Glucagon-recognizing peptide and glucagon detection method

Abstract

The purpose is to improve the specificity of a probe for detecting glucagon. A probe for detecting glucagon wherein a peptide moiety (P) comprises: a sequence selected from the group consisting of TPANTTANISCPWYL (SEQ ID NO: 2), LLLALAILGGLSKLH (SEQ ID NO: 3) and IFVRIVQLLVAKLRA (SEQ ID NO: 4), or a deletion sequence thereof is provided to improve specificity of the probe for detecting glucagon.

Description

Glucagon-recognition peptides and glucagon detection methods

[0001] The present invention relates to the technical field of glucagon detection.

[0002] Glucagon is a peptide hormone that acts as an antagonist to insulin, increasing blood glucose levels (blood glucose levels). Glucagon is secreted from alpha cells in the pancreas in response to a drop in blood glucose levels, such as during fasting. Liver cells sense glucagon and release glucose into the blood by breaking down glycogen, resulting in an increase in blood glucose levels. In recent years, the glucagon-centric hypothesis has been proposed, which suggests that glucagon plays a greater role in blood glucose control than insulin. While various studies focusing on insulin have been conducted on blood glucose control in the body, much knowledge has been accumulated, little is known about glucagon. One reason for this is that glucagon has been extremely difficult to detect until now. To elucidate the role of glucagon in the body, a simple, rapid, highly sensitive, and highly specific glucagon detection method is needed.

[0003] One method for detecting glucagon is the enzyme-linked immunosorbent assay (ELISA) using antibodies against glucagon. However, because glucagon is matured by processing from a precursor, there are many analogs with the same amino acid sequence. Therefore, antibody-based detection methods may also detect peptides other than glucagon, resulting in the problem of cross-reactivity.

[0004] Therefore, in recent years, highly specific glucagon detection ELISA kits have been commercially available that simultaneously use multiple antibodies that recognize both the N-terminus and C-terminus of glucagon. However, when these kits sold by various companies are used to detect glucagon in the blood of the same patient, it has been reported that the measurement results vary from kit to kit, and the detection ability is still insufficient (Non-Patent Document 1). Furthermore, antibody-based detection methods require specialized skills, and in order to detect glucagon with as high specificity as possible, the antibody reaction must be carried out at a low temperature for several tens of hours or more (usually about 20 hours), making them unsuitable for high-throughput measurements using a large number of samples.

[0005] Because antibody-based glucagon detection methods have the problems described above, accurate measurement of glucagon in blood generally requires time-consuming analytical methods such as HPLC or LC-MS. However, because glucagon has a short half-life in blood, these analytical methods are not suitable, and a measurement method that enables accurate measurement in a short time is desired.

[0006] The target recognition region of the glucagon receptor, where glucagon interacts with the glucagon receptor, has been identified (Non-Patent Document 3). Focusing on the specific binding of this target recognition region of the glucagon receptor, research has been conducted on glucagon detection probes in which a label is bound to a peptide created based on the glucagon receptor (Patent Document 1: Japanese Patent No. 7418788).

[0007] Patent No. 7418788

[0008] Bak, MJ et al. Specificity and sensitivity of commercially availables for glucagon and oxyntomodulin measurement in humans. Eur. J. Endocrinol. 170, 529-538 (2014).Siu, FY et al. Structure of the human glucagon class B G-protein-coupled receptor.Nature (2013)vol. 499, 444-449

[0009] In the development of a glucagon detection probe that enables accurate measurement of glucagon in a short time, there is a problem of cross-reactivity, and improvement of specificity is desired.

[0010] The present inventors have conducted extensive research to achieve higher specificity in glucagon detection probes and have discovered a new amino acid sequence in the glucagon receptor that has high binding affinity to glucagon but low binding affinity to the glucagon analog oxyntomodulin. Based on this finding, we have obtained a new glucagon-binding peptide that can specifically recognize glucagon. By attaching a label to this new glucagon-binding peptide, we have developed a glucagon detection probe that enables detection via the interaction between glucagon and the glucagon-binding peptide, and have established a glucagon detection method using the glucagon detection probe.

[0011]

[0013] Therefore, the present invention relates to the following: [1] A probe for detecting glucagon, comprising a peptide portion (P) containing a partial peptide of a glucagon receptor, a label portion (T) that enables detection by interaction between glucagon and the peptide portion (P), and a linking portion (L) that links the peptide portion (P) and the label portion (T), the probe having the following formula:

[0014] The glucagon detection probe according to item 1, wherein the peptide portion (P) comprises a sequence selected from the group consisting of the following: TPANTTANISCPWYL (SEQ ID NO: 2) LLLALAILGGLSKLH (SEQ ID NO: 3) IFVRIVQLLVAKLRA (SEQ ID NO: 4) or a deletion sequence thereof, wherein the deletion sequence is a sequence in which 1 to 10 amino acids are deleted from the N-terminus and / or C-terminus of the original sequence, and a peptide of the deletion sequence is a sequence that interacts with glucagon. [2] The glucagon detection probe according to item 1, wherein the peptide portion comprises the sequence of TPANTTANISCPWYL (SEQ ID NO: 2) or a deletion sequence thereof, and the deletion sequence is a sequence in which 1 to 10 amino acids are deleted from the N-terminus. [3] The glucagon detection probe according to item 1, wherein the peptide portion comprises the sequence LLLALAILGGLS (SEQ ID NO: 3) or a deletion sequence thereof, and the deletion sequence is a sequence in which 1 to 7 amino acids are deleted from the C-terminus. [4] The glucagon detection probe according to item 1, wherein the peptide portion comprises the sequence IFVRIVQLLVA (SEQ ID NO: 4) or a deletion sequence thereof, and the deletion sequence is a sequence in which 1 to 6 amino acids are deleted from the C-terminus. [5] The glucagon detection probe according to any one of items 1 to 4, wherein the peptide portion comprises at least one sequence consisting of 5 to 30 consecutive residues contained in the amino acid sequence of the glucagon receptor represented by SEQ ID NO: 1, linked to the sequence or the deletion sequence thereof. [6] The glucagon detection probe according to item 5, wherein the sequence consisting of 5 to 30 consecutive residues is a sequence adjacent to the sequence or the deletion sequence thereof in the amino acid sequence of the glucagon receptor. [7] The probe according to any one of items 1 to 6, wherein the labeling portion comprises one or more fluorescent labels. [8] The fluorescent labels are any of the following: (In the formula, R each independently represents, but is not limited to, a hydrogen atom; a linear or branched alkyl group having 1 to 15 carbon atoms; a linear or branched ether group having 1 to 10 carbon atoms; a phenyl group; a phenyl group in which a portion of the phenyl group is substituted with an amino group, a halogen, or a nitro group; an amino group; a cyano group; a nitro group; a carboxylic acid or a salt, ester, or amide thereof; a sulfonic acid or a salt, ester, or amide thereof; a thiol group; a hydroxyl group or a salt thereof; a ketone; a halogen; or a sugar; [9] The probe according to Item 7, wherein the fluorescent label is selected from the group consisting of: In the case of A-1, n is an integer of 1 to 9, in the case of A-2, n is an integer of 1 to 9, in the case of A-3, n is an integer of 1 to 5, in the case of A-4, n is an integer of 1 to 7, in the case of A-5, n is an integer of 1 to 6, in the case of A-6, n is an integer of 1 to 4, in the case of A-7, n is an integer of 1 to 7, and in the case of A-8, n is an integer of 1 to 5, and when n is 2 or more, each R may be the same or different; * represents a bond, which is bonded to the linking moiety (L) at the bond. (wherein * represents a bond, and is bonded to the linking moiety (L) at the bond).

[10] The probe according to item 8, wherein the fluorescent label is selected from the group consisting of the following: (wherein R represents, without limitation, independently of each other, a hydrogen atom; a linear or branched alkyl group having 1 to 15 carbon atoms; a linear or branched ether having 1 to 10 carbon atoms; a phenyl group; a phenyl group in which a portion of the phenyl group is substituted with an amino group, a halogen, or a nitro group; an amino group; a cyano group; a nitro group; a carboxylic acid or a salt thereof, an ester, or an amide; a sulfonic acid or a salt thereof, an ester, or an amide; a thiol group; a hydroxyl group or a salt thereof; a ketone; a halogen; or a sugar. n is an integer of 1 to 2, and each R may be the same or different. * represents a bond to the peptide.) The probe according to item 8,

[12] A method for measuring the glucagon concentration in a sample, comprising the steps of: contacting the sample with the probe according to item 7; irradiating the sample with excitation light and measuring fluorescence from the excited fluorescent label; and determining the glucagon concentration based on the fluorescence at a wavelength changed by the interaction between glucagon and the peptide portion (P) of the probe or a change in fluorescence intensity at the same wavelength.

[13] The method according to item 12, wherein the sample is a blood sample.

[14] The method according to item 12, wherein the glucagon concentration is determined based on a calibration curve showing the relationship between the fluorescence at the changed wavelength or the change in fluorescence intensity at the same wavelength and the glucagon concentration.

[15] A kit for determining glucagon concentration, comprising the probe according to any one of items 1 to 6.

[0012] In preparing a glucagon detection probe, specificity for glucagon is improved by using a glucagon-binding peptide that has high binding affinity to glucagon but low binding affinity to oxyntomodulin.

[0013] Figure 1 shows the positions of amino acid substitutions in the glucagon receptor that reduce the binding affinity of glucagon to the glucagon receptor, as disclosed in Non-Patent Document 3. Figure 2 shows the results of peptide screening based on binding to glucagon and oxyntomodulin in a peptide array. Peptides with high binding affinity to glucagon but low binding affinity to oxyntomodulin were screened. Figure 3 shows the results of glucagon detection using glucagon detection probes containing glucagon-binding peptides No. 1 to 3. It was shown that the fluorescence at 600 nm increased in the presence of glucagon (100 nM). Figure 4 shows calibration curves generated using each glucagon detection probe containing glucagon-binding peptides No. 1 to 3. Figure 5 shows the cross-reactivity of each glucagon detection probe containing glucagon-binding peptides No. 1 to 3 with glucagon, miniglucagon, oxyntomodulin, and glicentin. Figure 6 shows the cross-reactivity of each glucagon detection probe containing glucagon-binding peptides No. 1 to 3 with glucagon, miniglucagon, oxyntomodulin, and glicentin. The binding to glucagon of each glucagon detection probe containing peptides of the deletion sequences 1 to 3 is shown.

[0014] The glucagon detection probe of the present invention comprises a peptide portion (P) containing a partial peptide of the glucagon receptor, a label portion (T), and a linking portion (L). The label portion (T) and the peptide portion (P) are bound via the linking portion (L). An example of the glucagon detection probe is a compound represented by the following formula [I]: The labeling moiety (T) is a labeling moiety that enables detection by interaction between glucagon and the peptide moiety. When glucagon interacts with the peptide moiety, a change occurs in the labeling moiety, making it possible to detect it.

[0015] The glucagon receptor is a seven-transmembrane receptor, with approximately 130 residues at the N-terminus exposed to the outside of the cell. Of the seven transmembrane regions, three extracellular loops (ECLs) (approximately positions 200-224, approximately positions 284-302, and positions 370-380) are exposed to the outside of the cell, and three intracellular loops exist within the cytoplasm. The glucagon receptor modified by single amino acid substitution has been examined for its binding to glucagon, and regions capable of interacting with glucagon have been identified as shown in Figure 1 (Non-Patent Document 3). Meanwhile, the peptides identified in the present invention that interact with glucagon correspond to positions 71-85, 156-170, and 321-335 of SEQ ID NO: 1, and these peptides relate to regions not recognized in Non-Patent Document 2 as having glucagon-binding properties. In particular, positions 156-170 and 321-335 correspond to intracellular loop regions and are not expected to be involved in binding to extracellular glucagon. Peptides identified by the present invention that have the ability to bind to or interact with glucagon are referred to as "glucagon-binding peptides" in the present invention. The glucagon-binding peptides of the present invention consist of a sequence selected from the group consisting of the following: TPANTTANISCPWYL (SEQ ID NO: 2), LLLALAILGGLSKLH (SEQ ID NO: 3), and IFVRIVQLLVAKLRA (SEQ ID NO: 4), or a deletion sequence thereof. The binding or interaction with glucagon is preferably specific to glucagon, but may have low binding or interaction, i.e., nonspecific binding or interaction, with other proteins, such as miniglucagon, glicentin, and oxyntomodulin. The specificity for glucagon is preferably higher than that of the prior art.

[0016] The peptide portion (P) containing a partial peptide of the glucagon receptor comprises the glucagon-binding peptide of the present invention. As a result, the peptide portion (P) is a peptide capable of interacting with glucagon. Therefore, the peptide portion (P) comprises a glucagon-binding peptide consisting of a sequence selected from the group consisting of: TPANTTANISCPWYL (SEQ ID NO: 2), LLLALAILGGLSKLH (SEQ ID NO: 3), and IFVRIVQLLVAKLRA (SEQ ID NO: 4), or a deletion sequence thereof. The length of the peptide portion (P) is not particularly limited, but is preferably 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 18 or more, or 20 or more residues from the viewpoint of ensuring interaction with glucagon. From the viewpoint of ease of peptide synthesis, the length is preferably 50 or less, preferably 40 or less, more preferably 35 or less, and even more preferably 30 or less. Of the sequences of the peptide portion (P), sequences other than the glucagon-binding peptide sequence are preferably partial sequences contained in the glucagon receptor. Here, the partial sequence contained in the glucagon receptor may be any sequence contained in the amino acid sequence of the glucagon receptor represented by SEQ ID NO: 1, and may be, for example, at least one sequence consisting of 5 to 30 consecutive residues. Such a partial sequence may be directly linked to the sequence of the glucagon-binding peptide or a deletion sequence thereof, or may be indirectly linked via any sequence.

[0017] In an even more preferred embodiment, the peptide portion (P) may consist of a glucagon-binding peptide consisting of a sequence selected from the group consisting of: TPANTTANISCPWYL (SEQ ID NO: 2), LLLALAILGGLSKLH (SEQ ID NO: 3), and IFVRIVQLLVAKLRA (SEQ ID NO: 4), or a deletion sequence thereof.

[0018] The deleted sequence is a sequence in which 1 to 10 amino acids are deleted from the N-terminus and / or C-terminus of the original sequence, and a peptide consisting of the deleted sequence interacts with glucagon.

[0019] An example of a deleted sequence of TPANTTANISCPWYL (SEQ ID NO: 2) is a sequence in which 1 to 4 amino acids have been deleted from the C-terminus and / or a sequence in which 1 to 10 amino acids have been deleted from the N-terminus. For example, the deleted sequence of SEQ ID NO: 2 may be any of SEQ ID NOs: 5 to 24. From the viewpoint of maintaining the interaction with glucagon, the deleted sequence may be a sequence in which 1 or 2, particularly 1, amino acids have been deleted from the C-terminus ( FIG. 6A ). Examples of deleted sequences that have a particularly high interaction with glucagon include the following: CPWYL (SEQ ID NO: 24), SCPWYL (SEQ ID NO: 23), and ISCPWYL (SEQ ID NO: 22).

[0020] An example of a deleted sequence of LLLALAILGGLSKLH (SEQ ID NO: 3) is a sequence in which 1 to 10 amino acids have been deleted from the C-terminus. For example, the deleted sequence of SEQ ID NO: 3 may be any of the sequences of SEQ ID NOs: 25 to 44. From the viewpoint of maintaining the interaction with glucagon, a sequence in which 3 to 10 amino acids have been deleted from the C-terminus is preferred ( FIG. 6B ). That is, the deleted sequence is the sequence of LLLALAILGGLS (SEQ ID NO: 29) or a sequence in which 1 to 7 amino acids have been deleted from the C-terminus. Examples of deleted sequences that have a particularly high interaction with glucagon include the following: LLLALAIL (SEQ ID NO: 31), LLLALAI (SEQ ID NO: 32), LLLALA (SEQ ID NO: 33), and LLLAL (SEQ ID NO: 34).

[0021] An example of a deleted sequence of IFVRIVQLLVAKLRA (SEQ ID NO: 4) is a sequence in which 1 to 10 amino acids have been deleted from the C-terminus. For example, the deleted sequence of SEQ ID NO: 4 may be any of SEQ ID NOs: 45 to 64. From the viewpoint of maintaining the interaction with glucagon, a sequence in which 4 to 10 amino acids have been deleted from the C-terminus is preferred ( FIG. 6C ). That is, the deleted sequence is the sequence of IFVRIVQLLVA (SEQ ID NO: 51) or a sequence in which 1 to 6 amino acids have been deleted from the C-terminus. Examples of deleted sequences that have a particularly high interaction with glucagon include the following: IFVRIVQLLVA (SEQ ID NO: 48) IFVRIVQLLV (SEQ ID NO: 49) IFVRIVQLL (SEQ ID NO: 50) IFVRIVQL (SEQ ID NO: 51) IFVRIVQ (SEQ ID NO: 52) IFVRIV (SEQ ID NO: 53) IFVRI (SEQ ID NO: 54).

[0022] The labeling moiety (T) may be a fluorescent label. The interaction between glucagon and the peptide moiety (P) containing a partial peptide of the glucagon receptor changes the fluorescence wavelength of the fluorescent label. Glucagon can be detected by detecting this change in fluorescence wavelength, and the concentration of glucagon can be determined by measuring the intensity of the changed fluorescence wavelength. Examples of such fluorescent labels include polycyclic aromatic compounds or heterocyclic compounds such as naphthalene, anthracene, pyrene, biphenyl, coumarin, benzothiazole, fluorescein, rhodamine, bipyridine, quinoline, phenanthroline, and cyanopyranyl. More specifically, the fluorescent label is a compound represented by the following chemical formula: (In the formula, R each independently represents, but is not limited to, a hydrogen atom; a linear or branched alkyl group having 1 to 15 carbon atoms; a linear or branched ether group having 1 to 10 carbon atoms; a phenyl group; a phenyl group in which a portion of the phenyl group is substituted with an amino group, a halogen, or a nitro group; an amino group; a cyano group; a nitro group; a carboxylic acid or a salt, ester, or amide thereof; a sulfonic acid or a salt, ester, or amide thereof; a thiol group; a hydroxyl group or a salt thereof; a ketone; a halogen; or a sugar (glucose, mannose, melibiose, galactose, fructose, etc.); In the case of A-1, n is an integer of 1 to 9, in the case of A-2, n is an integer of 1 to 9, in the case of A-3, n is an integer of 1 to 5, in the case of A-4, n is an integer of 1 to 7, in the case of A-5, n is an integer of 1 to 6, in the case of A-6, n is an integer of 1 to 4, in the case of A-7, n is an integer of 1 to 7, in the case of A-8, n is an integer of 1 to 5, and in the case of A-9, n is an integer of 1 to 2, and when n is 2 or more, each R may be the same or different; * indicates a bond.) Here, the bond can be bonded to the linking moiety (L).

[0023] More specifically, the fluorescent label is a compound represented by the following chemical formula: (wherein * denotes a bond) Here, the bond can be bonded to the linking moiety (L). Particularly preferably, the fluorescent label used is a compound represented by formula a-9.

[0024] The linking moiety (L) linking the labeling moiety (T) and the peptide moiety (P) can be any linking moiety as long as it allows the labeling moiety (T) and the peptide moiety (P) to be linked by a covalent bond. Any linker can be used as the linking moiety as long as the label of the labeling moiety changes upon interaction between the peptide moiety and glucagon. From the viewpoint of easily linking the labeling moiety (T) to the synthesized peptide moiety (P), the linking moiety may be linked via any reactive functional group of the peptide moiety as long as it does not interfere with the interaction between the peptide moiety and glucagon. Examples of reactive functional groups include terminal amino and carboxy groups, as well as amino groups, carboxy groups, hydroxyl groups, sulfhydryl groups, and the like within the chain. The linking moiety generated by reaction with the amino group of the peptide moiety preferably contains, for example, a carbonyl group or a sulfonyl group, and can be represented by the following formula: (In the formula, R 2 is absent or -(CH2) m -(m is an integer of 1 to 5). The linking moiety formed by reaction with the carboxy group of the peptide moiety can be bonded via an amide bond, for example: (In the formula, R 2 is absent or -(CH2) m - (m is an integer of 1 to 5). When binding to a sulfhydryl group of the peptide moiety, the linkage can be achieved by a linking group containing maleimide or the like. From the viewpoint of facilitating the binding of the labeling moiety to the target, fluorescent dyes containing a linking moiety (L) are commercially available as the labeling moiety (T), and such fluorescent dyes may also be used. As an example, sulforhodamine 101, a fluorescent dye, is represented by the following formula: The compound has the formula (I) and can be bound to a target substance via a sulfonic acid group. Here, a binding group such as a sulfonic acid group corresponds to the linking moiety referred to in the present invention. The labeling moiety (T) and the linking moiety (L) may be pre-bound to form a salt, and a probe for detecting glucagon may be formed by reacting the salt with a synthesized peptide. As used in the examples, the acid chloride salt of sulforhodamine 101 can react with the amino group of a peptide to bind to the peptide.

[0025] The linking moiety is bound to the N-terminus, C-terminus, or side chain of the peptide moiety (P). From the viewpoint of not interfering with the interaction between the peptide moiety and glucagon, it is preferably bound to the N-terminus or C-terminus. Furthermore, from the viewpoint of ease of production, the linking moiety is more preferably bound to the N-terminus. When the linking moiety (L) is bound to the N-terminus, the linking moiety forms an amide bond or a sulfonamide bond.

[0026] In one embodiment, the labeling portion (T) is a compound represented by formula (a-9), and the linking portion (L) is a sulfonyl group. Specifically, the compound represented by the following formula: (wherein * represents a bond, which is bonded to the linking portion (L)).

[0027] In addition to the label of the labeling moiety, another label may be attached to the peptide moiety. Such labels include fluorescent labels, luminescent labels, and quenching labels, and can be configured so that the label of the labeling moiety changes when the peptide moiety binds to glucagon. For example, when a fluorescent label is attached, the fluorescence may change due to the binding of glucagon to the peptide moiety based on the principle of FRET. When a luminescent label is attached, the fluorescence may change due to the binding of glucagon to the peptide moiety based on the principle of BRET by further adding a luminescent substrate to the solution.

[0028] In another aspect, the present invention relates to a method for measuring glucagon using a glucagon detection probe, the method for measuring glucagon comprising the following steps: contacting a sample with the glucagon detection probe; irradiating the sample with excitation light and measuring fluorescence from the excited fluorescent label; and determining the glucagon concentration based on the fluorescence intensity at a wavelength changed by the interaction between glucagon and the peptide portion (P) of the glucagon detection probe. The determination of the glucagon concentration is based on a calibration curve showing the relationship between fluorescence intensity and concentration.

[0029] The sample used in the glucagon measurement method of the present invention may be any solution containing glucagon. One example is a blood sample. The blood sample is a sample derived from blood, and blood, serum, or plasma may be used directly or after treatment such as precipitation or dilution.

[0030] The glucagon measurement method of the present invention measures fluorescence using a fluorometer. The wavelengths of the excitation light and the fluorescence emitted upon excitation can be determined depending on the type of label in the glucagon detection probe. For example, when a fluorescent label containing a compound represented by formula (a-9) is used, excitation light with a wavelength of 550 to 580 nm (e.g., 568 nm) can be used, emitting fluorescence at 600 nm. However, the fluorescence wavelength modulated by the interaction between the peptide moiety and glucagon can be measured at 580 to 700 nm. The transition in fluorescence wavelength is thought to be due to an increase in fluorescence intensity at shorter wavelengths caused by the interaction between the peptide moiety and glucagon, which changes the environment around the probe from a hydrophilic to a hydrophobic environment. Because the modulated fluorescence wavelength can vary depending on the type of peptide moiety, the peak fluorescence wavelength must be determined for each probe. The glucagon concentration can be determined based on the intensity ratio between the original fluorescence wavelength and the modulated fluorescence wavelength. Determining the glucagon concentration using the intensity ratio allows for more stable glucagon measurement, in principle, independent of the fluorescence intensity of the probe.

[0031] The glucagon measurement method using the glucagon detection probe of the present invention enables highly specific measurement of glucagon. As shown in Figure 5, the glucagon detection probes prepared in the Examples, each containing glucagon-binding peptide Nos. 1 to 3, were reactive only with glucagon, but not with miniglucagon, glicentin, or oxyntomodulin (Figure 5). Conventional glucagon measurement methods, such as the ELISA method (Figure 5), require 20 to 48 hours for the reaction between the measurement sample and the antibody solution. However, the glucagon detection probe of the present invention allows measurement within 30 minutes, preferably within 15 minutes, and particularly preferably within 5 to 10 minutes. Furthermore, the ELISA method has a high cross-reactivity, resulting in detection of not only oxyntomodulin but also miniglucagon, resulting in a problem of specificity.

[0032] In yet another aspect, the present invention relates to a glucagon measurement kit comprising a glucagon detection probe. The glucagon measurement kit may further comprise a product manual, a calibration curve based on fluorescence intensity, and the like. Since the glucagon detection probe of the present invention binds to free glucagon, it can also be used for bioimaging. The present invention may also relate to a glucagon detection kit used for bioimaging.

[0033] All documents mentioned in this specification are incorporated herein by reference in their entirety. The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications of the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, can be made without departing from the spirit of the present invention.

[0034] Example 1: Preparation of Peptide Arrays Peptide arrays were prepared using a peptide synthesizer (MultiPep Rsi, Intavis AG, Koln, Germany). Peptides containing 15 amino acids were attached to a cellulose membrane (grade 542; Whatman, Maidstone, UK) activated with β-alanine, and amino acid coupling was performed using the following procedure, so that 10 amino acids overlapped from the N-terminus of the full-length receptor. Amino acids modified with Fmoc protecting groups were deprotected using 20% ​​piperidine, and the peptides were activated with 1.1 M hydroxybenzotriazole and 1.1 M N,N-diisopropylcarbodiimide, followed by coupling. After the elongation reaction was completed, the peptides were capped with 4% acetic anhydride and washed with N,N-dimethylformamide (DMF) and ethanol. Finally, the protecting groups of the synthesized peptide array were deprotected using DMF containing 20% ​​piperidine, Milli-Q water, triisopropylsilane, and trifluoroacetic acid (2:3:95), and the array was washed sequentially with DMF, ethanol, and PBS before being used for subsequent experiments.

[0035] Example 2: Screening of Glucagon-Binding Peptides We screened for peptides that bind to glucagon but have low binding affinity to oxyntomodulin, a glucagon analog. The prepared peptide arrays were blocked with 1% BSA, and glucagon modified with Cy3 at the N-terminus and oxyntomodulin modified with Cy3 at the N-terminus were added to separate peptide arrays and incubated at room temperature for 1 hour. After washing with TBS, fluorescent images were captured using a 3D-Gene Scanner (TORAY, Tokyo, Japan). The fluorescence intensity of each spot on the peptide array was quantified using ImageJ. Based on the fluorescence intensity, peptides with glucagon-binding ability but low binding affinity to oxyntomodulin were selected. The results are shown in Figure 2. The screened glucagon-binding peptides were designated glucagon-binding peptides No. 1, 2, and 3, respectively.

[0036] Example 3: Modification of glucagon-binding peptides with fluorescent dyes. Glucagon-binding peptides screened by peptide array were modified with fluorescent dyes. Sulforhodamine 101 acid chloride (Dojindo Laboratories) was used as the fluorescent dye to modify glucagon. Glucagon-binding peptides with the amino acid sequences shown in Table 1 below were synthesized (Peptide Institute, Inc.). 2 mg of each synthesized peptide was dissolved in 50 μL of water or dimethyl sulfoxide (DMSO) and mixed with 2 μL of triethylamine. 25 mg of sulforhodamine 101 acid chloride dried under reduced pressure was dissolved in 200 μL of DMSO. 20 μL of sulforhodamine 101 acid chloride solution was added to the peptide solution, stirred at room temperature for 2 hours, and then allowed to stand overnight at 4°C. The next day, the peptide solution was distilled under reduced pressure, 1 mL of toluene was added, and the mixture was distilled under reduced pressure again. 1 mL of toluene was added again, distilled under reduced pressure, and then 1 mL of dichloromethane was added to remove excess fluorescent dye for purification. After thorough drying by distillation under reduced pressure, each peptide solution modified with a fluorescent dye was dissolved in water or DMSO to prepare a probe solution containing the glucagon-binding peptide.

[0037] Example 4: Analysis using glucagon detection probe Glucagon (Peptide Institute) was dissolved in 0.01% aqueous acetic acid and then diluted with physiological saline (PBS) to obtain a glucagon solution with a concentration of 100 μM. The 100 nM probe solution synthesized in Example 3 was mixed with a glucagon solution at 25°C and measured using a fluorescence spectrometer (Hitachi High-Tech Science, excitation wavelength 568 nm). As a control, a glucagon-free solution was mixed with the probe solution and measured. The results for the probes containing glucagon-binding peptides No. 1 to 3 are shown in Figure 3 (A: Glucagon-binding peptide No. 1, B: Glucagon-binding peptide No. 2, C: Glucagon-binding peptide No. 3).

[0038] Next, to confirm the concentration-dependent change in fluorescence, glucagon was dissolved in 0.01% aqueous acetic acid and diluted with physiological saline (PBS) to 0.0005 nM, 0.005 nM, 0.05 nM, 0.5 nM, 5 nM, and 10 nM concentrations to obtain glucagon solutions. The 100 nM probe solution synthesized in Example 3 was mixed with the glucagon solution at 25°C, and the fluorescence spectrum was measured using a fluorescence spectrometer (Hitachi High-Tech Science Corporation, excitation wavelength 568 nm). A concentration-dependent increase in fluorescence intensity at 600 nm was observed. Based on the obtained fluorescence spectra, the ratio of the fluorescence intensity at 600 nm in the absence of glucagon to the fluorescence intensity at each glucagon concentration was calculated, and calibration curves were prepared for the glucagon detection probes containing glucagon-binding peptides No. 1, 2, and 3. The results are shown in Figures 4A to 4C (A: glucagon-binding peptide No. 1, B: glucagon-binding peptide No. 2, C: glucagon-binding peptide No. 3).

[0039] Example 5: Analysis of Cross-Reactivity (Specificity) with Glucagon Analogs To analyze the reactivity of the probes (Nos. 1-3) containing the glucagon-binding peptides of the present invention with glucagon analogs, miniglucagon, oxyntomodulin, and glicentin having the sequences shown in Table 2 were dissolved in PBS under the same conditions as above, instead of glucagon, to prepare 10 nM solutions of glucagon analogs. Furthermore, a probe solution containing 100 nM of each glucagon-binding peptide was mixed with a glucagon solution or a glucagon analog solution at 25°C, and the fluorescence spectrum immediately after mixing was measured using a fluorescence spectrometer (Hitachi High-Tech Science, excitation wavelength 568 nm). Each probe had high specificity for glucagon analogs other than glucagon (Figure 5). Table 3 shows a comparison of glucagon specificity with existing methods.

[0040] Example 6: Evaluation of glucagon detection probes composed of deletion sequences of glucagon-binding peptides. The glucagon-binding activity of glucagon detection probes prepared from peptides composed of deletion sequences of glucagon-binding peptides was evaluated. Peptides composed of deletion sequences of glucagon-binding peptides with the amino acid sequences shown in Tables 4 to 6 below were synthesized (Peptide Institute, Inc.). 2 mg of each synthesized peptide was dissolved in 50 μL of water or dimethyl sulfoxide (DMSO) and mixed with 2 μL of triethylamine. 25 mg of sulforhodamine 101 acid chloride dried under reduced pressure was dissolved in 200 μL of DMSO. 20 μL of sulforhodamine 101 acid chloride solution was added to the peptide solution, stirred at room temperature for 2 hours, and then allowed to stand overnight at 4°C. The next day, the peptide solution was distilled under reduced pressure, 1 mL of toluene was added, and the mixture was distilled under reduced pressure again. 1 mL of toluene was added again, and the mixture was distilled under reduced pressure. Purification was then carried out by adding 1 mL of dichloromethane and removing excess fluorescent dye. After thorough drying by vacuum distillation, each fluorescent dye-modified peptide solution was dissolved in water or DMSO to prepare a probe solution containing glucagon-binding peptide. Glucagon (Peptide Institute) was dissolved in 0.01% aqueous acetic acid and then diluted with physiological saline (PBS) to obtain a glucagon solution with a concentration of 100 μM. The 100 nM probe solution and glucagon solution were mixed at 25°C and measured using a fluorescence spectrometer (Hitachi High-Tech Science, excitation wavelength 568 nm). The results are shown in Figure 6.

Claims

1. A probe for detecting glucagon, comprising a peptide portion (P) containing a partial peptide of a glucagon receptor, a label portion (T) that enables detection by interaction between glucagon and the peptide portion (P), and a linking portion (L) that links the peptide portion (P) and the label portion (T), the probe having the following formula: wherein the peptide portion (P) comprises a sequence selected from the group consisting of the following: TPANTTANISCPWYL (SEQ ID NO: 2) LLLALAILGGLSKLH (SEQ ID NO: 3) IFVRIVQLLVAKLRA (SEQ ID NO: 4), or a deletion sequence thereof, wherein the deletion sequence is a deletion sequence in which 1 to 10 amino acids are deleted from the N-terminus and / or C-terminus of the original sequence, and a peptide of the deletion sequence is a sequence that interacts with glucagon.

2. The glucagon detection probe according to claim 1, wherein the peptide portion comprises the sequence TPANTTANISCPWYL (SEQ ID NO: 2) or a deletion sequence thereof, and the deletion sequence is a sequence in which 1 to 10 amino acids are deleted from the N-terminus.

3. A glucagon detection probe according to claim 1, wherein the peptide portion comprises the sequence LLLALAILGGLS (SEQ ID NO: 3) or a deletion sequence thereof, and the deletion sequence is a sequence in which 1 to 7 amino acids are deleted from the C-terminus.

4. The glucagon detection probe according to claim 1, wherein the peptide portion comprises the sequence IFVRIVQLLVA (SEQ ID NO: 4) or a deletion sequence thereof, and the deletion sequence is a sequence in which 1 to 6 amino acids are deleted from the C-terminus.

5. A glucagon detection probe according to claim 1, wherein the peptide portion comprises at least one sequence consisting of 5 to 30 consecutive residues contained in the amino acid sequence of the glucagon receptor represented by SEQ ID NO: 1, linked to the sequence or a deletion sequence thereof.

6. The glucagon detection probe according to claim 5, wherein the sequence consisting of 5 to 30 consecutive residues is a sequence adjacent to the sequence or its deletion sequence in the amino acid sequence of the glucagon receptor.

7. The probe according to any one of claims 1 to 6, wherein the labeling moiety comprises one or more fluorescent labels.

8. The fluorescent label is one of the following: (In the formula, R each independently represents, but is not limited to, a hydrogen atom; a linear or branched alkyl group having 1 to 15 carbon atoms; a linear or branched ether group having 1 to 10 carbon atoms; a phenyl group; a phenyl group in which a portion of the phenyl group is substituted with an amino group, a halogen, or a nitro group; an amino group; a cyano group; a nitro group; a carboxylic acid or a salt, ester, or amide thereof; a sulfonic acid or a salt, ester, or amide thereof; a thiol group; a hydroxyl group or a salt thereof; a ketone; a halogen; or a sugar; The probe according to claim 7, wherein n is an integer of 1 to 9 in the case of A-1, n is an integer of 1 to 9 in the case of A-2, n is an integer of 1 to 9 in the case of A-3, n is an integer of 1 to 5 in the case of A-4, n is an integer of 1 to 7 in the case of A-5, n is an integer of 1 to 6 in the case of A-6, n is an integer of 1 to 4 in the case of A-7, n is an integer of 1 to 7, and in the case of A-8, n is an integer of 1 to 5, and when n is 2 or more, each R may be the same or different; * represents a bond, which bonds to the linking moiety (L).

9. The fluorescent label is one of the following: The probe according to claim 8, wherein the probe is selected from the group consisting of: (wherein * represents a bond, which is bonded to the linking moiety (L) at the bond).

10. The fluorescent label is one of the following: (wherein R represents, without limitation, and independently of one another, a hydrogen atom; a linear or branched alkyl group having 1 to 15 carbon atoms; a linear or branched ether having 1 to 10 carbon atoms; a phenyl group; a phenyl group in which a portion of the phenyl group is substituted with an amino group, a halogen, or a nitro group; an amino group; a cyano group; a nitro group; a carboxylic acid or a salt, ester, or amide thereof; a sulfonic acid or a salt, ester, or amide thereof; a thiol group; a hydroxyl group or a salt thereof; a ketone; a halogen; or a sugar. n is an integer of 1 to 2, and each R may be the same or different. * indicates a bond to the peptide.) 11. The probe of claim 1, wherein the linking moiety (L) comprises a carbonyl group or a sulfonyl group.

12. A method for measuring the concentration of glucagon in a sample, comprising the steps of: contacting the sample with the probe described in claim 7; irradiating the sample with excitation light and measuring the fluorescence from the excited fluorescent label; and determining the concentration of glucagon based on the fluorescence at a wavelength changed by the interaction between glucagon and the peptide portion (P) of the probe or on a change in fluorescence intensity at the same wavelength.

13. The method of claim 12, wherein the sample is a blood sample.

14. The method of claim 12, wherein the glucagon concentration is determined based on a calibration curve showing the relationship between the fluorescence at the changed wavelength or the change in fluorescence intensity at the same wavelength and the glucagon concentration.

15. A kit for determining glucagon concentrations, comprising a probe according to any one of claims 1 to 6.

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