Reagent for L-biotin measurement, method for measuring L-biotin-containing samples, method for determining the number of labels on an L-biotin-labeled substance, and method for producing a solid phase immobilized with optically isomerized avidins.

Abstract

To provide means that enables measurement of L-biotin in a sample.SOLUTION: The above challenge is cleared by an L-biotin measurement reagent containing optically isomeric avidins and an azobenzene derivative represented by a formula (I) below. In the formula, each of R1 through R10 independently represents a hydrogen atom or a group selected from a group consisting of a hydroxy group, carboxy group, C1-C6 dialkylamino group having a non-substituent or substituent group, C1-C6 alkyl group having a non-substituent or substituent group, and C1-C6 alkoxy group having a non-substituent or substituent group, where at least one of R1 through R5 is a hydroxy group or C1-C6 dialkylamino group having a non-substituent or substituent group, and at least one of R6 through R10 is a carboxy group.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This invention relates to a reagent for measuring L-biotin. This invention relates to a method for measuring a sample containing L-biotin. This invention relates to a method for determining the number of labels in an L-biotin-labeled substance. This invention relates to a method for producing a solid phase on which optically isomerized avidins are immobilized. [Background technology]

[0002] Avidin and streptavidin have very high affinity for D-biotin and are therefore commonly used in immunological measurements. For example, when measuring a target substance in a biological sample using a D-biotin-labeled antibody and a solid phase immobilized with avidin, the target substance captured by the antibody is immobilized on the solid phase via the binding of D-biotin to avidin. By detecting this target substance on the solid phase, for example using chemiluminescence, the target substance can be measured with high sensitivity.

[0003] In immunological measurements using D-biotin-labeled antibodies, the number of D-biotin molecules bound to a single antibody molecule affects the measurement result. Therefore, it is necessary to quantify the D-biotin bound to the antibody. For example, Non-Patent Literature 1 describes a biotin quantification kit capable of measuring D-biotin bound to proteins such as antibodies, as well as free D-biotin. This kit includes a reagent containing a mixture of 4'-hydroxyazobenzene-2-carboxylic acid (hereinafter also referred to as "HABA") and avidin. This kit utilizes the fact that HABA and avidin bind to form a complex having a light absorption at a central wavelength of 500 nm, and that HABA in this complex readily substitutes for D-biotin, reducing the absorption at 500 nm. Since the degree of absorption reduction depends on the biotin concentration in the sample, the biotin measurement method using this kit measures the biotin concentration based on the absorbance at 500 nm. Such a biotin measurement method is also called the HABA method. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Instructions for use with the Pierce® Biotin Quantitation Kit (Product No.: 28005), Thermo Fisher Scientific. [Overview of the project] [Problems that the invention aims to solve]

[0005] In biological samples such as blood, free D-biotin may be present in high concentrations due to factors such as the ingestion of biotin-containing supplements. Free D-biotin in biological samples may compete with D-biotin-labeled antibodies and affect the measurement results. To solve this problem, the present inventors have developed an immunoassay method using a capture molecule labeled with L-biotin, an optical isomer of D-biotin, and a solid phase immobilized with optically isomerized avidins that do not substantially bind to D-biotin but do bind to L-biotin. However, even in this method, it is necessary to control the number of L-biotin molecules bound to each capture molecule. However, no methods or reagents are known that can measure L-biotin bound to proteins or free L-biotin. The reagent kit described in Non-Patent Literature 1 can measure D-biotin, but not L-biotin. Therefore, the present invention aims to provide a means that enables the measurement of L-biotin. [Means for solving the problem]

[0006] The inventors of the present invention have discovered that L-biotin in a sample can be measured in the same manner as the HABA method using a reagent containing optically isomerized avidins and HABA, and have completed the present invention. Accordingly, the present invention provides a reagent for measuring L-biotin, comprising optically isomerized avidins and an azobenzene derivative represented by the following formula (I).

[0007] [ka] (In the formula, R1~R 10Each of these groups is independently selected from the group consisting of a hydrogen atom, a hydroxyl group, a carboxyl group, an unsubstituted or substituted C1-C6 dialkylamino group, an unsubstituted or substituted C1-C6 alkyl group, and an unsubstituted or substituted C1-C6 alkoxy group. However, at least one of R1 to R5 is a hydroxyl group, or an unsubstituted or substituted C1 to C6 dialkylamino group, and R6 to R 10 (At least one of them is a carboxyl group.)

[0008] The present invention provides a method for measuring L-biotin-containing samples, comprising the steps of preparing a measurement sample by mixing a sample containing L-biotin with the above-mentioned reagent for measuring L-biotin, and measuring the absorbance of the measurement sample, wherein the measured absorbance serves as an indicator of the concentration of L-biotin in the sample.

[0009] The present invention provides a method for determining the number of labels on an L-biotin-labeled substance, comprising the steps of: preparing a sample by mixing a sample containing an L-biotin-labeled substance with the above-mentioned reagent for measuring L-biotin; measuring the absorbance of the sample; determining the concentration of L-biotin in the sample based on the measured absorbance; and determining the number of L-biotin groups per molecule of the L-biotin-labeled substance based on the concentration of L-biotin and the concentration of the substance in the sample.

[0010] The present invention provides a method for producing a solid phase immobilized with optical isomers of avidin, comprising the steps of preparing a measurement sample by mixing a sample separated from a liquid containing an L-biotin-labeled polypeptide with the above-mentioned reagent for measuring L-biotin; measuring the absorbance of the measurement sample; determining the concentration of L-biotin in the measurement sample based on the measured value of the absorbance; determining the number of L-biotin groups per molecule of the L-biotin-labeled polypeptide in the sample based on the concentration of L-biotin and the concentration of the polypeptide; when the number of L-biotin groups is within a predetermined range, contacting the liquid with a solid phase capable of binding to the polypeptide to immobilize the L-biotin-labeled polypeptide on the solid phase; and contacting the solid phase immobilized with the L-biotin-labeled polypeptide with optical isomers of avidin to immobilize the optical isomers of avidin on the solid phase.

Advantages of the Invention

[0011] According to the present invention, a means for measuring L-biotin in a sample is provided.

Brief Description of the Drawings

[0012] [Figure 1A] It is a diagram showing an example of the reagent of the present embodiment in the form of a single reagent. [Figure 1B] It is a diagram showing an example of the reagent of the present embodiment in the form of a two-reagent. [Figure 2] It is a diagram showing the principle of L-biotin measurement using the reagent of the present embodiment. [Figure 3] It is a diagram showing an example of a solid phase immobilized with optical isomers of avidin. [Figure 4A] It is an example of a calibration curve created using the D-biotin measurement reagent 1 of Example 1. [Figure 4B] It is an example of a calibration curve created using the D-biotin measurement reagent 2 of Example 1. [Figure 4C] It is an example of a calibration curve created using the L-biotin measurement reagent of Example 1. [Figure 4D] It is an example of a calibration curve created using the L-biotin measurement reagent of Example 1. [Figure 5] This graph shows the correlation between the measurement results using the reagent kit in Example 2 and the measurement results using a commercially available reagent kit. [Modes for carrying out the invention]

[0013] The reagent for measuring L-biotin in this embodiment (hereinafter also referred to as "the reagent of this embodiment") contains optically isomerized avidins and an azobenzene derivative represented by the above formula (I). The reagent of this embodiment enables the measurement of L-biotin using a principle similar to that of the HABA method. Furthermore, the optically isomerized avidins contained in the reagent of this embodiment bind to L-biotin but substantially do not bind to D-biotin. Therefore, even if D-biotin is mixed into the sample, the impact on the measurement results of L-biotin is minimized.

[0014] In this specification, "avidins" includes avidins and their analogues. Avidins and their analogues are polypeptides that have a high affinity for biotins. In this specification, "polypeptide" includes proteins and their fragments. Avidins may be deglycosylated polypeptides. Examples of deglycosylated avidins include neutraavidin. Neutraavidin is a deglycosylated avidin.

[0015] In this specification, "biotins" include biotin, its analogues, and their optical isomers. Examples of biotin analogues include desthiobiotin and biocitin. Each biotin and its analogues has optical isomers. For example, biotin theoretically has eight optical isomers. In this specification, "L-biotin" includes free 3aR,4R,6aS-L-biotin and 3aR,4R,6aS-L-biotin groups attached to any substance. Also, "D-biotin" includes free 3aS,4S,6aR-D-biotin and 3aS,4S,6aR-D-biotin groups attached to any substance. "Biotin group" refers to the heterocyclic portion of the biotin chemical structure that includes at least an imidazolidined ring.

[0016] Examples of avidin analogs include streptavidin, avidin-like protein derived from Pleurotus ostreatus, bladavidin, and rizavidin. In this specification, avidin analogs include chimeric and modified avidins. A chimeric avidin is a modified polypeptide formed by fusing all or part of the polypeptides constituting each of several types of avidins. A modified avidin is a modified polypeptide represented by an amino acid sequence in which at least one amino acid residue is substituted, deleted, or added to the amino acid sequence of a predetermined avidin. The amino acid sequence of a modified avidin is a polypeptide having 90% or more, preferably 95%, and more preferably 99% sequence identity with the amino acid sequence of the original avidin.

[0017] In this specification, avidin analogs include optically isomerized avidins. Generally, naturally occurring avidins are L-type avidins. In this specification, "L-type avidins" refers to polypeptides among avidins in which all amino acid residues other than glycine are L-amino acid residues, which bind to D-biotin but do not substantially bind to L-biotin. In this specification, "optically isomerized avidins" refers to polypeptides that have some or all of the amino acid sequence of L-type avidins, in which 90% or more of the amino acid residues other than glycine in that amino acid sequence are D-amino acid residues, which bind to L-biotin but do not substantially bind to D-biotin. That is, optically isomerized avidins contain the same amino acid sequence as L-type avidins, but 90% or more of the amino acid residues constituting the polypeptide represented by that amino acid sequence are D-amino acid residues. Hereinafter, optically isomerized avidins corresponding to a given L-type avidin will also be called "D-type polypeptides." In this specification, the notations "D-" and "L-" for amino acid residues are based on the DL notation method.

[0018] Optically isomerized avidins may have 90% or more of the amino acid residues other than glycine in the amino acid sequence of L-type avidins, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or all of them be D-amino acid residues. In preferred embodiments, the optically isomerized avidins have D-amino acid residues in the amino acid sequence of L-type avidins, with the amino acid residues other than glycine being D-amino acid residues.

[0019] Avidins have a binding site for biotins. The amino acid sequence of the polypeptide constituting the biotin binding site in avidins is also referred to as the "core sequence." In preferred embodiments, optically isomerized avidins have at least the core sequence of an L-type avidin, and in that core sequence, more than 90% of the amino acid residues other than glycine are D-amino acid residues. That is, the binding site for L-biotin in optically isomerized avidins is composed of a polypeptide in which more than 90% of the amino acid residues other than glycine in the core sequence are D-amino acid residues. Because more than 90% of the amino acid residues other than glycine in the core sequence are D-amino acid residues, the three-dimensional structure of the L-biotin binding site in optically isomerized avidins is considered to be enantiomerized with respect to the D-biotin binding site in L-type avidins. Therefore, optically isomerized avidins are considered to not substantially bind to D-biotin and to have a high affinity for L-biotin.

[0020] Optical isomer avidins have at least the core sequence of an L-type avidin, and in that core sequence, 90% or more of the amino acid residues other than glycine, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, may be D-amino acid residues, or all of the amino acid residues other than glycine in the core sequence may be D-amino acid residues. In a preferred embodiment, the optical isomer avidins have at least the core sequence of an L-type avidin, and in that core sequence, the amino acid residues other than glycine consist of D-amino acid residues.

[0021] Examples of optically isomerized avidins include D-type polypeptides such as streptavidin, avidin, avidin-like protein derived from Pleurotus ostreatus, bladavidin, zavidin, and other L-type avidins, as well as D-type polypeptides corresponding to their chimeric and modified forms. "Corresponding D-type polypeptide" refers to a D-type polypeptide having some or all of the amino acid sequences of the aforementioned L-type avidins and their chimeric and modified forms, wherein 90% or more of the amino acid residues other than glycine in the amino acid sequence are D-amino acid residues, and which binds to L-biotin but does not substantially bind to D-biotin. The D-type polypeptide corresponding to streptavidin (hereinafter referred to as "optically isomerized streptavidin") is a polypeptide that includes a core sequence consisting of at least positions 19 to 133 of the amino acid sequence of Sequence ID No. 1, wherein 90% or more of the amino acid residues other than glycine in the core sequence are D-amino acid residues. The amino acid sequence of Sequence ID No. 1 is the amino acid sequence of streptavidin. Preferably, the optically isomerized streptavidin is a polypeptide comprising a subsequence of at least the 13th to 133rd amino acid sequences of SEQ ID NO: 1, wherein 90% or more of the amino acid residues other than glycine in the subsequence are D-amino acid residues. More preferably, the optically isomerized streptavidin is a polypeptide comprising the amino acid sequences of SEQ ID NO: 1, wherein 90% or more of the amino acid residues other than glycine in the amino acid sequences are D-amino acid residues.

[0022] A D-type polypeptide corresponding to avidin (hereinafter referred to as "optically isomerized avidin") is a polypeptide that includes a core sequence consisting of at least positions 2 to 128 of the amino acid sequence of SEQ ID NO: 2, wherein 90% or more of the amino acid residues other than glycine in the core sequence are D-amino acid residues. The amino acid sequence of SEQ ID NO: 2 is the amino acid sequence of avidin. Preferably, the optically isomerized avidin is a polypeptide that includes the amino acid sequence of SEQ ID NO: 2, wherein 90% or more of the amino acid residues other than glycine in the amino acid sequence are D-amino acid residues.

[0023] An example of an avidin-like protein derived from Tamogitake mushroom is Tamavidin®. Tamavidin® is an L-type avidin discovered in Tamogitake mushroom, possessing high affinity for biotin and superior thermal stability compared to avidin (see International Publication No. 2002 / 072817). The D-type polypeptide corresponding to Tamavidin® (hereinafter referred to as "optically isomerized Tamavidin") is a polypeptide that includes a core sequence consisting of at least positions 4 to 129 of the amino acid sequence of Sequence ID No. 3, and in this core sequence, 90% or more of the amino acid residues other than glycine are D-amino acid residues. The amino acid sequence of Sequence ID No. 3 is the amino acid sequence of Tamavidin® 1. Preferably, the optically isomerized avidin is a polypeptide that includes the amino acid sequence of Sequence ID No. 3, and in this amino acid sequence, 90% or more of the amino acid residues other than glycine are D-amino acid residues.

[0024] Alternatively, the optically isomerized tamavidin may be a polypeptide comprising a core sequence consisting of at least positions 4 to 127 of the amino acid sequence of SEQ ID NO: 4, wherein 90% or more of the amino acid residues other than glycine in the core sequence are D-amino acid residues. The amino acid sequence of SEQ ID NO: 4 is the amino acid sequence of Tamavidin® 2. Preferably, the optically isomerized avidin is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, wherein 90% or more of the amino acid residues other than glycine in the amino acid sequence are D-amino acid residues.

[0025] An example of a streptavidin variant is the variant described in Qureshi MH. et al., J. Biol. Chem., vol.276, No.49, pp.46422-46428, 2001 (hereinafter referred to as "streptavidin variant 1"). The amino acid sequence of streptavidin variant 1 (SEQ ID NO: 5) has the substitutions S45A, T90A, and D128A compared to the amino acid sequence of SEQ ID NO: 1. The D-type polypeptide corresponding to streptavidin variant 1 (hereinafter referred to as "optically isomerized streptavidin variant 1") is a polypeptide that includes a core sequence consisting of at least positions 19 to 133 of the amino acid sequence of SEQ ID NO: 5, and in this core sequence, more than 90% of the amino acid residues other than glycine are D-amino acid residues. Preferably, the optically isomerized streptavidin variant 1 is a polypeptide comprising a subsequence of at least the 13th to 133rd amino acid sequences of SEQ ID NO: 5, wherein 90% or more of the amino acid residues other than glycine in the subsequence are D-amino acid residues. More preferably, the optically isomerized streptavidin variant 1 is a polypeptide comprising the amino acid sequences of SEQ ID NO: 5, wherein 90% or more of the amino acid residues other than glycine in the amino acid sequences are D-amino acid residues.

[0026] Another example of a streptavidin variant is the variant described in Wu SC. and Wong SL., J. Biol. Chem., vol.280, No.24, pp.23225-23231, 2005 (hereinafter referred to as "Streptavidin Variant 2"). The amino acid sequence of Streptabidin Variant 2 (SEQ ID NO: 6) has the substitutions V55T, T76R, L109T, and V125R compared to the amino acid sequence of SEQ ID NO: 1. The D-type polypeptide corresponding to Streptabidin Variant 2 (hereinafter referred to as "Optical Isomerized Streptabidin Variant 2") is a polypeptide that includes a core sequence consisting of at least positions 19 to 133 of the amino acid sequence of SEQ ID NO: 6, and in this core sequence, more than 90% of the amino acid residues other than glycine are D-amino acid residues. Preferably, the optically isomerized streptavidin variant 2 is a polypeptide comprising a subsequence of at least the 13th to 133rd amino acid sequences of SEQ ID NO: 6, wherein 90% or more of the amino acid residues other than glycine in the subsequence are D-amino acid residues. More preferably, the optically isomerized streptavidin variant 2 is a polypeptide comprising the amino acid sequences of SEQ ID NO: 6, wherein 90% or more of the amino acid residues other than glycine in the amino acid sequences are D-amino acid residues.

[0027] Another example of a streptavidin variant is the variant described in Lim KH et al., Biotech.Bioeng., vol.110, No.1, pp.57-67, 2013 (hereinafter referred to as "streptavidin variant 3"). The D-type polypeptide corresponding to streptavidin variant 3 (hereinafter referred to as "optically isomerized streptavidin variant 3") is a polypeptide that contains the amino acid sequence of SEQ ID NO: 7, and in that amino acid sequence, more than 90% of the amino acid residues other than glycine are D-amino acid residues. The amino acid sequence of SEQ ID NO: 7 is the amino acid sequence of streptavidin variant 3.

[0028] Another example of a streptavidin variant is the variant described in Sano T. et al., Proc. Natl. Acad. Sci. USA, vol. 94, pp. 6153-6158, 1997 (hereinafter referred to as "streptavidin variant 4"). The amino acid sequence of streptavidin variant 4 (sequence number 8) has a substitution of H127D and deletions from G113 to W120 compared to the amino acid sequence of sequence number 1. The D-type polypeptide corresponding to streptavidin variant 4 (hereinafter referred to as "optically isomerized streptavidin variant 4") is a polypeptide that includes a core sequence consisting of at least positions 19 to 125 of the amino acid sequence of sequence number 8, and in this core sequence, more than 90% of the amino acid residues other than glycine are D-amino acid residues. Preferably, optically isomerized streptavidin variant 4 is a polypeptide comprising a subsequence of at least positions 13-125, at least positions 19-131, or at least positions 13-131 of the amino acid sequence of SEQ ID NO: 8, wherein 90% or more of the amino acid residues other than glycine in the subsequence are D-amino acid residues. More preferably, optically isomerized streptavidin variant 2 is a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, wherein 90% or more of the amino acid residues other than glycine in the amino acid sequence are D-amino acid residues.

[0029] Another example of a streptavidin variant is the variant described in International Publication No. 2006 / 058226 (hereinafter referred to as "Streptavidin Variant 5"). The D-type polypeptide corresponding to streptavidin variant 5 (hereinafter referred to as "optically isomerized streptavidin variant 5") is a polypeptide that includes a core sequence consisting of at least positions 1-20, 35-196, and 213-261 of the amino acid sequence of SEQ ID NO: 9, and in this core sequence, 90% or more of the amino acid residues other than glycine are D-amino acid residues. The amino acid sequence of SEQ ID NO: 9 is the amino acid sequence of streptavidin variant 5. Preferably, optically isomerized streptavidin variant 5 is a polypeptide that includes a subsequence of at least positions 1-24, 29-202, and 207-261 of the amino acid sequence of SEQ ID NO: 9, and in this subsequence, 90% or more of the amino acid residues other than glycine are D-amino acid residues. More preferably, optically isomerized streptavidin variant 5 is a polypeptide comprising the amino acid sequence of SEQ ID NO: 9, wherein 90% or more of the amino acid residues other than glycine in the amino acid sequence are D-amino acid residues.

[0030] Avidins, including optically isomerized avidins, generally exist in the form of polymers composed of multiple subunits. These polymers may be, for example, dimers, tetramers, or octamers. Polymers are formed by the association of polypeptide molecules of a given avidin. For example, if the reagent of this embodiment contains an optically isomerized avidin or an optically isomerized streptavidin, then a tetrameric optically isomerized avidin or streptavidin may be present in the reagent. A tetrameric optically isomerized avidin or streptavidin can bind to four L-biotin molecules.

[0031] Optically isomerized avidins can be produced by known polypeptide synthesis methods. The peptide synthesis method is not particularly limited, but examples include liquid-phase synthesis, solid-phase synthesis, and cell-free synthesis using artificial tRNA. Furthermore, if the number of amino acid residues in the resulting polypeptide is above a certain number (generally 30 residues or more), it can be produced by synthesizing two or more peptide fragments and then linking them using a known ligation reaction.

[0032] Optically isomerized avidins can be synthesized, for example, by the following solid-phase synthesis method. (1) Using a protecting group, the carboxyl group of the first amino acid residue, whose nitrogen atom of the amino group is protected, is bonded to the resin. (2) The protecting group of the reactant obtained by the bonding reaction in (1) above is removed using a deprotecting agent, and then washed with a solvent to form free amino acids. (3) The free amino acid obtained in (2) above is condensed with any amino acid whose amino group is protected by a protecting group using a condensing agent. (4) The protecting group of the product of (3) above is removed using a deprotecting agent to form a free amino acid. (5) By repeating steps (2) to (4) above, a polypeptide can be obtained in which any amino acid is linked with a resin bonded to the C-terminus. (6) Wash with a solvent at any point after step (5) above, and when the resin to which the desired polypeptide has been bound has been produced. (7) After the polypeptide to which the resin has been washed in (6) above has been bound is protected by a protecting group to which the N-terminal amino group is protected, and then the resin is cleaved with an acid to obtain any polypeptide to which the protecting group has been bound.

[0033] The resin used in (1) above can be any known resin used in solid-phase synthesis. As the resin supplied with an amide group at the C-terminus, it is preferable to use, for example, Rink-Amide-resin (Merck KGaA), Rink-Amide-PEGA-resin (Merck KGaA), and Fmoc-NH-SAL-resin (Watanabe Chemical Industry Co., Ltd.) which are functionalized with amino groups. Alternatively, an Fmoc-NH-SAL-resin-linker (Watanabe Chemical Industry Co., Ltd.) may be bonded to an amino-functionalized AMINO-PEGA-resin (Merck KGaA), etc.

[0034] Furthermore, when the C-terminus is a carboxylic acid, resins such as chlorine-functionalized 2-chlorotrityl chloride resin (Merck KGaA), amino group-functionalized AMINO-PEGA-resin (Merck KGaA), NovaSyn TGT alcohol resin with a hydroxyl group (Merck KGaA), Wang-resin (Merck KGaA), and HMPA-PEGA-resin (Merck KGaA) can be used. A linker may also be present between the AMINO-PEGA-resin and the amino acid. Examples of such linkers include 4-hydroxymethylphenoxyacetic acid (HMPA) and 4-(4-hydroxymethyl-3-methoxyphenoxy)-butylacetic acid (HMPB). H-Cys(Trt)-TritylNovaPEG resin (Merck KGaA), in which the C-terminal amino acid is pre-bonded to the resin, can also be used.

[0035] When using a resin containing a hydroxyl group or a resin functionalized with chlorine, the bond between the resin and an amino acid whose nitrogen atom of the amino group is protected by a protecting group is formed by bonding the carboxyl group of the amino acid to the resin via an ester bond. Alternatively, when using a resin functionalized with an amino group, the carboxyl group of the amino acid is bonded to the resin via an amide bond.

[0036] Any known protecting group may be used as the protecting group. For example, carbonate-based or amide-based protecting groups such as 9-fluorenylmethoxycarbonyl (Fmoc) group, t-butyloxycarbonyl (Boc) group, benzyl group, allyloxycarbonyl group, and acetyl group can be used. When introducing a protecting group to an amino acid, for example, when introducing an Fmoc group, it can be introduced by adding 9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate and carrying out the reaction. The reaction temperature is 0 to 50°C, preferably room temperature, and the reaction time is 1 to 5 hours, preferably 3 hours.

[0037] Commercially available amino acids may be used as amino acids in which the nitrogen atom of the amino group is protected using a protecting group. Examples include Fmoc-Ser-OH, Fmoc-Asn-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-AIa-OH, Fmoc-Tyr-OH, Fmoc-Gly-OH, Fmoc-Lys-OH, Fmoc-Arg-OH, Fmoc-His-OH, Fmoc-Asp-OH, Fmoc-Glu-OH, Fmoc-Gln-OH, Fmoc-Thr-OH, Fmoc-Cys-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-SeMet-OH, and Fmoc-3-(methylseleno)-Ala-OH.

[0038] Furthermore, amino acids protected with a protecting group, in which a protecting group is introduced to the side chain, may also be used. For example, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(tBu)-OH, Fmoc-Cys(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(B oc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Sec(Trt)-OH, Examples include Fmoc-Sec(pMeOBzl)-OH, Fmoc-Sec(pMeBzl)-OH, Fmoc-HomoSec(pMeBzl)-OH, and Fmoc-HomoSec(Mob)-OH.

[0039] When using a resin containing a hydroxyl group, for example, HMPB resin can be used as an esterification catalyst. In this case, known dehydration condensation agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC) can be used as the dehydration condensation agent. The ratio of amino acid to dehydration condensation agent used is usually 1 to 10 equivalents, preferably 1 to 5 equivalents, of the dehydration condensation agent per equivalent of amino acid.

[0040] The esterification reaction is preferably carried out by, for example, providing the resin on a solid-phase column, washing it with a solvent, and adding an amino acid solution. Examples of washing solvents include dimethylformamide (DMF), 2-propanol, and dichloromethane (DCM). Examples of solvents for dissolving amino acids include dimethyl sulfoxide (DMSO), DMF, and DCM. The reaction temperature for the esterification reaction is 0 to 50°C, preferably room temperature. The reaction time is 10 minutes to 30 hours, preferably 15 minutes to 24 hours. At this time, it is preferable to cap any unreacted functional groups on the solid phase by acetylation using acetic anhydride or the like.

[0041] The lipophilic protecting group can be removed, for example, by treatment with a base. Examples of bases include piperidine and morpholine. Treatment with a base is preferably carried out in the presence of a solvent. Examples of solvents include DMF, DMSO, and methanol.

[0042] The amidation reaction between a free amino group and the carboxyl group of any amino acid whose amino group nitrogen is protected by a protecting group is preferably carried out in the presence of an activator, a base, and a solvent.

[0043] Examples of activators include DIC, DCC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), diphenylphosphoryl azide (DPPA), carbonyldiimidazole (CDI), diethylcyanophosphonate (DEPC), benzotriazole-1-yloxy-trispirolidinophosphonium hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HOBt), hydroxysuccinimide (HOSu), dimethylaminopyridine (DMAP), 1-hydroxy-7-azabenzotriazole (HOAt), hydroxyphthalimide (HOPht), and pentafluorophenol (Pfp-). Examples include O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphonate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (DHBT), and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM).

[0044] The amount of activator used is 1 to 20 equivalents, preferably 1 to 10 equivalents, and more preferably 1 to 5 equivalents, relative to any amino acid whose amino group nitrogen is protected by a protecting group.

[0045] As the base, a base that can coexist with the alkylation reaction is preferred. Examples include, but are not limited to, N-ethyldiisopropylamine (DIPEA), 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), DMAP,1,4-diazabicyclo[2.2.2]octane (DABCO), 2,6-dimethylpyridine, triethylamine (TEA), and 1,5-diazabicyclo[4.3.0]nona-5-ene (DBN).

[0046] Examples of solvents include DMF, DMSO, and DCM. The reaction temperature is 0 to 50°C, preferably room temperature. The reaction time is 10 minutes to 30 hours, preferably 15 minutes to 24 hours. The removal of protecting groups can be carried out in the same manner as described above. To cleave the peptide chain from the resin, it is preferable to treat it with an acid. Examples of acids include trifluoroacetic acid (TFA).

[0047] As a known ligation reaction for linking two or more peptide fragments, native chemical ligation (NCL method) (Dawson PE. et al., Science, vol.266, pp.776-779, 1994) can be used. The NCL method is a chemoselective reaction between a first peptide having an α-carboxythioester moiety at the C-terminus and a second peptide having a cysteine ​​residue at the N-terminus. The thiol group (also called the SH group or sulfhydryl group) of the cysteine ​​side chain selectively reacts with the carbonyl carbon of the thioester group, and an initial thioester bond intermediate is generated by a thiol exchange reaction. This intermediate spontaneously undergoes intramolecular rearrangement, giving a natural amide bond to the linking site, while simultaneously regenerating the cysteine ​​side chain thiol.

[0048] In the NCL method, the cysteine ​​binding site of the second peptide having a cysteine ​​residue at the N-terminus can also be replaced with alanine after the ligation reaction by a desulfurization reaction (Yan LZ. and Dawson PE., J.Am.Chem.Soc., vol.123, pp.526-533, 2001). In other words, the site that is originally alanine can be replaced with cysteine ​​during synthesis and used as the binding site for the ligation reaction.

[0049] Separation and / or purification steps may be included before or after the peptide synthesis or ligation reaction. Known methods may be used for purification, such as column chromatography. Examples of column chromatography include normal-phase chromatography, reverse-phase chromatography, gel filtration chromatography, and affinity chromatography. Depending on the substance to be separated and purified, the solvent, column packing material, detection methods for the substances to be separated and purified, temperature conditions, pressure conditions, etc., can be appropriately selected.

[0050] The peptide synthesis reaction, ligation reaction, or separation and / or purification step may include known washing, drying, dilution, and concentration steps as appropriate before and after the reaction.

[0051] If polypeptides do not fold correctly, refolding is preferable. Refolding can be performed by methods such as dilution refolding, dialysis refolding, solid-phase refolding, size exclusion chromatography refolding, and surfactant refolding (Arakawa T. and Ejima D., Antibodies, vol.3, pp.232-241, 2014).

[0052] The azobenzene derivative represented by formula (I) above is a dye capable of binding to the L-biotin binding site of optically isomerized avidins. When optically isomerized avidins exist in the reagent of this embodiment in the form of a polymer, multiple molecules of the azobenzene derivative bind to the L-biotin binding site of the optically isomerized avidin polymer. It is believed that a complex of the azobenzene derivative and the optically isomerized avidin is formed in the reagent of this embodiment by binding of the azobenzene derivative to the optically isomerized avidin polymer. This complex, due to the presence of the azobenzene derivative represented by formula (I) above, has light absorption at wavelengths of approximately 400 nm to 600 nm at a pH near neutral (pH 6.5 to 7.5).

[0053] In this specification, "C1-C6 alkyl group" refers to a monovalent group that is a straight-chain or branched saturated hydrocarbon chain having 1 to 6 carbon atoms. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl(t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and hexyl.

[0054] In this specification, "C1-C6 alkoxy group" refers to a group in which the above-mentioned C1-C6 alkyl group is bonded to an oxy group. Examples of C1-C6 alkoxy groups include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, tert-butoxy group, isobutoxy group, sec-butoxy group, n-pentyloxy group, isopentyloxy group, and n-hexyloxy group.

[0055] In this specification, "C1-C6 dialkylamino group" means a group in which two of the above C1-C6 alkyl groups replace two hydrogen atoms of an amino group. The two C1-C6 alkyl groups may be the same or different. Examples of C1-C6 dialkylaminos include dimethylamino, diethylamino, N,N-diisopropylamino, N-methyl-N-ethylamino, and N-isopropyl-N-ethylamino. Preferably, the C1-C6 dialkylamino group is dimethylamino or diethylamino.

[0056] The substituents of C1-C6 alkyl groups, C1-C6 alkoxy groups, and C1-C6 dialkylamino groups are independently selected from halogen atoms, cyano groups, nitro groups, carboxyl groups, carbamoyl groups, amino groups, and hydroxyl groups. The halogen atom is fluorine, chlorine, bromine, or iodine.

[0057] In a preferred embodiment, in formula (I) above, one of R1 to R5 is a hydroxyl group, or an unsubstituted dimethylamino group or diethylamino group, and R6 to R 10 One of these is a carboxyl group.

[0058] Examples of azobenzene derivatives represented by formula (I) above include HABA, 4'-hydroxyazobenzene-4-carboxylic acid, and 4'-dimethylaminoazobenzene-2-carboxylic acid. These azobenzene derivatives are shown in formulas (II) to (IV) below. Among these, HABA is particularly preferred.

[0059] [ka]

[0060] [ka]

[0061] [ka]

[0062] The azobenzene derivative represented by formula (I) above can be obtained by known synthesis methods, such as diazo coupling reactions. Alternatively, commercially available azobenzene derivatives may be used. For example, as shown in the reaction equation below, HABA can be obtained by reacting anthranilic acid with sodium nitrite under cooling conditions to produce a diazonium salt, and then reacting the diazonium salt with phenol.

[0063] [ka]

[0064] In the above reaction equation, if p-aminobenzoic acid is used instead of anthranilic acid, 4'-hydroxyazobenzene-4-carboxylic acid can be obtained. Also, in the above reaction equation, if dimethylaniline is used instead of phenol, 4'-dimethylaminoazobenzene-2-carboxylic acid can be obtained.

[0065] The reagent of this embodiment can be prepared by dissolving optically isomerized avidins and the azobenzene derivative represented by formula (I) above in a suitable solvent. Alternatively, the reagent of this embodiment can be prepared by mixing a solution of optically isomerized avidins with a solution of the azobenzene derivative represented by formula (I) above. The solvent is preferably an aqueous solvent having a pH near neutral (pH 6.5 to 7.5), such as water, physiological saline, or a buffer solution with a buffering effect at a pH near neutral. Examples of buffer solutions include phosphate-buffered saline (PBS), phosphate buffer, borate buffer, Tris-HCl buffer, BES buffer, HEPES buffer, MOPS buffer, and TES buffer.

[0066] The pH of the reagent in this embodiment is usually 6.5 to 7.5, preferably 7 to 7.5. The concentration of optically isomerized avidins in the reagent in this embodiment is not particularly limited, but for example, it is 50 μg / mL to 2000 μg / mL, preferably 100 μg / mL to 1000 μg / mL, and more preferably 200 μg / mL to 500 μg / mL. The concentration of the azobenzene derivative represented by formula (I) in the reagent in this embodiment is not particularly limited, but for example, it is 5 μg / mL to 200 μg / mL, preferably 10 μg / mL to 100 μg / mL, and more preferably 20 μg / mL to 50 μg / mL.

[0067] The reagent of this embodiment may be in the form of a single reagent containing both optically isomerized avidins and the azobenzene derivative represented by formula (I) above. Alternatively, the reagent of this embodiment may be in the form of two reagents, comprising a first reagent containing optically isomerized avidins and a second reagent containing the azobenzene derivative represented by formula (I) above.

[0068] The reagents of this embodiment may be packaged in a box and provided to the user. The box may also include an accompanying document describing how to use the reagents. Figures 1A and 1B show examples of the reagents of this embodiment. Referring to Figure 1A, 10 shows the reagent in the form of a single reagent, 11 shows the first container containing the reagent, 12 shows the packaging box, and 13 shows the accompanying document. Referring to Figure 1B, 20 shows the reagent in the form of a double reagent, 21 shows the first container containing the first reagent, 22 shows the second container containing the second reagent, 23 shows the packaging box, and 24 shows the accompanying document.

[0069] The reagent of this embodiment may contain optically isomerized avidins and the azobenzene derivative represented by formula (I) above in a solid state (powder, particles, lyophilized product, etc.). Alternatively, the reagent of this embodiment may contain optically isomerized avidins and the azobenzene derivative represented by formula (I) above in a solution state. If the reagent is in the form of two reagents, either the optically isomerized avidins or the azobenzene derivative represented by formula (I) above may be in a solid state, and the other may be in a solution state. Preferably, both the optically isomerized avidins and the azobenzene derivative represented by formula (I) above are in a solution state.

[0070] The reagent of this embodiment may further include a calibrator. The calibrator is a solution containing L-biotin at a predetermined concentration and is used to create a calibration curve. Preferably, the calibrator consists of multiple solutions, each with a different L-biotin concentration. Preferably, the calibrator also includes a solution with an L-biotin concentration of 0, such as a buffer solution that does not contain L-biotin.

[0071] Another embodiment of the present invention is a method for measuring a sample containing L-biotin. Hereinafter, this method will also be referred to as "the measurement method of this embodiment." In the measurement method of this embodiment, first, a sample for measurement is prepared by mixing a sample containing L-biotin with the reagent of this embodiment. The sample containing L-biotin is not particularly limited as long as it contains free L-biotin and / or a substance having at least one L-biotin group. In the measurement method of this embodiment, the sample containing L-biotin will also be referred to as the "test sample."

[0072] A substance having at least one L-biotin group may be, for example, an L-biotin-labeled substance. In an L-biotin-labeled substance, it is preferable that the L-biotin and the substance are covalently bonded. The substance is not particularly limited and includes, for example, polypeptides, polynucleotides, lipids, and sugar chains. A preferred L-biotin-labeled substance is an L-biotin-labeled polypeptide. The type of polypeptide is not particularly limited and includes, for example, antibodies, albumin, and enzymes such as alkaline phosphatase.

[0073] In this specification, "antibody" also includes antibody fragments. Examples of antibody fragments include Fab, Fab', and F(ab')2. The antibody may be either a monoclonal antibody or a polyclonal antibody. The origin of the antibody is not particularly limited and may be an antibody derived from any mammal such as mouse, rat, hamster, rabbit, goat, horse, camel, or human. The antibody isotype may be any of IgG, IgM, IgE, or IgA, but IgG is preferred.

[0074] L-biotin labeling can be performed, for example, by reacting a substance with an L-biotin labeling reagent. The L-biotin labeling reagent can be obtained by derivatizing the valeric acid side chain of L-biotin using a known method and introducing a labeling functional group. The labeling functional group is not particularly limited, but examples include N-hydroxysuccinimide (NHS) ester and maleimide group.

[0075] Since the measurement method in this embodiment is preferably performed in solution, if the sample to be tested is not in liquid form, it is preferable to make it liquid beforehand. Here, "liquid" sample is not limited to a solution in which the solute is completely dissolved in the solvent, but also includes suspensions and sols in which fine solid particles are suspended. For example, if the sample to be tested contains a solid L-biotin-labeled substance, the substance can be dissolved in a suitable solvent to make a liquid sample. The solvent is the same as that described for the reagents in this embodiment.

[0076] The amount of L-biotin-containing sample mixed with the reagent of this embodiment and the final concentration of the reagent are not particularly limited and can be determined as appropriate. For example, in the measurement of the absorbance of the sample described later, if the absorbance value is lower than a predetermined value, it is considered that the L-biotin concentration in the test sample is high. In this case, it is preferable to dilute the test sample and prepare the measurement sample again from the diluted test sample. The above predetermined value can be determined as appropriate, but for example, when measuring absorbance using a cuvette, the predetermined value may be 0.3. Also, when measuring absorbance using a microplate, the predetermined value may be 0.15.

[0077] The temperature and time conditions for mixing the L-biotin-containing sample with the reagent of this embodiment are not particularly limited. For example, the mixture may be incubated at 4°C to 40°C, preferably room temperature (about 20°C) to 37°C, for 1 minute to 60 minutes, preferably 5 minutes to 30 minutes. During incubation, the mixture may be left to stand, stirred, or shaken.

[0078] In the measurement method of this embodiment, the absorbance of the prepared sample is measured. Specifically, the absorbance of the azobenzene derivative represented by formula (I) above is measured at a wavelength that can be measured (e.g., the maximum absorption wavelength). Such a wavelength can be appropriately determined from the range of about 400 nm to about 600 nm. For example, when HABA is used as the azobenzene derivative, the absorbance at a wavelength of 500 nm may be measured. When 4'-hydroxyazobenzene-4-carboxylic acid is used, the absorbance at a wavelength of 470 nm may be measured. When 4'-dimethylaminoazobenzene-2-carboxylic acid is used, the absorbance at a wavelength of 548 nm or 577 nm may be measured.

[0079] Referring to Figure 2, the principle of the measurement method of this embodiment will be explained. In the example shown in Figure 2, the reagent of this embodiment contains optically isomerized avidins and HABA, and the test sample contains an L-biotin-labeled polypeptide, but the present invention is not limited to this example. In this reagent, a complex 30 is formed between a tetramer 31 of optically isomerized avidins and HABA 32. The complex 30 has an absorption at a wavelength of 500 nm in the presence of HABA 32. When the reagent of this embodiment is mixed with a sample containing an L-biotin-labeled polypeptide 33, the complex 30 and the polypeptide 33 come into contact. Upon this contact, HABA 32 in the complex 30 substitutes for the L-biotin group of the polypeptide 33, and HABA 32 is released. This is because L-biotin has a higher affinity for optically isomerized avidins than HABA. When HABA32 is substituted for an L-biotin group of polypeptide 33, a complex 34 is formed in which some or all of the HABA32 that was bound to the optically isomerized avidin tetramer 31 is replaced by polypeptide 33. Compared to complex 30, complex 34 exhibits reduced absorption at a wavelength of 500 nm. The degree of absorption reduction depends on the concentration of L-biotin in the test sample. In the example shown in Figure 2, this concentration corresponds to the number of L-biotin groups present in all polypeptides 33 in the test sample. Therefore, in the measurement method of this embodiment, the absorbance measurement serves as an indicator of the concentration of L-biotin in a sample containing L-biotin.

[0080] In this embodiment, the measurement method allows for the measurement of L-biotin concentration in a sample containing L-biotin based on absorbance measurements. For example, multiple calibrators containing free L-biotin at predetermined concentrations are measured in the same manner as the test sample to create a calibration curve showing the relationship between absorbance measurements and L-biotin concentration. Using the obtained calibration curve, the value of L-biotin concentration in the test sample can be determined from the absorbance measurements of the test sample.

[0081] Another embodiment of the present invention is a method for determining the number of L-biotin-labeled substances. Hereinafter, this method will also be referred to as "the determination method of this embodiment." In this specification, "number of L-biotin-labeled substances" refers to the number of L-biotin groups per molecule of L-biotin-labeled substance. Depending on the type of substance and L-biotin-labeling reagent, one or more L-biotin groups may be attached to the substance. For example, if the substance is an antibody, the number of L-biotin groups per molecule of L-biotin-labeled antibody may contribute to the results of assays using L-biotin-labeled antibodies. According to the determination method of this embodiment, it is possible to control the quality of L-biotin-labeled antibodies.

[0082] In the determination method of this embodiment, first, a sample containing an L-biotin-labeled substance and the reagent of this embodiment are mixed to prepare a measurement sample. The details of preparing the L-biotin-labeled substance and the measurement sample are the same as those described in the measurement method of this embodiment. In the determination method of this embodiment, the sample containing the L-biotin-labeled substance is also called the "test sample".

[0083] In the determination method of this embodiment, the absorbance of the prepared sample is measured. Based on the absorbance measurement, the concentration of L-biotin in the sample is determined. The details of the absorbance measurement and the determination of the L-biotin concentration are the same as those described in the measurement method of this embodiment. Here, the L-biotin concentration determined based on the absorbance measurement corresponds to the total number of L-biotin groups in the L-biotin-labeled substance in the sample. Therefore, by comparing the determined L-biotin concentration with the concentration of the substance in the sample, the number of L-biotin groups per molecule of the L-biotin-labeled substance can be determined. In other words, in the determination method of this embodiment, the number of L-biotin groups per molecule of the L-biotin-labeled substance is determined based on the concentration of L-biotin and the concentration of the substance in the sample.

[0084] The determination method of this embodiment may further include a step of determining the concentration of a substance in the test sample. The method for determining the concentration of a substance can be appropriately selected depending on the type of substance. For example, if the L-biotin-labeled substance is an L-biotin-labeled polypeptide, the polypeptide concentration in the test sample can be determined by a known method for quantifying total protein. As a method for quantifying total protein, a quantitative method based on absorbance is preferred, such as ultraviolet spectrophotometric analysis (280 nm method), BCA method, Bradford method, Lowry method, or a method using the Pierce® 660 nm Protein Assay Kit (Thermo Fisher Scientific). Also, if the L-biotin-labeled substance is an L-biotin-labeled polynucleotide, the polynucleotide concentration in the test sample can be determined by a known method for quantifying polynucleotides. As a method for quantifying polynucleotides, for example, absorbance analysis based on absorbance at 260 nm and 280 nm, fluorescence analysis using a fluorescent dye or fluorescent probe, etc.

[0085] The number of L-biotin groups per molecule of an L-biotin-labeled substance corresponds to the ratio of L-biotin molecules to the number of molecules of the substance. Therefore, the concentration of L-biotin and the concentration of the substance in the test sample are expressed as molar concentrations (units: mol / L, mol / dm³). 3 It is preferable that it be either (or M). When the concentration of a substance in the test sample is determined by a quantitative method based on absorbance, it is preferable to calculate the molar concentration from the absorbance value. For example, the absorbance of a standard solution of a substance of known concentration can be measured, and the molar concentration of the substance in the test sample can be calculated from the absorbance of the substance using the absorbance of the standard solution, the concentration of the substance, and the molecular weight of the substance.

[0086] In a preferred embodiment, the number of L-biotin groups per molecule of L-biotin-labeled substance is calculated using the following formula. X = A / B (In the formula, X is the number of L-biotin groups per molecule of L-biotin-labeled substance, A is the molar concentration of L-biotin in the test sample. (B is the molar concentration of the substance in the test sample.)

[0087] Another embodiment of the present invention is a method for producing a solid phase on which optically isomerized avidins are immobilized. Hereinafter, this method will also be referred to as "the production method of this embodiment." In the production method of this embodiment, the number of L-biotin groups per molecule of L-biotin-labeled polypeptide in the liquid is determined using a sample separated from a liquid containing L-biotin-labeled polypeptide. When the determined number of L-biotin groups is within a predetermined range, a solid phase on which optically isomerized avidins are immobilized is produced using the same liquid.

[0088] Referring to Figure 3, the solid phase produced by the manufacturing method of this embodiment will be described. In the example shown in Figure 3, the solid phase 40 is spherical particles, but the present invention is not limited thereto. The solid phase 40 is a particle whose surface can bind to the polypeptide portion 41 of the L-biotin-labeled polypeptide. In the manufacturing method of this embodiment, after binding the solid phase 40 to the polypeptide portion 41 of the L-biotin-labeled polypeptide, the L-biotin group 42 of the L-biotin-labeled polypeptide is bound to the optically isomerized avidins 43. In the example shown in Figure 3, the optically isomerized avidins 43 are tetramers. Also, in the example shown in Figure 3, since there are 4 L-biotin groups per molecule of the L-biotin-labeled polypeptide, 4 optically isomerized avidins 43 are bound. Thus, Figure 3 shows a solid phase in which multiple optically isomerized avidins are immobilized via the binding of the L-biotin group of the immobilized L-biotin-labeled polypeptide to the optically isomerized avidins. However, the present invention is not limited to this example.

[0089] In the manufacturing method of this embodiment, first, a sample separated from a liquid containing L-biotin-labeled polypeptide is mixed with the L-biotin measurement reagent of this embodiment to prepare a measurement sample. In the manufacturing method of this embodiment, the sample separated from the liquid containing L-biotin-labeled polypeptide is also called the "test sample". The details of preparing the L-biotin-labeled substance and the measurement sample are the same as those described in the measurement method of this embodiment. The amount of the test sample is not particularly limited, but it is preferably the minimum amount necessary for measuring absorbance.

[0090] Next, the absorbance of the sample is measured, and the concentration of L-biotin in the sample is determined based on the absorbance measurement. The details of the absorbance measurement and the determination of the L-biotin concentration are the same as those described in the measurement method of this embodiment. Then, based on the L-biotin concentration and the polypeptide concentration, the number of L-biotin groups per molecule of L-biotin-labeled polypeptide in the test sample is determined. The details of the determination of the number of L-biotin groups are the same as those described in the determination method of this embodiment.

[0091] The manufacturing method of this embodiment may include a step of determining whether the number of L-biotin groups per molecule of L-biotin-labeled polypeptide in the test sample is within a predetermined range. In the manufacturing method of this embodiment, as shown in Figure 3, optically isomerized avidins are immobilized on the solid phase via the binding of L-biotin groups of the L-biotin-labeled polypeptide to optically isomerized avidins. The number of optically isomerized avidins immobilized on the solid phase depends on the number of L-biotin groups of the L-biotin-labeled polypeptide. Therefore, the number of L-biotin groups affects the quality of the solid phase obtained by the manufacturing method of this embodiment.

[0092] In the manufacturing method of this embodiment, when the number of L-biotin groups per molecule of L-biotin-labeled polypeptide in the test sample is within a predetermined range, the above liquid is brought into contact with a solid phase capable of binding to the polypeptide to immobilize the L-biotin-labeled polypeptide on the solid phase. The liquid may be used entirely or partially. The contact conditions are not particularly limited, but for example, incubation may be carried out at 4°C to 40°C, preferably at room temperature (about 20°C) to 37°C, for 10 minutes to 4 hours, preferably 30 minutes to 3 hours. If the solid phase is particles, the mixture of the solid phase and the L-biotin-labeled polypeptide may be left to stand, stirred, or shaken during incubation.

[0093] If the number of L-biotin groups per molecule of L-biotin-labeled polypeptide in the test sample is not within the specified range, the L-biotin-labeled polypeptide should be prepared again, and a new liquid should be prepared. Alternatively, if multiple liquids are available, the number of L-biotin groups per molecule of L-biotin-labeled polypeptide may be determined for one of the liquids.

[0094] The solid phase can be any insoluble carrier capable of binding to the polypeptide portion of the L-biotin-labeled polypeptide. The mode of binding between the solid phase and the polypeptide portion of the L-biotin-labeled polypeptide is not particularly limited and includes, for example, physical adsorption or covalent bonding to the solid phase surface. As for covalent bonding between the solid phase surface and the polypeptide, for example, functional groups such as NHS esters or maleimide groups can be imparted to the solid phase surface, and the polypeptide portion can be covalently bonded to the solid phase surface by these functional groups. Alternatively, a divalent crosslinking agent may be used to covalently bond the polypeptide portion to the solid phase surface.

[0095] The solid phase material is not particularly limited and can be selected from, for example, organic polymers, inorganic compounds, and biopolymers. Examples of organic polymers include latex, polystyrene, and polypropylene. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, and ferrite, etc.), silica, alumina, and glass. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, and cellulose. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited and can be, for example, particles, films, microplates, microtubes, or test tubes. Among these, particles and microplates are preferred. Magnetic particles are particularly preferred as particles.

[0096] After contacting the solid phase with the L-biotin-labeled polypeptide, bound / free (B / F) separation may be performed to remove unreacted free components before further contact with optically isomerized avidins. Unreacted free components may include, for example, L-biotin-labeled polypeptide that was not immobilized on the solid phase. If the solid phase is particles, B / F separation can be performed by centrifugation to precipitate the particles and removing the supernatant containing unreacted free components. If the solid phase is magnetic particles, B / F separation can be performed by magnetically confining the magnetic particles with a magnet, for example, and removing the liquid containing unreacted free components. If the solid phase is a container such as a microplate, B / F separation can be performed by removing the liquid containing unreacted free components from the container. After B / F separation, the solid phase on which the L-biotin-labeled polypeptide is immobilized may be washed with a suitable aqueous medium such as PBS.

[0097] In the production method of this embodiment, a solid phase on which L-biotin-labeled polypeptides are immobilized is brought into contact with optically isomerized avidins to immobilize the optically isomerized avidins on the solid phase. As shown in Figure 3, the optically isomerized avidins are immobilized on the solid phase via the binding of the L-biotin group of the L-biotin-labeled polypeptide to the optically isomerized avidins. The contact conditions are not particularly limited, but for example, incubation may be carried out at 4°C to 40°C, preferably at room temperature (about 20°C) to 37°C for 1 minute to 1 hour, preferably 5 minutes to 30 minutes. If the solid phase is particles, the mixture of the solid phase and the optically isomerized avidins may be left to stand, stirred, or shaken during incubation. After contact between the solid phase and the optically isomerized avidins, B / F separation may be performed. By contacting the solid phase with the optically isomerized avidins, a solid phase on which the optically isomerized avidins are immobilized can be obtained.

[0098] By combining the immobilized solid phase containing optically isomerized avidins obtained by the manufacturing method of this embodiment with an L-biotin-labeled antibody, a reagent kit containing a solid phase and a capture agent for use in enzyme-linked immunosorbent assay (ELISA) can be obtained.

[0099] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0100] Example 1: Preparation and use of reagent for L-biotin measurement A method for measuring biotin in a sample is known that utilizes the fact that a complex of avidins and HABA has absorption at a wavelength of 500 nm, and that HABA in the complex readily replaces free or bound D-biotin. In this measurement method, we investigated whether L-biotin in a sample can be measured when optically isomerized avidins are used instead of avidins. Specifically, we prepared an L-biotin measurement reagent containing optically isomerized streptavidin and HABA, and used this reagent to measure L-biotin-labeled polypeptides and determine the number of biotin labels. For comparison, we also prepared a D-biotin measurement reagent and performed the same experiment.

[0101] 1. Preparation of reagents for biotin measurement (1.1) Preparation of reagents for L-biotin measurement PBS was prepared by mixing 0.1 M phosphate buffer (pH 7.5, 200 mL) and sodium chloride (1.75 g). HABA solution was obtained by mixing HABA (1.12 mg: Tokyo Chemical Industry Co., Ltd.) with PBS (20 mL). Optically isomerized streptavidin was obtained by peptide synthesis commissioned to the Glycotechnology Research Institute, Inc. The obtained optically isomerized streptavidin had D-amino acid residues other than glycine, and its amino acid sequence was identical to that of the polypeptide consisting of amino acid sequences from position 13 to 133 of the amino acid sequence of SEQ ID NO: 1. Optically isomerized streptavidin (1.28 mg) was dissolved in PBS (2 mL) to obtain an optically isomerized streptavidin solution. A reagent for L-biotin measurement was prepared by mixing the optically isomerized streptavidin solution (368 μL) and the HABA solution (432 μL). In the reagent in question, the concentration of optically isomerized streptavidin was 294 μg / mL, and the concentration of HABA was 30 μg / mL.

[0102] (1.2) Preparation of reagent 1 for D-biotin measurement HABA (1 mg) and PBS (33 mL) were mixed to obtain an HABA solution. Avidin (1 mg: Fujifilm Wako Pure Chemical Industries, Ltd.) and the HABA solution (4 mL) were mixed to prepare reagent 1 for D-biotin measurement. In this reagent, the concentration of avidin was 250 μg / mL and the concentration of HABA was 30 μg / mL.

[0103] (1.3) Preparation of reagent 2 for D-biotin measurement HABA (1.12 mg) and PBS (20 mL) were mixed to obtain an HABA solution. Streptavidin (1.28 mg: Roche) and PBS (2 mL) were mixed to obtain a streptavidin solution. D-biotin measurement reagent 2 was prepared by mixing streptavidin solution (368 μL) and HABA solution (432 μL). In this reagent, the concentration of streptavidin was 194 μg / mL and the concentration of HABA was 30 μg / mL.

[0104] 2. Preparation of biotin standard solution as a calibrator (2.1) Preparation of L-biotin standard solution L-biotin was obtained as follows: A mixture of D-biotin and L-biotin was synthesized according to the method described in Lavielle S. et al., J.Am.Chem.Soc., vol.100, pp.1558-1563, 1978. The mixture was then optically resolved by liquid chromatography at the request of Daicel Corporation to obtain L-biotin. A CHIRALPAK® IG column (Φ46×50 mm: Daicel Corporation) was used, and a mixed solvent of methanol and acetic acid (100:0.1 (v / v)) was used as the mobile phase. Optical resolution was performed under conditions of a flow rate of 1.0 mL / min, column temperature of 40°C, and detection wavelength of 205 nm. A 100 μM L-biotin standard solution was prepared by mixing the obtained L-biotin (1 mg) with 0.1 M phosphate buffer (pH 7.5, 40.9 mL). This standard solution was diluted with 0.1 M phosphate buffer (pH 7.5) to further prepare 25 μM and 50 μM L-biotin standard solutions.

[0105] (2.2) Preparation of D-biotin standard solution A 100 μM D-biotin standard solution was prepared by mixing D-biotin (1 mg: Kishida Chemical Co., Ltd.) with 0.1 M phosphate buffer (pH 7.5, 40.9 mL). This standard solution was further diluted with 0.1 M phosphate buffer (pH 7.5) to prepare 25 μM and 50 μM D-biotin standard solutions.

[0106] 3. Sample preparation (3.1) Preparation of L-biotin-labeled reagent To prepare L-biotin-labeled polypeptides, L-biotin with an N-hydroxysuccinimide (NHS) ester (L-biotin-AC5-NHS) and L-biotin with a maleimide group (L-biotin-PE-maleimide) were synthesized using the L-biotin obtained in (2.1) above, as described below. D-biotin-AC5-NHS and D-biotin-PE-maleimide were purchased from Dojin Chemical Laboratories Co., Ltd. (Biotin-AC5-OSu: product code B305, Biotin-PEAC5-maleimide: product code B299). In this specification, the notation NHS in compound names and chemical formulas is synonymous with Osu.

[0107] (3.1.1) Synthesis of L-biotin-AC5-NHS Using L-biotin (compound 1), 2,5-dioxopyrrolidine-1-yl 5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate (compound 2) was synthesized according to the following synthetic scheme. Specifically, the procedure was as follows: L-biotin (123 mg, 0.503 mmol) and NHS (69.4 mg, 0.603 mmol) were dissolved in N,N-dimethylformamide (DMF) (3.4 mL), and ethyl (dimethylaminopropyl)carboxydiimide (EDC) (116 mg, 0.603 mmol) was added, and the mixture was stirred at room temperature (rt) for 24 hours. The solvent was evaporated, and the resulting residue was recrystallized from ethanol:acetic acid:water (95:1:4 v / v) to obtain compound 2 (166 mg).

[0108] [ka]

[0109] Using compound 2, 6-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide)hexanoic acid (compound 3) was synthesized according to the following synthesis scheme. Specifically, the following steps were taken: Compound 2 (166 mg, 0.458 mmol) was dissolved in DMF (2.6 mL). 6-aminohexanoic acid (63.0 mg, 0.481 mmol) was dissolved in 0.25 M aqueous sodium carbonate solution (1.0 mL). This solution was added to the DMF solution of compound 2 and stirred at room temperature for 24 hours. The solvent was evaporated, and the resulting residue was dissolved in water. The aqueous layer was acidified with 4 M hydrochloric acid at 0°C, and the precipitate was collected by filtration to obtain compound 3 (159 mg).

[0110] [ka]

[0111] Using compound 3, L-biotin-AC5-NHS, i.e., 2,5-dioxopyrrolidine-1-yl 6-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide)hexanoate ester (compound 4), was synthesized according to the following synthesis scheme. Specifically, the procedure was as follows: Compound 3 (159 mg, 0.408 mmol) and NHS (93.9 mg, 0.816 mmol) were dissolved in DMF (7.6 mL), and EDC (157 mg, 0.816 mmol) was added and the mixture was stirred at room temperature. The reaction mixture was stirred at 35°C for 23 hours. The solvent was evaporated, and the resulting residue was purified by flash column chromatography (CH2Cl2 / methanol (8:1 v / v)) to obtain compound 4 (141 mg).

[0112] [ka]

[0113] The results of the NMR and mass spectrometry analysis of compound 4 were as follows: 1H NMR (400 MHz, DMSO-d6) δ 7.75 (t, J = 1.8 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 4.32-4.28 (m, 1H), 4.14-4.10 (m, 1H), 3.12-3.07 (m, 1H), 3.04-2.98 (m, 2H), 2.85-2.78 (m, 1H), 2.81 (s, 4H), 2.65 (t, J = 7.2 Hz, 2H), 2.57 (d, J = 12.8 Hz, 1H), 2.04 (t, J = 7.2 Hz, 2H), 1.65-1.22 (m, 12H). ESI-MS m / z 455.1967 [M+H] + (calcd for C 20 H 31 N4O6S, 455.1959).

[0114] (3.1.2) Synthesis of L-biotin-PE-maleimide (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-4,7-epoxyisobenzofuran-1,3-dione (compound 7) was synthesized using maleic anhydride (compound 5) and furan (compound 6) according to the following synthesis scheme. Specifically, the following steps were taken: Maleic anhydride (4.0 g, 40.0 mmol) was dissolved in ethyl acetate (20.0 mL), and furan (4.0 mL, 63.0 mmol) was added and the mixture was vigorously stirred. The reaction mixture was stirred at room temperature for 48 hours. The precipitate was collected by filtration and washed with ethyl acetate to obtain compound 7 (4.03 g).

[0115] [ka]

[0116] (3aR,4S,7R,7aS)-2-(2-(piperazine-1-yl)ethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3-(2H)dione (compound 9) was synthesized using compound 7 and N-(2-aminoethyl)piperazine (compound 8) according to the following synthesis scheme. Specifically, the following was performed: Compound 7 (600 mg, 3.61 mmol) was dissolved in ethanol (6.0 mL), compound 8 (0.520 mL, 3.97 mmol), triethylamine (0.520 mL, 3.97 mmol), and ethanol (1.2 mL) were added, and the mixture was stirred at 0°C for 30 minutes. The reaction mixture was heated to 70°C and stirred at 70°C for 15 hours. The solvent was evaporated, and the resulting residue was dissolved in ethyl acetate. The organic layer was washed with water, dried over Na2SO4, and filtered through Celite. The filtered product was concentrated to obtain compound 9 (518 mg).

[0117] [ka]

[0118] Using L-biotin (compound 1) and compound 9, (3aR,4S,7R,7aS)-2-(2-(4-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)piperazine-1-yl)ethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)dione (compound 10) was synthesized according to the following synthetic scheme. Specifically, it was as follows: L-biotin (89.6 mg, 0.367 mmol), compound 9 (123 mg, 0.442 mmol), and 4-(N,N-dimethylamino)pyridine (DMAP) (8.60 mg, 70.0 μmol) were dissolved in DMF (2.4 mL), and EDC (84.7 mg, 0.442 mmol) was added. The mixture was stirred at room temperature for 15 hours. The solvent was evaporated, and the resulting residue was purified by flash column chromatography (CH2Cl2 / methanol (8:1 v / v)) to obtain compound 10 (102 mg).

[0119]

Chem.

[0120] Using Compound 10, L-biotin-PE-maleimide, i.e., 1-(2-(4-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)piperazin-1-yl)ethyl)-1H-pyrrole-2,5-dione (Compound 11) was synthesized according to the following synthetic scheme. Specifically, it was as follows. Compound 10 (102 mg, 0.200 mmol) was dissolved in toluene (20.0 mL), and the solution was refluxed for 3 hours. The solvent was evaporated, and the resulting residue was purified by flash column chromatography (CH2Cl2 / methanol (8:1 v / v)) to obtain Compound 11 (70.0 mg).

[0121]

Chem.

[0122] The results of NMR analysis and mass spectrometry of Compound 11 were as follows. 1 H NMR (400 MHz, DMSO-d6) δ 7.01 (s, 2H), 6.42 (s, 1H), 6.34 (s, 1H), 4.28 - 4.24 (m, 1H), 4.11 - 4.08 (m, 1H), 3.55 (t, J = 6.4 Hz, 2H), 3.37 - 3.26 (m, 4H), 3.08 - 3.03 (m, 1H), 2.80 - 2.76 (m, 1H), 2.58 - 2.52 (m, 1H), 2.43 (t, J = 6.4 Hz, 2H), 2.34 - 2.32 (m, 2H), 2.28 - 2.26 (m, 2H), 2.29 (t, J = 7.6 Hz, 2H), 1.61 - 1.24 (m, 6H). [α] 15 = -54.6° (C = 1.00, MeOH). ESI-MS m / z found 436.1995 [M+H]+ (calcd for C 20 H 31 N4O6S, 436.2013).

[0123] (3.2) Preparation of samples containing biotin-labeled polypeptides (3.2.1) Preparation of biotin-labeled BSA L-biotin-AC5-NHS (32.5 mg) was dissolved in DMF (0.65 mL) to obtain an L-biotin-AC5-NHS solution. BSA (5.63 g: Proliant) was dissolved in 0.1 M phosphate buffer (pH 7.5, 112.5 mL) to obtain a BSA solution. The BSA solution (0.65 mL) and the L-biotin-AC5-NHS solution (0.65 mL) were mixed and allowed to stand at 35°C for 1 hour. The reaction mixture was passed through a PD-10 column (Cytiva) equilibrated with 0.1 M phosphate buffer (pH 7.5), and 500 μL of each fraction was collected. The absorbance of each fraction was measured, and the fraction with an absorption peak at 280 nm was used as the sample containing L-biotin-labeled BSA for subsequent experiments. A sample containing D-biotin-labeled BSA was obtained in the same manner as described above, except that D-biotin-AC5-NHS was used instead of L-biotin-AC5-NHS.

[0124] (3.2.2) Preparation of biotin-labeled antibodies L-biotin-PE-maleimide (10 mg) was dissolved in dimethyl sulfoxide (DMSO) (1.7 mL) to obtain an L-biotin-PE-maleimide solution. Anti-TSH antibody F(ab')2 was obtained from mouse anti-thyroid-stimulating hormone (TSH) monoclonal antibody (Kitayama Labes Co., Ltd.) by a conventional method using pepsin. EDTA-2 sodium salt and sodium hydroxide were dissolved in ultrapure water to prepare a 0.1 M ethylenediaminetetraacetic acid (EDTA) solution. Phosphorylated monosodium dihydrate, EDTA-2 sodium salt, and sodium hydroxide were dissolved in ultrapure water to prepare a gel filtration buffer. The obtained anti-TSH antibody F(ab')2 was mixed with 0.1 M EDTA solution and gel filtration buffer to obtain a solution with an antibody concentration of 8 mg / mL. 0.3 M 2-mercaptoethylamine (MEA) solution was added to this solution to reduce the disulfide bonds of the antibody. The reaction mixture was passed through a PD-10 column (Cytiva) equilibrated with 0.1 M phosphate buffer (pH 7.5) to obtain a fraction with an absorption peak at 280 nm. The antibody-containing fraction (500 μL) was mixed with L-biotin-PE-maleimide (1.7 mL) and allowed to stand at 35°C for 1 hour. The reaction mixture was purified by ultrafiltration (cutoff molecular weight 30,000) and gel filtration to obtain a solution of L-biotin-labeled anti-TSH antibody Fab'. The obtained solution was used as a sample containing L-biotin-labeled antibody in subsequent experiments. A sample containing D-biotin-labeled antibody was obtained in the same manner as above, except that D-biotin-PE-maleimide was used instead of L-biotin-PE-maleimide.

[0125] 4. Measurement of the sample (4.1) Preparation of a calibration curve The reagent for L-biotin measurement (91.7 μL) was mixed with 0.1 M phosphate buffer (pH 7.5, 18.3 μL) or L-biotin standard solution (18.3 μL), and allowed to stand at room temperature for 5 minutes. The absorbance of the reaction mixture at 500 nm (A) was then measured. 500The absorbance was measured using a UV-2600 spectrophotometer (Shimadzu Corporation). Similar measurements were also performed using D-biotin reagents 1 and 2 and a D-biotin standard solution. A straight line was used as the calibration curve based on the least squares method. The absorbance measurement results are shown in Table 1. Examples of calibration curves for each reagent are shown in Figures 4A to 4D.

[0126] [Table 1]

[0127] As shown in Table 1, Figure 4A, and Figure 4B, avidin or streptavidin complexes with HABA exhibited absorption at a wavelength of 500 nm, and the absorbance decreased in a dependency of the D-biotin concentration in the standard solution. The coefficient of determination (R) of the calibration curve was calculated. 2 The coefficient of determination of the calibration curve for the L-biotin measurement reagent was 0.9997, indicating a good correlation between D-biotin concentration and absorbance. Therefore, D-biotin measurement reagents 1 and 2 were able to reproduce commercially available biotin quantification reagents as shown in Non-Patent Literature 1. Referring to Table 1, Figure 4C, and Figure 4D, the complex of optically isomerized streptavidin and HABA also exhibited absorption at a wavelength of 500 nm, and the absorbance decreased in a manner dependent on the L-biotin concentration of the standard solution. The coefficient of determination of the calibration curve for the L-biotin measurement reagent was also 0.9997, indicating a good correlation between L-biotin concentration and absorbance. Therefore, it was demonstrated that the concentration of free L-biotin in a sample can be quantified using a reagent containing optically isomerized streptavidin and HABA.

[0128] (4.2) Determination of biotin concentration in the sample The sample containing L-biotin-labeled BSA prepared in (3.2.1) above was diluted 100-fold with 0.1 M phosphate buffer (pH 7.5). The reagent for L-biotin measurement (91.7 μL) was mixed with the diluted sample containing L-biotin-labeled BSA (18.3 μL) or the sample containing L-biotin-labeled antibody (18.3 μL), and allowed to stand at room temperature for 5 minutes. Then, the reaction mixture A 500The amount of L-biotin (μM) in each sample was determined from the calibration curve (regression equation) for the L-biotin measurement reagent. Similarly, the D-biotin concentration (μM) in each sample was determined from the calibration curve by measuring with D-biotin measurement reagent 1 and a sample containing D-biotin-labeled BSA or D-biotin-labeled antibody. 500 The results of the biotin concentration measurements are shown in Table 2. For samples containing L-biotin-labeled BSA and D-biotin-labeled BSA, the biotin concentration was calculated by multiplying the value determined from the calibration curve by 100.

[0129] [Table 2]

[0130] As can be seen from Table 2, the D-biotin concentration in samples containing D-biotin-labeled BSA and D-biotin-labeled antibody could be measured using D-biotin measurement reagent 1. Similarly, the L-biotin concentration in samples containing L-biotin-labeled BSA and L-biotin-labeled antibody could be measured using the L-biotin measurement reagent. Therefore, it was suggested that the concentration of conjugated L-biotin in a sample can be measured using the L-biotin measurement reagent, similar to the D-biotin measurement reagent.

[0131] 5. Determination of the number of biotin groups in biotin-labeled polypeptides. To determine the protein concentration (BSA or Fab' concentration) of the above samples, the absorbance at 280 nm (A) of each sample was measured. 280 The A of BSA solution (1 mg / mL) was measured using a NanoDrop® spectrophotometer (Thermo Fisher Scientific). 280 Assuming 0.63, the A of the sample containing D-biotin-labeled BSA and the sample containing L-biotin-labeled BSA 280From the measured values, the BSA concentration (mg / mL) in each sample was calculated. Assuming the molecular weight of BSA is 66200, the BSA concentration (mg / mL) in each sample was converted to molar concentration (μM). Similarly, the A of Fab' solution (1 mg / mL) was calculated. 280 Using 1.38, the A of the sample containing D-biotin-labeled antibody and the sample containing L-biotin-labeled antibody 280 The Fab' concentration (mg / mL) in each sample was calculated from the measured values. Assuming a molecular weight of Fab' of 46000, the Fab' concentration (mg / mL) in each sample was converted to molar concentration (μM). For each sample, the number of biotin groups in one biotin-labeled polypeptide molecule (hereinafter also referred to as the number of labels) was calculated by dividing the biotin concentration determined in step 4 above by the molar concentration of BSA or Fab'. The results are shown in Table 3.

[0132] [Table 3]

[0133] As shown in Table 3, the number of D-biotin groups in a D-biotin-labeled polypeptide could be determined from the D-biotin concentration in the sample determined by D-biotin measurement reagent 1 and the protein concentration in the same sample. Similarly, the number of L-biotin groups in an L-biotin-labeled polypeptide could be determined from the L-biotin concentration in the sample determined by L-biotin measurement reagent and the protein concentration in the same sample. Therefore, it was suggested that the number of L-biotin-labeled substances in a sample can be measured using the L-biotin measurement reagent, similar to the D-biotin measurement reagent.

[0134] Example 2: Manufacturing and Use of Immunological Measurement Reagent Kits An immunoassay reagent kit was prepared using L-biotin-labeled BSA and L-biotin-labeled antibody, which were confirmed to meet the specified number of labels in Example 1. The TSH level in the sample was then measured using this reagent kit.

[0135] 1. Preparation of a solid phase immobilized with optically isomerized streptavidin. Magnetic particles MAG2201 (2 g: JSR Corporation) were washed with 20 mM phosphate buffer (pH 6.0, 4 mL). These magnetic particles had carboxyl groups as functional groups on their surface. A solution of an activator to convert the carboxyl groups to NHS esters was prepared by dissolving N-hydroxysuccinimide (6.2 g) and carbodiimide (19.2 g) in 20 mM phosphate buffer (pH 6.0, 2000 mL). The magnetic particles were magnetically separated, the supernatant was removed, and the activator solution (4 mL) was added. The mixture was stirred at 300 rpm for 15 minutes at 15°C to 30°C. The magnetic particles were magnetically separated, the supernatant was removed, and the magnetic particles were washed with 20 mM phosphate buffer (pH 6.0, 4 mL). The magnetic particles were magnetically separated, the supernatant was removed, and 20 mM phosphate buffer (pH 7.5, 3.6 mL) was added and the mixture was stirred. To a suspension of magnetic particles, L-biotin-labeled albumin (10 mg / mL, 0.4 mL) prepared in Example 1 was added and the mixture was stirred at 300 rpm for 120 minutes at 15°C to 30°C. This caused the NHS ester on the magnetic particles to bind to the albumin portion of the L-biotin-labeled albumin, labeling the surface of the magnetic particles with biotin. The magnetic particles were magnetically separated, the supernatant was removed, and the magnetic particles were washed with 20 mM phosphate buffer (pH 7.5, 3.6 mL). 20 mM phosphate buffer (pH 7.5, 2 mL) was added to the magnetic particles. Optically isomerized streptavidin (544 mg) was dissolved in 20 mM phosphate buffer (pH 6.0, 1000 mL) to obtain an optically isomerized streptavidin solution. A suspension of magnetic particles was mixed with 3.6 mL of optically isomerized streptavidin solution and stirred at 300 rpm for 10 minutes at 15°C to 30°C. This caused L-biotin on the magnetic particles to bind to the optically isomerized streptavidin, immobilizing the optically isomerized streptavidin on the surface of the magnetic particles. The magnetic particles were magnetically separated, the supernatant was removed, and the magnetic particles were washed with magnetic particle preservation buffer (MES buffer (pH 6.5)). Magnetic particle preservation buffer was added to the magnetic particles to obtain 18 mL of magnetic particles immobilized with optically isomerized streptavidin.

[0136] 2. Preparation of each reagent in the immunoassay reagent kit. As the R1 reagent (capture antibody reagent), a solution of the L-biotin-labeled anti-TSH antibody Fab' prepared in Example 1 was used. As the R2 reagent (solid phase), a suspension of magnetic particles immobilized with optically isomerized streptavidin, prepared in 1. above, was used. As the R3 reagent (detection antibody reagent), HISCL™ TSH R3 reagent (Sysmex Corporation), containing an ALP-labeled mouse anti-TSH antibody monoclonal antibody, was used. As the R4 reagent (measurement buffer), HISCL R4 reagent (Sysmex Corporation) was used. As the R5 reagent (substrate solution), HISCL R5 reagent (Sysmex Corporation), containing CDP-Star™, was used. As the dilution buffer, HISCL sample diluent (Sysmex Corporation) was used. As the washing solution, HISCL washing solution (Sysmex Corporation) was used. For comparison, a commercially available TSH measurement kit, HISCL TSH reagent (Sysmex Corporation), was used. This reagent included HISCL TSH R1 reagent containing D-biotin-labeled anti-TSH antibody, HISCL TSH R2 reagent containing streptavidin-immobilized magnetic particles, HISCL® TSH R3 reagent, HISCL R4 reagent, and HISCL R5 reagent.

[0137] 3. Measurement of TSH The HISCL TSH calibrator (Sysmex Corporation) was used as the sample. The calibrator is a kit consisting of six samples containing six different concentrations of TSH. The TSH in each sample was measured using the HISCL-5000 fully automated immunoassay analyzer (Sysmex Corporation). The specific procedure was as follows: Reagent R1 (30 μL) and diluted serum (30 μL) were mixed and incubated at 42°C for 2 minutes. Reagent R2 (30 μL) was added to the reaction mixture and mixed, and incubated at 42°C for 2.5 minutes. Reagent R3 (30 μL) was added to the reaction mixture and mixed, and incubated at 42°C for 2.5 minutes. After that, magnetic particles were separated by magnetic separation and the supernatant was removed, and the magnetic particles were washed with washing solution (300 μL). Washing was performed four times. After removing the supernatant, R4 reagent (50 μL) was added to the magnetic particles and mixed, then R5 reagent (50 μL) was added and mixed, and the mixture was incubated at 42°C for 5 minutes. The luminescence intensity of the reaction mixture was then measured. For comparison, TSH was measured using HISCL TSH reagent and HISCL-5000 according to the instructions provided with the reagents. Each sample constituting the calibrator was measured three times consecutively. The average value of the luminescence count obtained from the three measurements was calculated and used as the measured value.

[0138] 4.Results Figure 5 shows a graph plotting the measured values ​​obtained by measuring the calibrator using the reagent kit of Example 2, which contains L-biotin-labeled anti-TSH antibody Fab' and magnetic particles immobilized with optically isomerized streptavidin, against the measured values ​​obtained using the HISCL TSH reagent. As can be seen from Figure 5, the measurement results using the reagent kit of Example 2 showed good linearity, similar to the measurement results using commercially available reagent kits. [Explanation of symbols]

[0139] 10, 20: Reagents 11, 21: 1st container 12, 23: Packaging box 13, 24: Package insert 22: Second container 30: Complex of optically isomerized avidins (tetramers) and HABA 31: Optical isomer avidin (tetramer) 32: HABA 33: L-biotin-labeled polypeptide 34: Complex of optically isomerized avidins (tetramers) with HABA and L-biotin-labeled polypeptides 40: Spherical particles 41: polypeptide portion of L-biotin-labeled polypeptide 42: L-biotin group 43: Optical isomer avidin (tetramer)

Claims

1. A reagent for measuring L-biotin, comprising optically isomerized avidins and 4'-hydroxyazobenzene-2-carboxylic acid, The optically isomerized avidins are - A polypeptide comprising a subsequence consisting of at least the 13th to 133rd amino acid residues of the amino acid sequence of SEQ ID NO: 1, wherein all amino acid residues other than glycine in the subsequence are D-amino acid residues. - A polypeptide comprising a core sequence consisting of at least the 2nd to 128th amino acid residues of the amino acid sequence of Sequence ID No. 2, wherein all amino acid residues other than glycine in the core sequence are D-amino acid residues. - A polypeptide comprising a core sequence consisting of at least the 4th to 129th amino acid residues of the amino acid sequence of Sequence ID No. 3, wherein all amino acid residues other than glycine in the core sequence are D-amino acid residues. - A polypeptide comprising a core sequence consisting of at least the 4th to 127th amino acid residues of the amino acid sequence of Sequence ID No. 4, wherein all amino acid residues other than glycine in the core sequence are D-amino acid residues. - A polypeptide comprising a subsequence consisting of at least the 13th to 133rd amino acid residues of the amino acid sequence of Sequence ID No. 5, wherein all amino acid residues other than glycine in the subsequence are D-amino acid residues. - A polypeptide comprising a partial sequence consisting of at least the 13th to 133rd amino acid residues of the amino acid sequence of Sequence ID No. 6, wherein all amino acid residues other than glycine in the partial sequence are D-amino acid residues. - A polypeptide comprising the amino acid sequence of Sequence ID No. 7, wherein all amino acid residues other than glycine in the sequence are D-amino acid residues. - A polypeptide comprising a subsequence of at least positions 13 to 125, at least positions 19 to 131, or at least positions 13 to 131 of the amino acid sequence of Sequence ID No. 8, wherein all amino acid residues other than glycine in the subsequence are D-amino acid residues, and - A polypeptide comprising a subsequence of at least positions 1-24, 29-202, and 207-261 of the amino acid sequence of Sequence ID No. 9, wherein all amino acid residues other than glycine in the subsequence are D-amino acid residues. A reagent for measuring L-biotin, which is at least one selected from the group consisting of the following.

2. A step of preparing a sample for measurement by mixing a sample containing L-biotin with the L-biotin measurement reagent described in claim 1, A step of measuring the absorbance of the sample to be measured, The absorbance measurement is an indicator of the concentration of L-biotin in the sample. Method for measuring samples containing L-biotin.

3. The measurement method according to claim 2, further comprising the step of determining the concentration of L-biotin in the sample based on the absorbance measurement value.

4. A step of preparing a measurement sample by mixing a sample containing an L-biotin-labeled substance with the L-biotin measurement reagent described in claim 1, A step of measuring the absorbance of the sample to be measured, A step of determining the concentration of L-biotin in the sample based on the absorbance measurement, A step of determining the number of L-biotin groups per molecule of the L-biotin-labeled substance based on the concentration of L-biotin in the sample and the concentration of the substance, A method for determining the number of labels on an L-biotin-labeled substance, including [specific substance].

5. The determination method according to claim 4, further comprising the step of determining the concentration of the substance in the sample.

6. The method for determining the number of L-biotin groups per molecule of the L-biotin-labeled substance according to claim 4 or 5, wherein the number of L-biotin groups per molecule is calculated by the following formula. X = A / B (In the formula, X is the number of L-biotin groups per molecule of the L-biotin-labeled substance, A is the molar concentration of L-biotin in the sample, (B is the molar concentration of the substance in the sample.)

7. The method according to any one of claims 4 to 6, wherein the L-biotin-labeled substance is an L-biotin-labeled polypeptide.

8. The method according to claim 7, wherein the L-biotin-labeled polypeptide is an L-biotin-labeled antibody.

9. A step of preparing a measurement sample by mixing a sample separated from a liquid containing an L-biotin-labeled polypeptide with the L-biotin measurement reagent described in claim 1, A step of measuring the absorbance of the sample to be measured, A step of determining the concentration of L-biotin in the measurement sample based on the absorbance measurement value, A step of determining the number of L-biotin groups per molecule of the L-biotin-labeled polypeptide in the sample based on the concentration of L-biotin and the concentration of the polypeptide, When the number of L-biotin groups is within a predetermined range, the liquid is brought into contact with a solid phase capable of binding to the polypeptide to immobilize the L-biotin-labeled polypeptide onto the solid phase. A step of contacting the solid phase on which the L-biotin-labeled polypeptide is immobilized with optically isomerized avidins to immobilize the optically isomerized avidins on the solid phase, A method for producing a solid phase in which optically isomerized avidins are immobilized.

10. The manufacturing method according to claim 9, further comprising the step of determining the concentration of the polypeptide in the sample.

11. The manufacturing method according to claim 9 or 10, wherein the number of L-biotin groups per molecule of the L-biotin-labeled substance is calculated by the following formula. X = A / B (In the formula, X is the number of L-biotin groups per molecule of the L-biotin-labeled substance, A is the molar concentration of L-biotin in the sample, (B is the molar concentration of the substance in the sample.)

12. The method for producing an L-biotin-labeled polypeptide according to any one of claims 9 to 11, wherein the L-biotin-labeled polypeptide is L-biotin-labeled albumin.

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