]

[0010] (In the formula, R ３ is a hydrogen atom, a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group. ２ is any one of groups represented by the following formula (ii-1), (ii-2), (ii-3), (ii-4) or (ii-5).

[0011]

[0012] [2] The method for evaluating the denatured state of an antibody according to [1], wherein the compound represented by general formula (1) is a compound represented by any one of the following formulas:

[0013]

[0014] [3] The method for evaluating the denatured state of an antibody according to [1] or [2], wherein the test sample is evaluated to contain a denatured antibody if the maximum emission wavelength upon the contact changes by a predetermined reference value or more compared to the maximum emission wavelength upon contact of the compound represented by general formula (1) or a salt or solvate thereof with a standard sample comprising an undenatured antibody. [4] The method for evaluating the denatured state of an antibody according to any one of [1] to [3], wherein the antibody is IgA, IgE, IgG, or IgM.

[0015] The present invention provides a method for evaluating the denatured state of an antibody, which is easy to operate. The evaluation method of the present invention can detect a denatured antibody simply by contacting a specific compound with a test sample, thereby enabling simple and rapid evaluation of the denatured state of an antibody.

[0016] FIG. 1 shows the results of Example 1. FIG. 2 shows the results of Example 2. FIG. 3 shows the results of Examples 3-1 and 3-2. FIG. 4-1 shows the results of Examples 4-1 and 4-2. FIG. 4-2 shows the results of Example 4-3. FIG. 5-1 shows the results of Example 5. FIG. 5-2 shows the results of Example 5. FIG. 6 shows the results of Example 6. FIG. 7 shows the results of Example 7. FIG. 8 shows the results of Example 7.

[0017] [Method for Evaluating the Denatured State of an Antibody] The method for evaluating the denatured state of an antibody of the present invention comprises: (A) contacting a test sample containing an antibody with a compound represented by the following general formula (1) or a salt or solvate thereof; and (B) measuring the maximum emission wavelength resulting from the contact:

[0018] <Denatured State of Antibody> The denatured state of an antibody is evaluated according to the present invention. A denatured antibody is an antibody whose entire or partial structure has changed to form a non-native three-dimensional structure due to stress caused by acid, alkali, heating, freezing and thawing, stirring, electromagnetic waves including light, etc. Causes of denaturation include stress occurring during antibody production and the passage of time during storage, etc.

[0019] <Step (A)> Step (A) is a step of contacting the compound represented by the above general formula (1) or a salt or solvate thereof with a test sample containing an antibody.

[0020] (Compound) The compound used in step (i) is a compound represented by the following general formula (1) or a salt or solvate thereof. The compound represented by the following general formula (1) is a compound having a structure similar to Cypridina luciferin, which has an imidazopyrazinone skeleton. In this specification, the compound represented by the following general formula (1) may be simply referred to as a "luciferin compound."

[0021] [In the formula, R１ represents a methyl group or one of groups represented by the following formula (i-1) or (i-2):

[0022]

[0023] (In the formula, R ３ is a hydrogen atom, a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group. ２ is any one of groups represented by the following formula (ii-1), (ii-2), (ii-3), (ii-4) or (ii-5).

[0024]

[0025] The above formula (i-1) is a compound having an asymmetric carbon C ＊１ It has.

[0026]

[0027] C ＊１ The configuration of may be either the R configuration shown in the following formula (i-1-R) or the following formula (i-1-S). In addition, it may be either one configuration alone or a mixture thereof. A mixture of the R and S configurations includes a racemate containing equal amounts of each configuration.

[0028]

[0029] In the above formula (i-2), R ３ When R is a hydroxy group, a fluorine atom, a methoxy group, or a trifluoromethyl group, the bonding position to the benzene ring skeleton may be any of the ortho, meta, and para positions relative to the other methylene group, and is preferably the para position. ３ is preferably a hydrogen atom or a hydroxy group.

[0030] Preferred embodiments of the group represented by formula (i-2) above include groups represented by formulas (i-2-1) and (i-2-2) below.

[0031]

[0032] The groups represented by the above formulae (ii-1), (ii-2), (ii-3), (ii-4), and (ii-5) are all groups having a structure in which a substituted amino group or a pyrrolidin-1-yl group is bonded to a benzene ring group bonded to an imidazopyrazinone skeleton at the ortho-, meta-, or para-position with respect to the bond to the imidazopyrazinone skeleton. The bonding position of the substituted amino group or the pyrrolidin-1-yl group is preferably the para-position, and the groups represented by the above formulae (ii-1), (ii-2), (ii-3), (ii-4), and (ii-5) are preferably groups represented by the following formulae (ii-1-1), (ii-2-1), (ii-3-1), (ii-4-1), and (ii-5-1), respectively.

[0033]

[0034] R １ is preferably a group represented by the above formula (i-2-1) or (i-2-2).

[0035] R ２ is preferably a group represented by the above formula (ii-1-1) or (ii-3-1).

[0036] Preferred embodiments of the compound represented by the general formula (1) include compounds LIg3, LIg5 and LIg7 represented by the following formulae.

[0037]

[0038] The salt of the compound represented by the general formula (1) is not particularly limited. Specific examples include acid addition salts of hydrohalides (e.g., hydrochlorides, hydrobromides, and hydroiodides), inorganic acid salts (e.g., sulfates, nitrates, perchlorates, phosphates, carbonates, and bicarbonates), organic carboxylates (e.g., acetates, trifluoroacetates, maleates, tartrates, fumarates, and citrates), and organic sulfonates (e.g., methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates, and camphorsulfonates); and base addition salts such as quaternary amine salts, alkali metal salts (e.g., sodium salts and potassium salts), and alkaline earth metal salts (e.g., magnesium salts and calcium salts). Hydrates and solvates of the compound represented by the general formula (1) include, for example, solvates with ethanol.

[0039] (Production Method) The compound of the present invention can be produced, for example, as shown in Scheme 1, by condensing a coelenteramine derivative (x1) with a ketoacetal compound (x2-1) or methylglyoxal (x2-2).

[0040]

[0041]

[0042] [In the formula, R １ and R ２ is the same as above.]

[0043] Specifically, R １ is a group represented by the above formula (i-1) or (i-2), a ketoacetal compound (x2-1) can be used, and R １ When is a methyl group, methylglyoxal (x2-2) can be used.

[0044] The reaction proceeds advantageously in the presence of water, using about 1 to 4 moles of ketoacetal compound (x2-1) or methylglyoxal (x2-2) per mole of coelenteramine derivative (x1), and reacting in the presence of acid at a temperature of about 65 to 100°C for 1 to 5 hours. The reaction proceeds advantageously in a solvent, such as ethanol.

[0045] (Test Sample Containing Antibody) The test sample containing an antibody is not particularly limited as long as it is a sample containing an antibody. The origin of the antibody is not particularly limited, and examples thereof include antibodies derived from humans, mice, rats, rabbits, goats, etc., and preferably antibodies derived from humans. The type of antibody is not particularly limited, and examples thereof include immunoglobulin isotypes (IgA, IgE, IgG, IgM, IgG, etc.) and their isotype subclasses. More specifically, human immunoglobulins include IgA1, IgA2, IgE, IgG1, IgG2, IgG3, IgG4, and IgM. The antibody may be either a full-length immunoglobulin protein or a fragment thereof. Preferably, it is a full-length immunoglobulin protein. Examples of fragments of immunoglobulin proteins include F(ab') ２ Examples of such fragments include fragments containing variable regions such as Fab, Fab', and Fv, as well as fragments containing constant regions such as Fc.

[0046] Furthermore, the antibody is not limited to an antibody having a native amino acid sequence derived from a single animal species, but may also be an antibody formed by combining antibody fragments derived from different animal species. For example, it may be a chimeric antibody (a chimeric antibody containing the Fc region of a human antibody and sequences (variable region, CDR, etc.) derived from an antibody derived from another animal (e.g., a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody)), a humanized antibody, etc. Furthermore, the antibody may be a monoclonal antibody or a polyclonal antibody. Specific examples of test samples include research reagents, antibody drugs, and their production intermediates.

[0047] (Contact) Conditions for contacting the compound represented by general formula (1) or a salt or solvate thereof with a test sample containing an antibody can be appropriately determined by those skilled in the art. The temperature is, for example, 0 to 40°C, preferably 15 to 30°C. The pH is, for example, 4.0 to 10.0, preferably 6.0 to 9.0. The pH can also be, for example, 4.0 to 9.0, preferably 6.0 to 8.0. The contact time (reaction time) is, for example, 1 second to 30 minutes, preferably 1 to 30 seconds.

[0048] The step of contacting the compound represented by general formula (1) or a salt or solvate thereof with a test sample containing an antibody can be carried out in a buffer solution for maintaining an appropriate pH, such as Tris-HCl, PBS, or HEPES. The concentration of the compound represented by general formula (1) or a salt or solvate thereof during contact is not particularly limited, and is preferably 1 to 100 μM, more preferably 3 to 50 μM. A concentration equal to or higher than the above lower limit is preferred because it is believed that a sufficient luminescence reaction rate can be achieved. A concentration equal to or lower than the above upper limit is preferred from the viewpoint of the solubility of the compound, etc. The concentration of the antibody during contact is not particularly limited, and is preferably 0.001 mg / mL to 100 mg / mL.

[0049] <Step (B)> Step (B) is a step of measuring the maximum emission wavelength based on the contact. The maximum emission wavelength is also called the peak emission wavelength, and refers to the wavelength at which the emission intensity is maximum in the emission spectrum. The maximum emission wavelength can be measured by a method known to those skilled in the art. For example, the emission spectrum can be obtained using a known luminometer, spectrophotometer, etc., and the maximum emission wavelength can be calculated. The maximum emission wavelength can also be calculated using known analysis software.

[0050] The maximum emission wavelength upon contact is preferably compared with the maximum emission wavelength obtained when a standard sample is contacted with the compound represented by general formula (1) or its salt or solvate. The standard sample is not limited as long as it contains the same antibody as the test sample and the denatured state of the antibody is clear. Specific examples include a standard sample consisting of a native antibody and a standard sample consisting of a denatured antibody. A standard sample consisting of a native antibody is, for example, a standard sample that has not been subjected to any treatment that could denature the antibody and is essentially composed of only native antibody. A standard sample consisting of a denatured antibody is, for example, a standard sample consisting of an antibody that has been subjected to excessive denaturation treatment. Examples of such excessive denaturation treatment include heating at approximately 75°C for 15 minutes or more. In a preferred embodiment of the present invention, the test sample is evaluated to contain a denatured antibody if the maximum emission wavelength upon contact is changed by more than a predetermined reference value compared to the maximum emission wavelength obtained when a standard sample consisting of a native antibody is contacted with the compound represented by general formula (1) or its salt or solvate. A native antibody refers to an antibody that has a native three-dimensional structure. The change in maximum emission wavelength is also referred to as a spectral shift. The predetermined reference value or greater is preferably 1 nm or greater, more preferably 10 nm or greater, and even more preferably 15 nm or greater. For example, when the compound represented by the general formula (1) is the following compounds LIg3, LIg5, or LIg7, the maximum emission wavelength when contacted with a sample containing a modified antibody is shorter than the maximum emission wavelength when contacted with a standard sample consisting of an unmodified antibody.

[0051]

[0052] Thus, the denatured state of the antibody is evaluated. The method of the present invention for evaluating the denatured state of an antibody can be used, for example, for quality control of various products containing antibodies. Examples of products containing antibodies include research reagents and antibody drugs.

[0053] While not wishing to be bound by theory, similar to the bioluminescence reaction (also known as the luciferin-luciferase reaction) that proceeds through the reaction between the substrate luciferin and the enzyme luciferase, the antibody catalyzes a luminescence reaction using the compound represented by general formula (1) above, or a salt or solvate thereof, as a substrate. The emission wavelength of the compound represented by general formula (1) above, or a salt or solvate thereof, varies depending on the microenvironment, such as the structure and polarity, of the active site. Since antibody denaturation is expected to result in a change in the microenvironment of the active site, it is believed that the denatured state of the antibody contained in the test sample can be evaluated by measuring the change in the maximum emission wavelength.

[0054] [Kit for assessing the denatured state of an antibody] The present invention also provides a kit for assessing the denatured state of an antibody. The kit for assessing the denatured state of an antibody of the present invention comprises a compound represented by general formula (1) above, or a salt or solvate thereof. The compound represented by general formula (1) above, or a salt or solvate thereof may be in a solid form such as a lyophilized product, or in a liquid form dissolved or dispersed in an appropriate medium such as water.

[0055] The kit of the present invention can contain other components as needed. Examples of other components include a standard sample consisting of an undenatured antibody and tools such as test tubes. The kit can also contain a manual describing a method for assessing the denatured state of an antibody using the kit. The manual preferably describes the method for assessing the denatured state of an antibody of the present invention. The denatured state of an antibody can be assessed by carrying out the method for assessing the denatured state of an antibody of the present invention using the kit of the present invention.

[0056] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples.

[0057] [Synthesis Example] Each reagent was purchased from Fujifilm Wako Pure Chemical Industries, Ltd., Kanto Chemical Co., Ltd., Tokyo Chemical Industry Co., Ltd., BLD Pharma Tech, Inc., or Sigma-Aldrich, and was used as is without purification. １ H-NMR and １３C-NMR was performed using a Bruker Avance III-500 as the measuring device. Tetramethylsilane (TMS, 0 ppm) was used as the internal standard. Coupling constants (J) are expressed in Hz. The abbreviations s, d, t, q, m, and br represent singlet, doublet, triplet, quartet, multiplet, and broad line, respectively.

[0058] General Remarks In the following synthesis examples, as shown in Scheme 1 or 2, compounds were synthesized by condensing a coelenteramine derivative with a ketoacetal compound or methylglyoxal.

[0059]

[0060]

[0061] [In the formula, R １ and R ２ is the same as above.]

[0062] The coelenteramine derivative was synthesized according to the following reaction scheme 3.

[0063]

[0064] Synthesis Example 1: Synthesis of 2-benzyl-6-(4-(diethylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (LIg1)

[0065]

[0066] (1) Under an argon atmosphere, 5-bromopyrazin-2-amine (200.0 mg, 1.14 mmol, 1 eq.) and (4-(diethylamino)phenyl)boronic acid (355.0 mg, 1.83 mmol, 1.6 eq.) were dissolved in ethanol (3.5 mL) and toluene (24 mL). 1 M aqueous sodium carbonate solution (9 mL) was added and the mixture was stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added. The mixture was degassed under vacuum again and stirred at 100°C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate = 1 / 1) to give 5-(4-(diethylamino)phenyl)pyrazin-2-amine (261.9 mg, 94%). １ H-NMR (500MHz, CDCl ３ ): δ (ppm) = 8.36 (d, J = 1.4 Hz, 1H), 8.00 (d, J = 1.5 Hz, 1H), 7.74 (q, J = 3.0 Hz , 2H), 6.74 (d, J = 8.7Hz, 2H), 3.39 (q, J = 7.0Hz, 4H), 1.18 (t, J = 7.0Hz, 6H). １３ C-NMR (125MHz, CDCl ３ ): δ (ppm) = 12.68, 44.56, 111.98, 124.16, 126.83, 131.38, 137.86, 143.90, 147.80, 152.14.

[0067] (2) Under an argon atmosphere, 5-(4-(diethylamino)phenyl)pyrazin-2-amine (40.0 mg, 0.16 mmol, 1 eq.) obtained in (1) above and 1,1-diethoxy-3-phenylpropan-2-one (73 mg, 0.33 mmol, 2 eq.) were dissolved in ethanol (2 mL) and ultrapure water (Milli-Q) (0.2 mL) and cooled to 0°C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 mL) was added, and the mixture was stirred at 80°C for 4 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethyl acetate / methanol = 10 / 1) to obtain 2-benzyl-6-(4-(diethylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (LIg1) as a yellow solid (14.5 mg, 23%). １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 7.71 (s, 1H), 7.48 (s, 1H), 7.33 (d, J = 8.8Hz, 2H), 7.23-7.05 (m, 5H), 6.64 (d, J=8.9Hz, 2H), 4.04 (s, 2H), 3.31 (q, J=7.0Hz, 4H), 1.07 (t, J=7.0Hz, 6H).

[0068] Synthesis Example 2: Synthesis of 6-(4-(diethylamino)phenyl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one (LIg2)

[0069]

[0070] (1) 5-(4-(diethylamino)phenyl)pyrazin-2-amine was obtained in the same manner as in (1) of Synthesis Example 1. (2) Under an argon atmosphere, 5-(4-(diethylamino)phenyl)pyrazin-2-amine (40.0 mg, 0.16 mmol, 1 eq.) obtained in (1) above and methylglyoxal (23 mg, 0.33 mmol, 2 eq.) were dissolved in ethanol (2 mL) and ultrapure water (Milli-Q) (0.2 mL), and the solution was cooled to 0°C. The reaction solution was degassed under vacuum, and concentrated hydrochloric acid (0.1 mL) was added, followed by stirring at 80°C for 4 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane / methanol=10 / 1) to obtain 6-(4-(diethylamino)phenyl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one (LIg2) as a yellow solid (7.0 mg, 14%). １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 7.62 (s, 1H), 7.44 (s, 1H), 7.36 (d, J = 8.4Hz, 2H), 6.67 (d, J =8.7Hz, 2H), 3.34 (d, J = 7.0Hz, 4H), 2.38 (s, 3H), 1.11 (t, J = 7.0Hz, 6H).

[0071] Synthesis Example 3: Synthesis of 2-benzyl-6-(4-(diphenylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (LIg3)

[0072]

[0073] (1) Under an argon atmosphere, 5-bromopyrazin-2-amine (100.0 mg, 0.57 mmol, 1 eq.) and (4-(diphenylamino)phenyl)boronic acid (230.0 mg, 0.79 mmol, 1.4 eq.) were dissolved in ethanol (2 mL) and toluene (16 mL). 1 M aqueous sodium carbonate solution (6 mL) was added and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added. The solution was degassed under vacuum again and stirred at 100°C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: ethyl acetate / hexane = 1 / 1) to give 5-(4-(diphenylamino)phenyl)pyrazin-2-amine as a yellow solid (194.2 mg, 99%). １ H-NMR (500MHz, CDCl ３ ): δ (ppm) = 8.39 (d, J = 1.0 Hz, 1H), 8.02 (d, J = 1.3 Hz, 1H), 7.73 (d, J = 8.6 Hz, 2H), 7.27-7.01 (m, 12H), 4.62 (s, 2H).

[0074] (2) Under an argon atmosphere, 5-(4-(diphenylamino)phenyl)pyrazin-2-amine (40.0 mg, 0.12 mmol, 1 eq.) obtained in (1) above and 1,1-diethoxy-3-phenylpropan-2-one (53 mg, 0.24 mmol, 2 eq.) were dissolved in ethanol (2 mL) and ultrapure water (Milli-Q) (0.2 mL) and cooled to 0°C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 mL) was added, and the mixture was stirred at 80°C for 4 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane / methanol = 20 / 1) to obtain 2-benzyl-6-(4-(diphenylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (LIg3) as a yellow solid (13.0 mg, 23%). １ H-NMR (500MHz, CD ３OD): δ (ppm) = 7.74 (s, 1H), 7.58 (s, 1H), 7.39 (d, J = 8.2Hz, 2H), 7.24-6.97 (m, 18H), 4.05 (s, 2H).

[0075] Synthesis Example 4: Synthesis of 6-(4-(diphenylamino)phenyl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one (LIg4)

[0076]

[0077] (1) 5-(4-(diphenylamino)phenyl)pyrazin-2-amine was obtained in the same manner as in (1) of Synthesis Example 3. (2) Under an argon atmosphere, 5-(4-(diphenylamino)phenyl)pyrazin-2-amine (40.0 mg, 0.12 mmol, 1 eq.) obtained in (1) above and methylglyoxal (17 mg, 0.24 mmol, 2 eq.) were dissolved in ethanol (2 mL) and ultrapure water (Milli-Q) (0.2 mL), and the solution was cooled to 0°C. The reaction solution was degassed under vacuum, and concentrated hydrochloric acid (0.1 mL) was added, followed by stirring at 80°C for 4 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane / methanol=20 / 1) to obtain 6-(4-(diphenylamino)phenyl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one (LIg4) as a yellow solid (26.8 mg, 57%). １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 7.89 (s, 1H), 7.68 (s, 1H), 7.54 (d, J = 8.6Hz, 2H), 7.32-7.06 (m, 12H), 4.55 (s, 3H).

[0078] Synthesis Example 5: Synthesis of 2-benzyl-6-(4-(dimethylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (LIg5)

[0079]

[0080] (1) 5-(4-(dimethylamino)phenyl)pyrazin-2-amine was synthesized according to the description in the literature: Photochem. Photobiol. Sci. 2014, 13, 182-189. (2) Under an argon atmosphere, 5-(4-(dimethylamino)phenyl)pyrazin-2-amine (30.0 mg, 0.14 mmol, 1 eq.) obtained in (1) above and 1,1-diethoxy-3-phenylpropan-2-one (37 mg, 0.16 mmol, 2 eq.) were dissolved in ethanol (2 mL) and ultrapure water (Milli-Q) (0.2 mL) and cooled to 0°C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 mL) was added, and the mixture was stirred at 80°C for 4 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane / methanol=10 / 1) to obtain 2-benzyl-6-(4-(dimethylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (LIg5) as a yellow solid (7.2 mg, 14%). １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 7.83 (s, 1H), 7.57 (s, 1H), 7.43 (d, J = 8.7Hz, 2H), 7.23-7.07 (m, 5H), 6.74 (d, J = 8.7Hz, 2H), 4.03 (s, 2H), 2.90 (s, 6H).

[0081] Synthesis Example 6: Synthesis of 6-(4-(dimethylamino)phenyl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one (LIg6)

[0082]

[0083] (1) 5-(4-(dimethylamino)phenyl)pyrazin-2-amine was synthesized according to the description in the literature: Photochem. Photobiol. Sci. 2014, 13, 182-189. (2) 5-(4-(dimethylamino)phenyl)pyrazin-2-amine (40.0 mg, 0.18 mmol, 1 eq.) obtained in (1) above and methylglyoxal (27 mg, 0.37 mmol, 2 eq.) were dissolved in ethanol (2 mL) and ultrapure water (Milli-Q) (0.2 mL) and cooled to 0°C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 mL) was added, and the mixture was stirred at 80°C for 4 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: dichloromethane / methanol=20 / 1) to obtain 6-(4-(dimethylamino)phenyl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one (LIg6) as a yellow solid (41.6 mg, 83%). １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 7.87 (s, 1H), 7.70 (s, 1H), 7.65 (d, J = 7.7Hz, 2H), 6.98 (d, J = 8.9Hz, 2H), 3.506-3.500 (m, 6H), 2.63 (s, 3H).

[0084] Synthesis Example 7: Synthesis of 6-(4-(diphenylamino)phenyl)-2-(4-hydroxybenzyl)imidazo[1,2-a]pyrazin-3(7H)-one (LIg7)

[0085]

[0086] (1) 5-(4-(diphenylamino)phenyl)pyrazin-2-amine was obtained in the same manner as in (1) of Synthesis Example 1. (2) Under an argon atmosphere, 5-(4-(diphenylamino)phenyl)pyrazin-2-amine (40.0 mg, 0.11 mmol, 1 eq.) obtained in (1) above and 3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one (83 mg, 0.23 mmol, 2 eq.) were dissolved in ethanol (2 mL) and ultrapure water (Milli-Q) (0.2 mL), and the solution was cooled to 0°C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 mL) was added, and the mixture was stirred at 80°C for 4 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: chloroform / methanol=19 / 1) to give 6-(4-(diphenylamino)phenyl)-2-(4-hydroxybenzyl)imidazo[1,2-a]pyrazin-3(7H)-one (LIg7) as a yellow solid (13.78 mg, 23%). １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 8.28 (s, 1H), 7.84 (s, 1H), 7.58 (d, J = 8.0Hz, 2H), 7.37-6.60 (m, 16H), 3.26 (s, 2H).

[0087] [Examples] The structures of the luciferin compounds used in the following examples are shown below. Natural Cypridina luciferin was "Cypridina Luciferin" (ATTO Corporation, product number 3512055). HuLumiono1 was synthesized according to the description in the literature: Bioconjugate Chem., 2020, 31, 2679-2684.

[0088]

[0089] Example 1 Natural Cypridina luciferin, HuLumiono1, and the luciferin compounds LIg1 to LIg6 obtained in Synthesis Examples 1 to 6 were each reacted with a human IgG antibody, and the luminescence intensity was measured.

[0090] 5 μL of the human IgG antibody solution and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, manufactured by PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, manufactured by Promega) to determine the luminescence intensity ([RLU / min]) (n=4).

[0091] The luciferin compound-buffer solution used was a solution of natural Cypridina luciferin (manufactured by ATTO Corporation, product number 3512055), HuLumiono1, or the luciferin compounds LIg1 to LIg6 obtained in Synthesis Examples 1 to 6, dissolved in PBS buffer at a concentration of 20 μM. The human IgG antibody solution used was a solution of "Normal Human IgG, Whole Molecule, Purified Product" (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number 149-09503) dissolved in PBS at concentrations of 10 mg / mL and 0 mg / mL (control). The final concentration in the reaction solution was 1 mg / mL.

[0092] The results are shown in Figure 1. Figure 1(a) shows the measurement results of luminescence intensity, and Figure 1(b) shows the signal-to-noise ratio (S / N ratio) calculated relative to the control.

[0093] Natural Cypridina luciferin, HuLumiono1, and LIg1 to LIg6 reacted with human IgG antibodies, and luminescence was observed. Among them, LIg3, which has a triphenylamine group, exhibited significantly higher luminescence intensity. Furthermore, LIg1 had a low background signal and its S / N ratio was equivalent to that of LIg3.

[0094] Example 2 The compounds LIg3, LIg4 and LIg7 obtained in Synthesis Examples 3, 4 and 7 were reacted with human IgG antibody and the luminescence intensity was measured. LIg3, LIg4 and LIg7 are luciferin compounds having a triphenylamine group.

[0095] 5 μL of the human IgG antibody solution and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, manufactured by PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, manufactured by Promega) to determine the luminescence intensity ([RLU / min]) (n=4).

[0096] The luciferin compound-buffer solution used was a solution of LIg3, LIg4, and LIg7 obtained in Synthesis Examples 3, 4, and 7 dissolved in 10 mM Tris-HCl (pH 7.4) buffer at a concentration of 20 μM. The human IgG antibody solution used was a solution of "Normal Human IgG, Whole Molecule, Purified Product" (Fujifilm Wako Pure Chemical Industries, Ltd., product number 149-09503) dissolved in 10 mM Tris-HCl (pH 7.4) at concentrations of 10 mg / mL, 1 mg / mL, and 0 mg / mL (control). The final concentrations of the human IgG antibody in the reaction solution were 1 mg / mL, 0.1 mg / mL, and 0 mg / mL, respectively.

[0097] The results are shown in Figure 2. Figure 2(a) shows the structure of each compound measured, Figure 2(b) shows the measurement results of luminescence intensity, and Figure 2(c) shows the signal-to-noise ratio (S / N ratio) calculated relative to the control.

[0098] The luminescence intensity and S / N ratio were significantly higher for LIg3 and LIg7, while the values ​​were relatively low for LIg4. These results demonstrate that modifications with triphenylamine at position 6 and benzyl at position 2 of the imidazopyrazinone ring result in significantly higher luminescence intensity and S / N ratio upon reaction with human IgG.

[0099] Example 3-1 The compound LIg3 obtained in Synthesis Example 3 was reacted with various human IgG antibodies and fragments thereof, and the luminescence intensity was measured.

[0100] 5 μL of the protein solution and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, manufactured by PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (Spark Cyto, manufactured by TECAN) to measure the luminescence intensity ([RLU / min]) (n=3).

[0101] The luciferin compound-buffer solutions used were prepared by dissolving the compound LIg3 obtained in Synthesis Example 3 in 10 mM Tris-HCl (pH 7.4) buffer at concentrations of 0.03, 0.07, 0.15, 0.31, 0.62, 1.25, 2.5, and 5.0 μM. The protein solutions used were prepared by dissolving the following human IgG antibodies or fragments thereof in 10 mM Tris-HCl (pH 7.4) at a concentration of 20 μM. IgG: "Normal human IgG, whole molecule, purified product" (Fujifilm Wako Pure Chemical Corporation, product number 149-09503) Fc: "Human IgG-Fc fragment" (Jackson Immuno Research Laboratories, purchased from Fujifilm Wako Pure Chemical Corporation, product number 562-71603) Fab: "Human IgG-Fab fragment" (Jackson Immuno Research Laboratories, purchased from Fujifilm Wako Pure Chemical Corporation, product number 566-71623) F(ab')2: "Human IgG-F(ab')2 fragment" (Jackson Immuno Research Laboratories, purchased from Fujifilm Wako Pure Chemical Corporation, product number 566-71623) (manufactured by Fujifilm Laboratories, purchased from Fujifilm Wako Pure Chemical Industries, part number 569-71613)

[0102] The results are shown in Figures 3(a) and (b). Figure 3(a) shows the results of measuring luminescence intensity. Figure 3(b) shows the relative k between full-length IgG (2 µM) and each fragment (2 µM). ｃａｔ The value is shown as relative k. ｃａｔ The value is the k for full-length IgG calculated from the results in Figure 3(a). ｃａｔ The value was normalized to 1.0 and calculated.

[0103] The full-length IgG showed particularly high luminescence intensity across the entire range of LIg3 concentrations measured (Figure 3(a)). ｃａｔ The results of the values ​​show that the reaction rate of full-length IgG with LIg3 was approximately 7 times higher than that of the other fragments (Figure 3(b)). It is presumed that the full-length structure of IgG particularly contributes as a reaction field between the luciferin compound and the IgG antibody.

[0104] Example 3-2 The compound LIg3 obtained in Synthesis Example 3 was reacted with a human IgG antibody in the presence of Protein A, and the luminescence intensity was measured. Protein A is a protein derived from Staphylococcus aureus that specifically binds to the Fc region of immunoglobulin.

[0105] 5 μL of the protein mixture and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, manufactured by PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, manufactured by Promega), and the luminescence intensity ([RLU / min]) was measured (n=3).

[0106] The luciferin compound-buffer solution used was the compound LIg3 obtained in Synthesis Example 3 dissolved in 10 mM Tris-HCl (pH 7.4) buffer at a concentration of 20 μM. The protein mixture used was a solution of human IgG antibody and Protein A dissolved in 10 mM Tris-HCl (pH 7.4). The human IgG antibody was "Normal Human IgG, Whole Molecule, Purified Product" (Fujifilm Wako Pure Chemical Corporation, Product No. 149-09503) dissolved at a concentration of 10 mg / mL. The Protein A was "Protein A, Recombinant" (Fujifilm Wako Pure Chemical Corporation, Product No. 164-26194) dissolved at concentrations of 50 mg / mL, 40 mg / mL, 30 mg / mL, 20 mg / mL, 10 mg / mL, 5 mg / mL, 2.5 mg / mL, and 0 mg / mL (control).

[0107] The results are shown in FIG.

[0108] Even when Protein A was bound to a human IgG antibody in advance, no significant change was observed in the luminescence intensity upon reaction with a luciferin compound. It is presumed that the Fc region of IgG to which Protein A binds contributes little as a reaction site with the luciferin compound.

[0109] Example 4-1 The luciferin compound LIg3 obtained in Synthesis Example 3 was reacted with human IgG antibody and IgG antibody of a specific species, and the luminescence intensity was measured. 5 μL of the antibody solution and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, manufactured by PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (Spark Cyto, manufactured by TECAN), and the luminescence intensity ([RLU / min]) was calculated (n=4).

[0110] The luciferin compound-buffer solution used was the compound LIg3 obtained in Synthesis Example 3 dissolved in 10 mM Tris-HCl (pH 7.4) buffer at a concentration of 5 μM. The antibody solution used was an antibody dissolved in 10 mM Tris-HCl (pH 7.4). The antibody solutions used were the following human, mouse, rat, rabbit, and goat IgG antibodies dissolved in 10 mM Tris-HCl (pH 7.4) at a concentration of 20 μM. Human: "Normal human IgG, whole molecule, purified product" (Fujifilm Wako Pure Chemical Corporation, product number 149-09503) Mouse: "Normal mouse IgG, whole molecule, purified product" (Fujifilm Wako Pure Chemical Corporation, product number 140-09511) Rat: "Normal rat IgG, whole molecule, purified product" (Fujifilm Wako Pure Chemical Corporation, product number 147-09521) Rabbit: "Normal rabbit IgG, whole molecule, purified product" (Fujifilm Wako Pure Chemical Corporation, product number 148-09551) Goat: "Normal goat IgG, whole molecule, purified product" (Fujifilm Wako Pure Chemical Corporation, product number 144-09531)

[0111] The results are shown in Figures 4(a) and (b).

[0112] LIg3 reacted with both human IgG antibodies and IgG antibodies from other species, and showed significant luminescence signals.

[0113] Example 4-2 The luciferin compound LIg3 obtained in Synthesis Example 3 was reacted with various concentrations of human IgG antibody, and the luminescence intensity was measured. 5 μL of the human IgG antibody solution and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area OpticalPlate-96, PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, Promega), and the luminescence intensity ([RLU / min]) was measured (n=3).

[0114] The luciferin compound-buffer solution used was a solution of the compound LIg3 obtained in Synthesis Example 3 dissolved in 10 mM Tris-HCl (pH 7.4) buffer at a concentration of 20 μM. The human IgG antibody solution used was a solution of "Normal Human IgG, Whole Molecule, Purified Product" (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number 149-09503) dissolved in 10 mM Tris-HCl (pH 7.4) at IgG concentrations of 10 mg / mL, 7.5 mg / mL, 5 mg / mL, 3.75 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0 mg / mL.

[0115] The results are shown in FIG.

[0116] Example 4-3 The compound LIg3 obtained in Synthesis Example 3 was reacted with unmodified and modified human IgG antibodies, and the emission spectra were measured.

[0117] The luciferin compound-buffer solution used was a solution of the compound LIg3 obtained in Synthesis Example 3 dissolved in 10 mM Tris-HCl (pH 7.4) buffer at a concentration of 5 μM.

[0118] "Normal human IgG, whole molecule, purified product" (Fujifilm Wako Pure Chemical Industries, Ltd., product number 149-09503) was dissolved in 10 mM Tris-HCl (pH 7.4) at a concentration of 20 μM and used as a native human IgG antibody solution.

[0119] The modified antibody was prepared as follows. The unmodified human IgG antibody solution was heated at 75°C for 5, 10, 15, and 30 minutes using a block incubator (ATTO Corporation). After heating, the solution was cooled on ice for 20 minutes to obtain a modified IgG antibody solution. The modified antibody was analyzed by measuring the absorbance (OD ) at a wavelength of 400 nm using a luminescence plate reader (Spark Cyto, TECAN). ４００ ) and the peak value (θ) of the CD spectrum measured by a circular dichroism spectrometer (manufactured by JASCO Corporation). ２１６ x10 －３ [deg cm ２ The denaturation of the human IgG antibody depending on the heating time was measured by OD ４００ and θ ２１６ The solution obtained after heating for 5 minutes was used as a denatured IgG antibody solution in luminescence measurements.

[0120] Next, 5 μL of the human IgG antibody solution and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area OpticalPlate-96, manufactured by PerkinElmer), and the luminescence spectrum was immediately measured using a luminescence plate reader (Spark Cyto, manufactured by TECAN). (n=4) The maximum luminescence wavelength was calculated using medical statistics and graph creation software ("GraphPad Prism 9", manufactured by GraphPad Software). The luciferin compound-buffer solution used was a solution of the compound LIg3 obtained in Synthesis Example 3 dissolved in 10 mM Tris-HCl (pH 7.4) buffer at a concentration of 5 μM.

[0121] The results are shown in Figure 4(e). The native human IgG antibody (Native IgG) had a maximum emission wavelength (λ ｍａｘ) is 533 nm, and denatured human IgG antibody (Denatured IgG) has a maximum emission wavelength (λ ｍａｘ The emission spectrum is a normalized luminescence intensity where the emission intensity at the maximum emission wavelength is set to 1.0.

[0122] Example 5 Compounds LIg1, 3, 4, 5 and 7 obtained in Synthesis Examples 1, 3, 4, 5 and 7 were reacted with native and denatured human IgG antibodies, and the emission spectra were measured.

[0123] 5 μL of the human IgG antibody solution and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, manufactured by PerkinElmer), and the luminescence spectrum was immediately measured using a luminescence plate reader (Spark Cyto, manufactured by TECAN) (n=4). The maximum luminescence wavelength was calculated using medical statistics and graph creation software (GraphPad Prism 9, manufactured by GraphPad Software). Similarly, 5 μL of the human IgG antibody solution and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, manufactured by PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (Spark Cyto, manufactured by TECAN) to determine the luminescence intensity ([RLU / min]) (n=3).

[0124] The luciferin compound-buffer solutions used were prepared by dissolving each of the compounds LIg1, 3, 4, 5, and 7 obtained in Synthesis Examples 1, 3, 4, 5, and 7 in 10 mM Tris-HCl (pH 7.4) buffer at a concentration of 5 μM. The human IgG antibody solutions used were a native human IgG antibody solution and a denatured human IgG antibody solution heated at 75°C for 5 minutes, 10 minutes, 15 minutes, and 30 minutes. The native and denatured human IgG antibody solutions were prepared in the same manner as in Example 4-3.

[0125] The results are shown in Figure 5-1, Table 1, and Figure 5-2. Figure 5-1 shows the emission spectra when a native human IgG antibody and each modified human IgG antibody were used, with the emission intensity normalized by the emission intensity at the maximum emission wavelength (normalized luminescence). Table 1 shows the difference (spectral shift) between the maximum emission wavelength when each compound was reacted with a modified human IgG antibody heated for 30 minutes and the maximum emission wavelength when each compound was reacted with a native human IgG antibody. Figure 5-2 shows the change in emission intensity depending on the heating time when a native human IgG antibody and each modified human IgG antibody were used.

[0126] Particularly significant spectral shifts were observed for three compounds: LIg3, LIg5, and LIg7. The spectral shift for LIg7 was particularly large. Differences in emission intensity were also observed between native and modified IgG for LIg1, LIg3, LIg4, and LIg7.

[0127]

[0128] Example 6 The compound LIg3 obtained in Synthesis Example 3 was reacted with a mixed solution containing unmodified and / or modified human IgG antibodies, and the emission spectrum was measured.

[0129] 5 μL of the human IgG antibody mixed solution and 45 μL of the luciferin compound-buffer solution were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, manufactured by PerkinElmer), and the luminescence spectrum was immediately measured using a luminescence plate reader (Spark Cyto, manufactured by TECAN) (n=4). The maximum luminescence wavelength was calculated using medical statistics and graph creation software (GraphPad Prism 9, manufactured by GraphPad Software).

[0130] The luciferin compound-buffer solution used was the compound LIg3 obtained in Synthesis Example 3, dissolved in 10 mM Tris-HCl (pH 7.4) buffer at a concentration of 5 μM. The human IgG antibody solutions used were a native human IgG antibody solution and a denatured human IgG antibody solution heated at 75°C for 5 minutes. The native and denatured human IgG antibody solutions were prepared in the same manner as in Example 4-3. The human IgG antibody mixed solutions were prepared by mixing native human IgG:denatured human IgG (IgG:non-native IgG) in the following composition ratios: 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:30, 30:70, 20:80, 10:90, and 0:100.

[0131] The results are shown in Figure 6. Figure 6(a) shows the emission spectra of human IgG antibody mixed solutions of each composition, normalized by the emission intensity at the peak wavelength (normalized luminescence). Figure 6(b) shows the difference (Δλ) between the maximum emission wavelength when reacted with a mixed solution containing denatured human IgG antibody and the maximum emission wavelength when reacted with native human IgG antibody (native human IgG:denatured human IgG = 100:0). ｍａｘ The coefficient of determination R2 was 0.99 when the content ratio of denatured human IgG (Spiked non-native IgG [%]) was in the range of 0 to 100%.

[0132] As shown in FIGS. 6(a) and (b), the spectral shift increases as the content ratio of denatured human IgG (spiked non-native IgG [%]) increases.

[0133] Example 7 The compound LIg3 obtained in Synthesis Example 3 was reacted with a mixed solution containing unmodified and / or modified IgG2 antibodies, and the emission spectrum was measured.

[0134] The luciferin compound-buffer solution used was a solution of the compound LIg3 obtained in Synthesis Example 3 dissolved in 10 mM Tris-HCl (pH 9) buffer at a concentration of 5 μM.

[0135] Denosumab (product name: Ranmark Subcutaneous Injection 120 mg, manufactured and sold by Daiichi Sankyo Co., Ltd.) was used as the IgG2 antibody, and was dissolved in 10 mM Tris-HCl (pH 9) at a concentration of 20 μM to prepare a non-native IgG2 antibody solution. The non-native IgG2 antibody solution was prepared by heating at 75°C for 5 minutes in the same manner as in Example 4-3. Denosumab (Ranmark) is a commercially available antibody drug and is a human IgG2 monoclonal antibody.

[0136] Non-native IgG2:denatured IgG2 (IgG2:non-native IgG2) were mixed at the following composition ratios to prepare IgG2 antibody mixed solutions.

[0137] The results are shown in Figure 7. Figure 7(a) shows the emission spectra of IgG2 antibody mixed solutions of each composition, normalized by the emission intensity at the peak wavelength (normalized luminescence). Figure 7(b) shows the difference (Δλ) between the maximum emission wavelength when reacted with a mixed solution containing denatured human IgG2 antibody and the maximum emission wavelength when reacted with native human IgG2 antibody (native human IgG2:denatured human IgG2 = 100:0). ｍａｘ 8 shows the Δλ in the range of 0 to 20% of the content of denatured human IgG (Spiked non-native IgG2 [%]). ｍａｘ In this range, the coefficient of determination R = 0.96.

[0138] As shown in Figures 7(a), 7(b), and 8, the spectral shift increases with an increase in the content of denatured IgG2 (spiked non-native IgG2 [%]). This demonstrates that the method of the present invention for evaluating the denatured state of an antibody can be used for quality control of various antibody-containing products.

Claims

1. A method for evaluating the denatured state of an antibody, comprising the steps of: contacting a compound represented by the following general formula (1) or a salt or solvate thereof with a test sample containing an antibody; and measuring the maximum emission wavelength upon said contact: [In the formula, R １ represents a methyl group or any one of groups represented by the following formula (i-1) or (i-2): (In the formula, R ３ R is a hydrogen atom, a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group. ２ is any one of groups represented by the following formulas (ii-1), (ii-2), (ii-3), (ii-4) and (ii-5).

2. The method for evaluating the denatured state of an antibody according to claim 1, wherein the compound represented by general formula (1) is a compound represented by any one of the following formulas:

3. A method for evaluating the denatured state of an antibody according to claim 1, wherein the test sample is evaluated to contain a denatured antibody if the maximum emission wavelength upon said contact has changed by a predetermined reference value or more compared to the maximum emission wavelength upon contact of the compound represented by general formula (1) or a salt or solvate thereof with a standard sample containing an undenatured antibody.

4. The method for evaluating the denatured state of an antibody according to claim 1, wherein the antibody is IgA, IgE, IgG, or IgM.

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