Methods and reagent kits for measuring polypeptides, polymers, solid phases, and test substances.

Abstract

The present invention addresses the problem of providing a polypeptide with further reduced binding properties with respect to D-biotin, and which strongly binds to L-biotin. Provided is a polypeptide belonging to the avidin-streptavidin family, wherein of amino acid residues other than glycine in the polypeptide, not less than 90% of the amino acid residues include a D-amino acid residue, the polypeptide having binding capacity with respect to L-biotin and having substantially no binding capacity with respect to D-biotin.

Description

[Technical Field]

[0001] This invention relates to polypeptides, polymers, solid phases, methods for measuring test substances, and reagent kits. [Background technology]

[0002] The affinity between polypeptides belonging to the avidin-streptavidin family and biotin is very strong, and these polypeptides and biotin are used in various fields. For example, in the diagnostic field, in immunological assays, a capture substance is immobilized on a solid phase such as a plate or particles via streptavidin and biotin, and this is used to measure the target substance. In the therapeutic field, pretargeting therapy is known in which an antibody with an avidin tag and a drug with a biotin tag are administered to the patient (for example, Non-Patent Literature 1).

[0003] However, if the subject's blood contains naturally occurring D-biotin, this D-biotin (endogenous D-biotin) can bind to avidin or streptavidin. In particular, if the subject is taking supplements such as vitamins, the blood concentration of D-biotin may be high. In such cases, in immunological measurement methods using blood samples, biotinylated captures and endogenous D-biotin in the blood compete, and some captures may not be immobilized on the solid phase, potentially reducing measurement accuracy. Furthermore, in pretargeting therapy, when an antibody conjugated with avidin is administered to a patient, endogenous D-biotin in the blood may bind to the avidin. In this case, a drug with a biotin group attached to the antibody may not be able to bind, potentially reducing the amount of drug that reaches the affected area. As a means to solve these problems, for example, the technology described in Patent Document 1 is known. Patent Document 1 discloses a streptavidin variant with reduced affinity for D-biotin and a biotin variant that can bind to the variant. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent Application Publication No. 2016 / 0137704 [Non-patent literature]

[0005] [Non-Patent Document 1] Hnatowich et al., Journal of Nuclear Medicine 28.8(1987):1294-1302 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The streptavidin variant described in Patent Document 1 still maintains its ability to bind to D-biotin, and the affinity between the variant and the biotin variant is insufficient. The present invention aims to provide a polypeptide that does not substantially bind to D-biotin but strongly binds to L-biotin. The present invention also aims to provide a reagent kit containing a polymer of the polypeptide, a solid phase on which the polypeptide is immobilized, a method for measuring a test substance using the solid phase, and a capture body to which the solid phase and L-biotin are bound. [Means for solving the problem]

[0007] The present invention provides a polypeptide belonging to the avidin-streptavidin family, wherein 90% or more of the amino acid residues other than glycine in the polypeptide are D-amino acid residues, and which has the ability to bind to L-biotin but substantially lacks the ability to bind to D-biotin.

[0008] The present invention provides the polypeptide and the polypeptide polymer. The present invention also provides a solid phase on which the polypeptide and the polypeptide polymer are immobilized, a measurement method using the solid phase, and a reagent kit comprising a capture body to which the solid phase and L-biotin are bound. [Effects of the Invention]

[0009] The present invention provides a polypeptide that does not substantially bind to D-biotin but has the ability to bind to L-biotin. The present invention also provides a reagent kit comprising a polymer of the polypeptide, a solid phase to which the polypeptide is bound, a measurement method using the solid phase, and a capture body to which the solid phase and L-biotin are bound. [Brief explanation of the drawing]

[0010] [Figure 1] This is a schematic diagram showing an example of the measurement method of this embodiment. [Figure 2] This is a schematic diagram showing an example of the reagent kit of this embodiment. [Figure 3] This graph shows the absorbance transition at a wavelength of 280 nm in gel filtration chromatography. [Figure 4A] This is a sensorgram of surface plasmon resonance (SPR) between the polypeptide of Example 2 and L-biotin. [Figure 4B] This is a sensorgram of surface plasmon resonance (SPR) between the polypeptide of Example 2 and D-biotin. [Figure 5] This is a sensorgram of surface plasmon resonance (SPR) between natural core streptavidin and D-biotin-labeled albumin from Example 2. [Figure 6] This graph shows the results of Example 3. [Figure 7] This graph shows the results of Example 4. [Figure 8A] This is the structural model of Example 5 before substituting the 21st amino acid, which is involved in maintaining the structure, into the L-form. [Figure 8B] This is a structural model of Example 5 after substituting the L-form with the 21st amino acid, which is involved in maintaining the structure. [Figure 8C] This is a structural model obtained by Clean Geometry after substituting the 21st amino acid, which is involved in maintaining the structure of Example 5, with the L-isomer. [Figure 9A]Sensorgram of surface plasmon resonance (SPR) between D-Tamavidin (trademark) 2 of Example 7 and L-biotin-labeled albumin. [Figure 9B] Sensorgram of surface plasmon resonance (SPR) between D-Tamavidin (trademark) 2 of Example 7 and D-biotin-labeled albumin. [Figure 10] Graph showing the results of Example 8. [Figure 11] Graph showing the results of Example 9. [Figure 12A] Structure of L-biotin. [Figure 12B] Structure of D-biotin.

Mode for Carrying Out the Invention

[0011] 1. Polypeptide The polypeptide of the present embodiment belongs to the avidin-streptavidin family. Among the amino acid residues other than glycine in this polypeptide, 90% or more of the amino acid residues are D-amino acid residues. This polypeptide is a polypeptide having a binding ability to L-biotin and substantially no binding ability to D-biotin.

[0012] Polypeptides of the avidin-streptavidin family are composed of L-amino acid residues in nature, except for glycine which has no optical isomers. The polypeptide of the present embodiment may contain L-amino acid residues, but it is preferable that 90% or more of the amino acid residues other than glycine are D-amino acid residues. In a more preferred embodiment of the polypeptide, 95% or more of the amino acid residues other than glycine are D-amino acid residues, and in an even more preferred embodiment of the polypeptide, all amino acid residues other than glycine are D-amino acid residues. As a result, it is considered that at least the three-dimensional structure of the site that binds to D-biotin becomes an enantiomer. As a result, the binding property to natural D-biotin is reduced, and it becomes possible to bind to L-biotin which is an enantiomer of D-biotin.

[0013] The polypeptide of this embodiment does not substantially bind to D-biotin even if D-biotin is present in the subject's blood, thus reducing the influence of endogenous D-biotin in immunological assays. Furthermore, in pretargeting therapy, the influence of endogenous D-biotin is reduced, allowing for more appropriate delivery of the drug to the affected area.

[0014] In this specification, "polypeptide" includes proteins and their fragments. The length of the amino acids in the polypeptide of this embodiment is not particularly limited as long as it has the ability to bind to L-biotin, and is, for example, several tens to several hundred residues. The polypeptide of this embodiment preferably has at least a core sequence. Here, "core sequence" refers to the amino acid sequence of the polypeptide of this embodiment that is necessary for binding to biotin.

[0015] The "polypeptides belonging to the avidin-streptavidin family" include avidin, streptavidin, avidin-like protein derived from Pleurotus cornucopiae (hereinafter referred to as Tamavidin®), bradavidin, rhizavidin, chimeras and variants thereof. In this specification, D-type polypeptides (for example, D-type avidin (hereinafter referred to as "D-avidin" or "avidin of this embodiment")), D-type streptavidin (hereinafter referred to as "D-streptavidin" or "streptavidin of this embodiment"), D-type Tamavidin® (hereinafter referred to as "D-Tamavidin®" or "Tamavidin® of this embodiment"), D-type bradavidin (hereinafter referred to as "D-bradavidin"), and D-type rhizavidin are also included in the "polypeptides belonging to the avidin-streptavidin family".

[0016] Modified polypeptides include, for example, polypeptides with altered amino acid sequences and polypeptides that have undergone chemical treatment. Examples of amino acid sequence alteration include substitution, deletion, and addition of amino acid residues. Examples of chemical treatment include deglycosylation.

[0017] Streptoavidin is a biotin-binding protein derived from Streptomyces avidinii.

[0018] In a preferred embodiment, D-streptavidin contains the amino acid sequence from positions 19 to 133 of SEQ ID NO: 1. The amino acid sequence from positions 19 to 133 is known as the core sequence of D-streptavidin. In a more preferred embodiment, D-streptavidin contains the amino acid sequence from positions 13 to 133 or from positions 19 to 133. In an even more preferred embodiment, D-streptavidin contains the amino acid sequence from positions 13 to 139. In another embodiment, D-streptavidin contains the entire amino acid sequence of SEQ ID NO: 1. In this embodiment, streptavidin has low binding affinity to D-biotin and high binding affinity to L-biotin because more than 90% of the amino acid residues other than glycine are D-amino acid residues.

[0019] Avidin is a biotin-binding protein produced in birds and other animals. One embodiment of D-avidin may contain the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, D-avidin contains the amino acid sequence from the 2nd to the 128th amino acid sequence of SEQ ID NO: 2. The amino acid sequence from the 2nd to the 128th amino acid sequence is considered to be the core sequence of D-avidin. In another embodiment, D-avidin contains the entire amino acid sequence of SEQ ID NO: 2. In this embodiment, more than 90% of the amino acid residues other than glycine are composed of D-amino acid residues, so it has low binding affinity to D-biotin and high binding affinity to L-biotin.

[0020] Tamavidin® is a protein discovered in the mushroom *Pleurotus ostreatus*. It has characteristics such as high affinity for biotin and superior thermal stability compared to avidin (International Publication No. 2002 / 072817). Examples of the amino acid sequence of Tamavidin® include the sequences shown in SEQ ID NO: 3 (Tamavidin® 1) and SEQ ID NO: 4 (Tamavidin® 2).

[0021] In a preferred embodiment, D-Tamavidin® 1 contains the amino acid sequence from position 4 to 129 of SEQ ID NO: 3. The amino acid sequence from position 4 to 129 is considered to be the core sequence of Tamavidin® 1. D-Tamavidin® 2 contains the amino acid sequence from position 4 to 127 of SEQ ID NO: 4. The amino acid sequence from position 4 to 127 is considered to be the core sequence of Tamavidin® 2. Since more than 90% of the amino acid residues other than glycine in D-Tamavidin® 1 and D-Tamavidin® 2 are D-amino acid residues, they have low binding affinity to D-biotin and high binding affinity to L-biotin.

[0022] The core sequence of the polypeptide in this embodiment may contain L-amino acid residues, but it is preferable that 90% or more of the amino acid residues other than glycine are D-amino acid residues. In a more preferred embodiment, 95% or more of the amino acid residues other than glycine in the core sequence are D-amino acid residues, and in an even more preferred embodiment, all amino acid residues other than glycine are D-amino acid residues.

[0023] The polypeptide of this embodiment may be a polypeptide that includes modifications of amino acids, such as substitution, deletion, or addition, compared to the amino acid sequence described above. A preferred embodiment is a polypeptide having 90% or more homology to the amino acid sequence described above, and a more preferred embodiment is a polypeptide having 95% or more homology to the amino acid sequence described above.

[0024] A specific example of a polypeptide with a modified amino acid sequence is the streptavidin variant described in Qureshi et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol.276, No.49, Issue of December 7, pp.46422-46428, 2001 (hereinafter referred to as "streptavidin variant 1"). Streptabidin variant 1 has substitution mutations at S45A, T90A, and D128A compared to the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 5). The homology between the core sequence of streptavidin and the core sequence of streptavidin variant 1 is 97.4%. One embodiment of streptavidin is the D-form streptavidin variant 1 (hereinafter referred to as "D-streptavidin variant 1"), which contains the amino acid sequence from positions 19 to 133 of SEQ ID NO: 5. The amino acid sequence from position 19 to 133 is known as the core sequence of streptavidin variant 1. In a more preferred embodiment, D-streptavidin variant 1 includes either the amino acid sequence from position 13 to 133 or the amino acid sequence from position 19 to 139. In an even more preferred embodiment, D-streptavidin variant 1 includes the amino acid sequence from position 13 to 139. In another embodiment, D-streptavidin variant 1 includes the entire amino acid sequence of SEQ ID NO: 5.

[0025] Another example of a streptavidin variant is the streptavidin variant described in Wu et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol.280, No.24, Issue of June 17, pp.23225-23231, 2005 (hereinafter referred to as "streptavidin variant 2"). Streptabidin variant 2 has substitution mutations at T76R, V125R, V55T, and L109T compared to the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 6). The homology between the core sequence of streptavidin and the core sequence of streptavidin variant 2 is 96.5%. One embodiment of streptavidin is the D-form streptavidin variant 2 (hereinafter referred to as "D-streptavidin variant 2"), which contains the amino acid sequence from positions 19 to 133 of SEQ ID NO: 6. The amino acid sequence from position 19 to 133 is known as the core sequence of streptavidin variant 2. In a more preferred embodiment, D-streptavidin variant 2 includes either the amino acid sequence from position 13 to 133 or the amino acid sequence from position 19 to 139. In an even more preferred embodiment, D-streptavidin variant 2 includes the amino acid sequence from position 13 to 139. In another embodiment, D-streptavidin variant 2 includes the entire amino acid sequence of SEQ ID NO: 6.

[0026] Another example of a streptavidin variant is the streptavidin variant described in Lim et al., BIOTECHNOLOGY AND BIOENGINEERING, 2013 Jan;110(1):57-67 (hereinafter referred to as "streptavidin variant 3"). This protein has the amino acid sequence of SEQ ID NO: 7. In one embodiment, the streptavidin is preferably the D-streptavidin variant 3 (hereinafter referred to as "D-streptavidin variant 3"), which contains the entire amino acid sequence of SEQ ID NO: 7.

[0027] Another example of a streptavidin variant is the streptavidin variant described in Sano et al., Proc. Natl. Acad. Sci. USA Vol. 94, pp. 6153-6158, June 1997 (hereinafter referred to as "streptavidin variant 4"). Streptabidin variant 4 has a substitution mutation at H127D and a deletion mutation at G113-W120 compared to the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 8). The homology between the core sequence of streptavidin and the core sequence of streptavidin variant 2 is 92.2%. One embodiment of streptavidin is the D-form streptavidin variant 4 (hereinafter referred to as "D-streptavidin variant 4"), which contains the amino acid sequence from positions 19 to 125 of SEQ ID NO: 8. The amino acid sequence from position 19 to 125 is known as the core sequence of streptavidin variant 4. In a more preferred embodiment, D-streptavidin variant 4 includes either the amino acid sequence from position 13 to 125 or the amino acid sequence from position 19 to 131. In an even more preferred embodiment, D-streptavidin variant 4 includes the amino acid sequence from position 13 to 131. In another embodiment, D-streptavidin variant 4 includes the entire amino acid sequence of SEQ ID NO: 8.

[0028] Another example of a streptavidin variant is the streptavidin variant described in International Publication No. 2006 / 058226 (hereinafter referred to as "Streptavidin Variant 5"). Streptavidin Variant 5 has the amino acid sequence of Sequence ID No. 9. In one embodiment, streptavidin is the D-streptavidin variant 5 (hereinafter referred to as "D-Streptavidin Variant 5"), which includes the amino acid sequences of positions 1-20, 35-196, and 213-261 of Sequence ID No. 9. The amino acid sequences of positions 1-20, 35-196, and 213-261 are known as sequences corresponding to the core sequence of streptavidin variant 5. In a more preferred embodiment, D-Streptavidin Variant 5 includes positions 1-24, 29-202, and 207-261. In another embodiment, D-streptavidin variant 5 contains the entire amino acid sequence of SEQ ID NO: 9.

[0029] In this specification, the notation D- or L- indicates the stereochemistry of a compound based on IUPAC nomenclature. d-glyceraldehyde is used as the stereochemistry reference, and compounds that maintain this stereochemistry are designated as D-isomers, while their enantiomers are designated as L-isomers.

[0030] The L-biotin binding partner of the polypeptide in this embodiment is an enantiomer of D-biotin. In this specification, "L-biotin" is a concept that includes both free L-biotin and L-biotin groups attached to other substances such as capture groups. L-biotin can be synthesized by known methods, such as those described in non-patent literature (Journal of the American Chemical Society 1978, 100, 1558-1563), and obtained by optical resolution using chiral column chromatography or the like. As long as L-biotin is an enantiomer of D-biotin, the manufacturing method is not particularly limited, and commercially available products may be used.

[0031] The binding affinity of the polypeptide in this embodiment to L-biotin and D-biotin can be confirmed, for example, by the signal in a plate assay using a biotinylating enzyme and polypeptide-immobilized plate, the change in the sensorgram in surface plasmon resonance (SPR), and the dissociation constant (Kd value). For measuring the dissociation constant, known methods such as SPR analysis or isothermal titration calorimetry can be used. An example of a measuring device is the Biacore T200 (Cytiva).

[0032] The dissociation constant between the polypeptide and L-biotin in this embodiment is preferably 10. -7 M is less than or equal to 10 -10 M is less than or equal to 10 -13 It is M or less.

[0033] "Substantially lacking the ability to bind to D-biotin" means, for example, that the dissociation constant between the polypeptide of this embodiment and D-biotin is preferably 10 -6 M or higher, more comfortable 10 -4 M or greater, and more preferably 10 -2 The result should be M or greater. In the most preferred embodiment, the polypeptide of this embodiment and D - When the binding ability to biotin was measured using Biacore T200 (Cytiva), no specific sensorogram changes due to analyte binding were observed. Note that "substantially lacks binding ability to D-biotin" is synonymous with "substantially does not bind to D-biotin."

[0034] The method for determining binding affinity using the Biacore T200 system is as follows: Bovine serum albumin is immobilized at a target level of 400 RU on flow cells 1 and 3 of the Biacore T200 system's CM5 sensor chip (Cytiva) using an amine coupling method with an amine coupling kit (Cytiva). Similarly, L-biotin-labeled albumin and D-biotin-labeled albumin are immobilized at a target level of 400 RU on flow cells 2 and 4 using the amine coupling method. Each flow cell is blocked with 1 M ethanolamine solution, pH 8.5 (Cytiva). For D-biotin-labeled albumin, the number of D-biotin labels per albumin is estimated to be 0.4 by the HABA method, and for L-biotin-labeled albumin, the number of D-biotin labels is estimated to be similar by preparing it under the same conditions as the D-biotin-labeled albumin.

[0035] Using HBS-EP+ (Cytiva) as the running buffer, polypeptides belonging to the avidin-streptavidin family are flowed through each flow cell at a flow rate of 30 μL / min in the range of 100 pM to 100 nM using a single-cycle method, and data showing the change in sensograms during SPR for D-biotin-labeled albumin or L-biotin-labeled albumin is acquired using Biacore T200 Evaluation Software (Cytiva). Since the data acquired from the flow cell immobilized with bovine serum albumin represents background values, the data showing the change in sensograms during SPR for D-biotin-labeled albumin or L-biotin-labeled albumin may be normalized using the background values. The binding affinity of polypeptides belonging to the avidin-streptavidin family can be determined, for example, based on [data showing the binding affinity of L-biotin-labeled albumin to polypeptides belonging to the avidin-streptavidin family] (hereinafter referred to as "Data L1") and [data showing the binding affinity of D-biotin-labeled albumin to polypeptides belonging to the avidin-streptavidin family] (hereinafter referred to as "Data D1").

[0036] For example, if the value of data D1 divided by the value of data L1 is 1 / 10 or less, 1 / 100 or less, or 1 / 1000, then polypeptides belonging to the avidin-streptavidin family can be determined to "substantially not bind to D-biotin."

[0037] For example, if the value obtained by dividing the value of data L1 by the value of data D1 is 10 or greater, 100 or greater, or 1000 or greater, then polypeptides belonging to the avidin-streptavidin family can be determined to "substantially not bind to D-biotin."

[0038] For example, if the absolute value of the difference between the value of data L1 and the value of data D1, or the absolute value of the difference between the value of data D1 and the value of data L1, is less than or equal to 1 / 10, less than or equal to 1 / 1000, or less than or equal to 1 / 1000 of the value of data L1, then polypeptides belonging to the avidin-streptavidin family can be determined to "not substantially bind to D-biotin."

[0039] In a plate assay using biotinylating enzymes and polypeptide-immobilized plates, a signal-based method for determining binding affinity is used. For example, based on Example 3 described later, a biotin measurement system using the ELISA method is constructed, and the enzyme activity value when using L-biotin (data L2) and the enzyme activity value when using D-biotin (data D2) are obtained. Similar to the case using the Biacore T200 system described above, based on data L2 and data D2, it is determined whether polypeptides belonging to the avidin-streptavidin family substantially bind to D-biotin.

[0040] 2. Multimer Another embodiment is a polymer in which the above-described polypeptide is used as a monomer unit. This polymer may be formed by the association of multiple above-described polypeptides. In this polymer, the number of monomer units is not particularly limited. For example, this polymer may be a dimer, a tetramer, or an octamer.

[0041] 3. Method for synthesizing polypeptides The polypeptide of this embodiment can be produced by known peptide synthesis methods. The peptide synthesis method is not particularly limited as long as the desired polypeptide can be obtained. Examples include liquid-phase synthesis, solid-phase synthesis, and cell-free synthesis using artificial tRNA. Furthermore, if the number of amino acid residues of the product polypeptide is above a certain number (generally 30 to 50 residues or more), it can be produced by synthesizing two or more peptide fragments and then linking the polypeptides using a known ligation reaction. The polypeptide of this embodiment 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) After removing the protecting group of the reactant obtained by the bonding reaction in (1) or (3) using a deprotecting agent, the reactant is washed with a solvent to form free amino acids. (3) The free amino acid obtained in (2) above and any amino acid whose amino group is protected by a protecting group are condensed using a condensing agent. (4) The protecting group of the product of (3) above is removed using a deprotecting agent to form free amino acids. (5) By repeating steps (2) through (4), a polypeptide can be obtained in which any amino acid is linked to a resin bonded to the C-terminus. (6) Wash with a solvent at any point after step (5), and once the resin to which the desired polypeptide has been bound has been produced. (7) After washing the resin-bound polypeptide in (6), the N-terminal amino group is protected with a protecting group, and then the resin is cleaved with an acid to obtain any polypeptide to which a protecting group is attached.

[0042] Each step may be performed manually, or an automated synthesizer may be used. Alternatively, some steps may be performed manually, with automated synthesizers used as appropriate. Examples of automated synthesizers include, but are not limited to, Liberty Blue (CEM), Multipep2 (CEM), Initiator+Alstra (Biotage), and SyrII (Biotage).

[0043] The resin used in (1) may 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.

[0044] Furthermore, when the C-terminus is a carboxylic acid, resins such as chlorine-functionalized 2-chlorotrityl chloride resin (Merck KGaA), amino-functionalized Amino-PEGA resin (Merck KGaA), NovaSyn TGT alcohol resin with hydroxyl groups (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, and examples of such linkers include 4-hydroxymethylphenoxyacetic acid (HMPA) and 4-(4-hydroxymethyl-3-methoxyphenoxy)-butylacetic acid (HMPB). H-Cys(Trt)-Trityl NovaPEG resin (Merck KGaA), in which the C-terminal amino acid is pre-bonded to the resin, can also be used.

[0045] 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 such that the carboxyl group of the amino acid is bonded to the resin by 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 by an amide bond.

[0046] Any known protecting group may be used as the protecting group. For example, carbonate-based or amide-based protecting groups such as 9-fluorenyl methoxycarbonyl (Fmoc) group, t-butyloxylcarbonyl (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-fluorenyl methoxycarbonyl-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.

[0047] 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.

[0048] In addition, 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( Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Sec(Trt)-OH , Fmoc-Sec(pMeOBzl)-OH, Fmoc-Sec(pMeBzl)-OH, Fmoc-HomoSec(pMeBzl)-OH, and Fmoc-HomoSec(Mob)-OH.

[0049] When using a resin containing hydroxyl groups, 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 acids to dehydration condensation agents used is usually 1 to 10 equivalents, preferably 1 to 5 equivalents, of the latter to 1 equivalent of the former.

[0050] 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, and the reaction time is approximately 10 minutes to 30 hours, preferably 15 minutes to 24 hours.

[0051] At this time, it is preferable to acetylate and cap the unreacted functional groups on the solid phase using acetic anhydride or the like.

[0052] The removal of lipophilic protecting groups can be carried out, for example, by treatment with a base. Examples of bases include piperidine and morpholine. It is preferable to carry out this treatment in the presence of a solvent. Examples of solvents include DMF, DMSO, and methanol.

[0053] 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.

[0054] 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 Pentaful. Examples include olophenol (Pfp-OH), 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).

[0055] The amount of activator used is preferably 1 to 20 equivalents, more 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.

[0056] 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).

[0057] Examples of solvents include DMF, DMSO, and DCM. The reaction temperature is 0 to 50°C, preferably room temperature, and the reaction time is approximately 10 minutes to 30 hours, preferably 15 minutes to 24 hours. The removal of the protecting group can be carried out in the same manner as described above. To cleave peptide chains from the resin, treatment with an acid is preferable. An example of such an acid is trifluoroacetic acid (TFA).

[0058] Native chemical ligation (NCL) can be used as a known ligation reaction for linking two or more peptide fragments (Dawson et al., Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).

[0059] 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 (SH group, also called 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 at the linking site, while simultaneously regenerating the cysteine ​​side chain thiol.

[0060] 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 (Yanet al., J. Am. Chem. Soc. 123, 526 (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.

[0061] The peptide synthesis or ligation reaction may include separation and / or purification steps before and after the reaction. The purification method can be any known method, such as column chromatography. Examples of column chromatography include, but are not limited to, 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 method for the substances to be separated and purified, temperature conditions, pressure conditions, etc., can be appropriately selected.

[0062] The peptide synthesis reaction, ligation reaction, or separation and / or purification step may optionally include known washing, drying, dilution, and concentration steps before and after the reaction.

[0063] If the polypeptide does not fold correctly, refolding is preferable. Refolding can be performed, for example, by dissolving the purified polypeptide in a denaturing buffer containing urea, guanidine hydrochloride, etc., or by adding a denaturing agent such as urea, guanidine hydrochloride, etc., to the crude purified solution of the fusion polypeptide to dissolve the aggregation of the fusion polypeptide, and then by methods such as the dilution refolding method, dialysis refolding method, solid-phase refolding method, size exclusion chromatography refolding method, surfactant refolding method, etc., as described in non-patent literature (Arakawa et al., Antibodies 232 (2014)). Dialysis refolding is preferred.

[0064] 4.Measurement method The measurement method of this embodiment is a method for measuring a test substance in a sample in vitro using a solid phase on which the above-mentioned polypeptide or its polymer is immobilized, and a capture body immobilized on the solid phase.

[0065] As the capture agent, a substance that specifically binds to the test substance can be used. The capture agent is directly or indirectly attached to L-biotin or a variant thereof. Examples of capture agents include, but are not limited to, antibodies, antigens, aptamers, lectins, nucleic acids, and enzymes.

[0066] The "antibody" used as a capture agent or detection agent (described later) may be a full-length antibody or a fragment thereof. The antibody class may be any of IgG, IgA, IgM, IgD, and IgE, but preferably IgG. The subclass of IgG is not particularly limited and may be any of IgG1, IgG2, IgG3, and IgG4. Examples of antibody fragments include reduced IgG (rIgG), Fab, Fab', F(ab'), F(ab')2, Fv, single-chain antibody (scFv), diabody, triabody, etc. Methods for preparing these antibody fragments are well known. The antibody may be either a monoclonal antibody or a polyclonal antibody, but preferably a monoclonal antibody. The monoclonal antibody may be a chimeric antibody, a humanized antibody, a fully humanized antibody, etc. The antibody may also be derived from any animal such as a mouse, rat, hamster, rabbit, goat, horse, camel, alpaca, or chicken.

[0067] The test substance is the substance to be detected by the measurement method of this embodiment, and can be any substance that can be captured by the capture body. Examples of test substances include, but are not limited to, cells, extracellular vesicles, proteins, nucleic acids, polysaccharides, glycoproteins, and phospholipids.

[0068] A specimen is a sample containing the test substance or a sample suspected of containing the test substance. Examples of specimens include biological samples, environmental samples, and samples that have undergone pretreatment. Examples of biological samples include body fluids and excrement. Examples of body fluids are not limited to samples collected from living organisms, but include serum, plasma, blood, cerebrospinal fluid, semen, tissue, tissue fluid, lymph, saliva, nasopharyngeal swabs, sputum, and bronchoalveolar lavage fluid. Examples of excrement are not limited to urine and feces. Examples of environmental samples include sewage, river water, seawater, and soil.

[0069] The polypeptide or its polymer described above is immobilized on a solid phase. The manner of immobilization is not particularly limited. For example, the polypeptide or its polymer may be directly bonded to the solid phase or indirectly bonded to it. Examples of direct bondation include physical adsorption. Indirect bondation is a manner in which another substance is interposed between the polypeptide or its polymer and the solid phase. For example, if the surface of the solid phase is coated with a blocking agent such as bovine serum albumin or polyethylene glycol, the polypeptide or its polymer can be immobilized on the solid phase by bonding the polypeptide or its polymer to the solid phase and the blocking agent.

[0070] In the measurement method of this embodiment, the capture body and the solid phase may be provided to the user separately, or the capture body and the solid phase may be provided to the user already immobilized on the solid phase. When the capture body and the solid phase are provided to the user separately, the measurement method of this embodiment may include a step of contacting the sample, the solid phase, and the capture body. In this step, the order of contact between the sample, the solid phase, and the capture body is not particularly limited. Preferably, the sample and the capture body are brought into contact to form a complex between the test substance in the sample and the capture body, and then the complex is brought into contact with the solid phase to form the complex on the solid phase. In this case, even if the sample contains D-biotin, the polypeptide or polymer immobilized on the solid phase will not substantially bind to the D-biotin, but will be able to bind to the capture body containing the L-biotin group. In this embodiment, the measurement method is preferable because the capture body and the solid phase are provided separately to the user, as this allows for the use of a common solid phase for multiple types of test substances.

[0071] The measurement method of this embodiment may include a step of forming a complex (hereinafter also referred to as a "sandwich complex") on a solid phase that includes a capture body, a test substance, and a detector (hereinafter also referred to as a "complex formation step"), and a step of measuring a signal based on the detector contained in this complex (hereinafter also referred to as a "measurement step").

[0072] In the complex formation step, a sandwich complex can be formed on the solid phase by contacting the solid phase, the capture body, the sample containing the test substance, and the detector. The order of contact between the solid phase, the capture body, the sample, and the detector is not particularly limited. Preferably, the sample and the capture body are brought into contact to form a complex between the test substance in the sample and the capture body, then the complex is brought into contact with the solid phase to form the complex on the solid phase, and then the complex is brought into contact with the detector to form a sandwich complex on the solid phase. In this case, even if the sample contains D-biotin, the polypeptide or polymer immobilized on the solid phase does not substantially bind to D-biotin but can bind to the capture body containing L-biotin groups. After forming the complex of the test substance and the capture body on the solid phase, it is preferable to perform B / F separation to remove unreacted components before contacting the detector. The reaction conditions for each component in the complex formation step (e.g., solvent, temperature, pressure, reaction time, etc.) can be appropriately selected by those skilled in the art.

[0073] The detection substrate preferably includes a substance that binds to the test substance and a labeling substance. Examples of substances that bind to the test substance include antibodies, antigens, aptamers, lectins, and nucleic acids. The detection substrate may also include a primary substance that binds to the test substance and a secondary substance that contains a labeling substance and binds to the primary substance. Examples of primary and secondary substances include antibodies, antigens, aptamers, lectins, and nucleic acids. When both the primary and secondary substances are antibodies, they are called primary antibodies and secondary antibodies, respectively, and are widely used in this industry. When primary and secondary antibodies are used, a sandwich complex containing a labeling substance can be formed by binding the primary antibody to the test substance and the secondary antibody to the primary antibody. Specifically, the primary substance is a primary antibody derived from an animal such as a mouse or rabbit that specifically binds to the test substance, and the secondary substance is a secondary antibody that contains a labeling substance and specifically binds to the antibody of the animal.

[0074] The labeling substance is not particularly limited. For example, it includes substances that generate signals by themselves (hereinafter also referred to as "signal-generating substances") and substances that catalyze the reaction of other substances to generate signals. Examples of signal-generating substances include fluorescent substances, radioactive isotopes, chromogenic substances, etc. Examples of substances that catalyze the reaction of other substances to generate detectable signals include enzymes. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of radioactive isotopes include 125 I, 14 C, 32 P, etc. Examples of chromogenic substances include metal colloids such as gold nanocolloids. Examples of enzymes include alkaline phosphatase, peroxidase, β-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase, luciferase, etc. A preferred labeling substance is an enzyme, and alkaline phosphatase is particularly preferred.

[0075] In the measurement step, the analyte can be measured by measuring the signal based on the detected substance. Based on the value obtained in the measurement step, quantitative, qualitative or semi-quantitative detection of the analyte can be performed. Here, semi-quantitative detection means indicating the intensity of the signal stepwise, such as "no signal generation", "weak", "medium", "strong", etc.

[0076] The method for detecting the signal itself is known in the art. In this embodiment, a method corresponding to the type of signal derived from the above labeling substance may be appropriately selected. The signal detection device can use, for example, an absorbance meter, a spectrophotometer, a fluorometer, an infrared spectrophotometer, a Raman spectrophotometer, an SPR measurement device, a chemiluminescent enzyme immunoassay device, etc., but is not particularly limited.

[0077] In one embodiment, the test substance in the sample is measured immunologically. Examples of immunological measurement methods include ELISA, immunochromatography, and the immune complex transfer method described in Japanese Patent Publication No. 1-254868.

[0078] In another embodiment, a target nucleic acid in a sample is measured as the test substance. For example, a solid phase on which the polypeptide or polymer described above is immobilized and a nucleic acid probe that hybridizes to the target nucleic acid can be used as a capture agent. The nucleic acid probe is labeled with L-biotin and binds to the polypeptide or polymer of this embodiment on the solid phase. By bringing the solid phase, the target nucleic acid, and the sample into contact, the target nucleic acid in the sample is immobilized on the solid phase. On the solid phase, the DNA extension reaction is carried out using DNA polymerase and dNPTs, with the target nucleic acid as a template and the nucleic acid probe as a primer. DNA detection can be performed by known methods. For example, a synthesized amplicon can be labeled with an intercalator (e.g., SYBR® Green I dye, ethidium bromide, etc.) as a detector and the fluorescence can be measured. Alternatively, a fluorescent probe that can bind to the extension chain can be bound as a detector and the fluorescence can be measured. The TaqMan® method may also be used. An example of the measurement method of this embodiment will be described with reference to Figure 1.

[0079] First, the sample containing the test substance 81 and the R1 reagent are dispensed into container 90. The first reagent dispensing unit 551 dispenses the R1 reagent into container 90, and the sample dispensing unit 530 dispenses the sample into container 90. The R1 reagent contains a capture body 84 to which L-biotin is attached, which reacts with and binds to the test substance 81. After dispensing the sample and the R1 reagent, the sample in container 90 is heated to a predetermined temperature in the reaction unit 580, causing the capture body 84 to bind to the test substance 81.

[0080] Next, the R2 reagent is dispensed into the container 90 by the second reagent dispensing unit 552. The R2 reagent contains a solid phase 82. The solid phase 82 has the polypeptide or its polymer of this embodiment immobilized on it. After dispensing the R2 reagent, the sample in the container 90 is heated to a predetermined temperature in the reaction unit 580. As a result, the L-biotin and the polypeptide or its polymer of this embodiment on the solid phase bind together, thereby immobilizing the test substance 81 and the capture body 84 on the solid phase 82.

[0081] The test substance 81 and the capture body 84 formed on the solid phase 82, and the unreacted capture body 84 may be separated by a primary BF separation treatment using a BF separation device 100. The primary BF separation treatment removes unwanted components such as the unreacted capture body 84 from the container 90.

[0082] Next, the R3 reagent is dispensed into the container 90 by the third reagent dispensing unit 553. The R3 reagent contains a detector 83, which reacts with and binds to the test substance 81. After dispensing the R3 reagent, the sample in the container 90 is heated to a predetermined temperature in the reaction unit 580. As a result, a sandwich complex 85 containing the test substance 81, the detector 83, and the capture agent 84 is formed on the solid phase 82. In the example in Figure 1, the detector 83 is an enzyme-labeled antibody.

[0083] The sandwich complex 85 formed on the solid phase 82 and the unreacted labeled substance 83 are separated by a secondary BF separation treatment using the BF separation device 100. The secondary BF separation treatment removes unwanted components such as the unreacted detection substance 83 from the container 90.

[0084] Subsequently, reagents R4 and R5 are dispensed into container 90 by the fourth reagent dispensing section 554 and the fifth reagent dispensing section 555, respectively. Reagent R4 contains a buffer solution. The sandwich complex 85, bound to the solid phase 82, is dispersed in the buffer solution. In the example shown in Figure 1, reagent R5 contains a chemiluminescent substrate. The buffer solution contained in reagent R4 has a composition that promotes the reaction between the enzyme labeled on the detector 83 contained in the sandwich complex 85 and the chemiluminescent substrate contained in reagent R5. After dispensing reagents R4 and R5, the sample in container 90 is heated to a predetermined temperature in the reaction section 580. Light is generated by reacting the substrate with the detector 83, and the generated light is detected by the photodetector 521 of the detection section 520. The test substance 81 is measured based on the intensity of the detected light.

[0085] In this measurement method, reagents R1 and R2 are dispensed separately into container 90. However, reagents R1 and R2 may be mixed beforehand, and the mixture may be dispensed into container 90 with the capture body 84 fixed to the solid phase 82.

[0086] 5. Solid phase and reagent kits containing solid phase The solid phase in this embodiment is a solid phase on which the above-mentioned polypeptide or its polymer is immobilized.

[0087] In this embodiment, the solid phase is an insoluble carrier for immobilizing the captured material. The manner in which the polypeptide or its polymer is immobilized onto the solid phase is as described above. The solid phase can be selected from known solid phases according to the purpose. As the material of the solid phase, for example, polymer compounds, inorganic materials, etc. Multiple materials may be appropriately combined to form the solid phase.

[0088] Polymeric compounds include organic polymeric compounds, inorganic polymeric compounds, and semi-organic compounds. Examples of polymeric compounds include latex, rubber, polystyrene, polyethylene, polypropylene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), silicone, insoluble agarose, and insoluble dextran.

[0089] Inorganic materials include inorganic compounds and metals. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, cobalt, nickel, ferrite, magnetite, etc.), glass, silica, and alumina. Examples of metals include gold, silver, and materials containing them. The reagent kit of this embodiment includes a solid phase to which the above-mentioned polypeptide or its polymer is bound, and a capture body to which L-biotin is attached.

[0090] An example of the reagent kit of this embodiment is shown in Figure 2. In Figure 2, 11 shows the reagent kit, 12 shows a first container containing a reagent including particles to which the polypeptide or its polymer is bound, 13 shows a second container containing a reagent including a capture body to which L-biotin is attached, 14 shows the packaging box, and 15 shows the accompanying documentation. The accompanying documentation may describe the composition, usage, and storage methods of each reagent. The reagent kit may also contain other reagents, such as buffer solutions, calibrators, and detectors. [Examples]

[0091] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the examples. 1. Example 1: Preparation of D-streptavidin 1-1. Method

[0092] To synthesize the polypeptide (D-core streptavidin) consisting of amino acids 13 to 139 of the polypeptide shown in SEQ ID NO: 1, the polypeptide consisting of amino acids 13 to 71 of SEQ ID NO: 1 was synthesized, and the polypeptide consisting of amino acid 139 was synthesized from the peptide thioester consisting of amino acids 13 to 71 of SEQ ID NO: 1, and from the polypeptide in which the 72nd alanine residue was substituted with cysteine.

[0093] (1) DCM was added to HMPB-ChemMatrix resin (0.49 mmol / g, Biotage) (820 mg, 400 μmol) and allowed to swell at room temperature for at least 1 hour. Next, Fmoc-D-Thr(tBu)-OH (795 mg, 2.0 mmol), an amino acid with its side chain protected by a protecting group, MSNT (593 mg, 2.0 mmol) as a condensing agent, and 1-methylimidazole (160 μL, 2.0 mmol) as a capping agent for the 5' terminal hydroxyl group were added to DCM (12 mL) and mixed. This mixed solution was added to the swollen resin and stirred at room temperature for 3 hours.

[0094] (2) After removing the reaction solution, the resin was thoroughly washed with DCM and DMF. Next, acetic anhydride (2.0 mL, 20 mmol) and pyridine (1.6 mL, 20 mmol) were added to DMF (12 mL) and mixed well, and this was mixed with the resin and stirred at room temperature. After 30 minutes, the solution was removed and the resin was thoroughly washed with DMF.

[0095] (3) To the resin (100 μmol) condensed with Fmoc-D-Thr(tBu)-OH, Fmoc-Gly-OH (500 μmol), the activator DIC (500 μmol), the epitaxial inhibitor Oxyma Pure™ (500 μmol) (Merck KGaA), and the solvent N-methylpyrrolidone (NMP) (4 mL) were added and condensed. In the same procedure, amino acids with side chains protected by protecting groups were changed to Fmoc-D-Ser(tBu)-OH (500 μmol) and Fmoc-D-Asp(OtBu)-(Hmb)-Gly-OH (1st time: 150 μmol, 2nd time: 100 μmol), and condensed by manual synthesis. Fmoc-D-Asp(OtBu)-(Hmb)-Gly-OH was condensed by double coupling.

[0096] (4) The 28 residues from 39L to 66T were extended using a microwave-assisted automated peptide synthesizer (LibertyBlue, CEM). Fmoc-D-amino acids (500 μmol), DIC (500 μmol), Oxyma Pure (trademark) (500 μmol), and DIPEA (50 μmol) were used in the microwave-assisted automated peptide synthesizer. DMF was used for the condensation reaction and resin washing, and 20% piperidine / DMF was used for the removal of the Fmoc group. Microwave irradiation was used to accelerate the condensation reaction and the removal of the Fmoc group.

[0097] (5) The resin was recovered, and Fmoc-D-Ala-OH and Fmoc-D-Asp(OtBu)-(Hmb)-Gly-OH were synthesized again by hand, and the peptides were extended under the same reaction conditions as in (3). Here, double coupling of Fmoc-D-Asp(OtBu)-(Hmb)-Gly-OH was performed twice at 100 μmol each time. To the resin-bound polypeptide, acetic anhydride (167 μL, 1.8 mmol), DIPEA (78.8 μL, 0.45 mmol) and Oxyma Pure™ (5.3 mg, 37 μmol) were added to NMP (7 mL), mixed, stirred at room temperature for 15 minutes, and then thoroughly washed with NMP.

[0098] (6) Using a microwave-assisted automated peptide synthesizer, the 23 residues from 13A to 35A were extended to prepare a 59-residue polypeptide, 13A-71T, to which the resin was finally bound.

[0099] (7) The 59-residue polypeptide to which the resin was bound was recovered from the synthesizer with the N-terminal Fmoc group removed. Boc2O (115 μL, 500 μmol), DIPEA (87 μL, 500 μmol), and NMP (3 mL) were added to the recovered resin (100 μmol) and stirred at room temperature. After 1 hour, the solution was removed and the resin was thoroughly washed with NMP. This Bocization procedure was repeated twice.

[0100] (8) A 1% TFA / DCM solution (3 mL) was added to a 59-residue polypeptide (100 μmol) to which the resin was bound, and the mixture was stirred for 5 minutes. The filtrate was then collected in a round-bottom flask containing pyridine (585 μL). This procedure was repeated 13 times, and the resin was washed three times with trifluoroethanol / DCM (3:5) solution (3 mL). The washings were collected in the same round-bottom flask. After removing the solvent under reduced pressure, H2O (40 mL) was added to the obtained solid and the mixture was stirred. The mixture was centrifuged for 10 minutes (10000 rpm), and the supernatant was removed. This procedure was repeated three times, and after washing the solid, the obtained solid was freeze-dried to obtain a protective peptide (171 mg, 20 μmol).

[0101] (9) The obtained protected peptide (116 mg, 13.5 μmol) was dissolved in NMP (5 mL) and cooled to -15°C with stirring. Thiophenol (42 μL, 405 μmol), PyBOP (36.7 mg, 67.5 μmol), and DIPEA (12 μL, 67.5 μmol) were added and stirred overnight. After the reaction was complete, the reaction solution was poured into ice-cold diethyl ether (40 mL) and stirred. Centrifuge for 10 minutes (10000 rpm) and the supernatant was removed. After repeating this procedure three times, the resulting precipitate was air-dried to obtain the protected crude peptide thioester (thiophenyl ester) (113 mg). To the obtained crude product, a mixture of TFA:H2O:triisopropylsilane (TIPS):ethanedithiol (EDT) (92.5:2.5:2.5, v / v / v / v) (3 mL) was added and stirred at room temperature. After 3 hours, the reaction mixture was added to ice-cold diethyl ether (40 mL), and the resulting precipitate was air-dried to obtain the deprotected crude peptide (85.6 mg). To the obtained deprotected crude peptide, 0.2 M phosphate buffer (pH 7.0) (3 mL) containing 8 M guanidine hydrochloride and 0.2 M sodium 2-mercaptoethanesulfonate (MESNA) was added and the mixture was stirred at room temperature. After stirring for more than 1 hour, the mixture was purified by RP-HPLC (Proteonavi C1, 4.6 × 250 mm, Linear gradient of B: 17.5-21.5, 1.0 mL / min, 12 min, 60°C, 220 nm, A: aqueous solution containing 0.1% TFA, B: 90% MeCN, 10% H2O containing 0.09% TFA, manufactured by Shiseido) to obtain a 59-residue peptide thioester (13A-71T, MESNA thioester) (6.6 mg, 1.09 μmol).

[0102] (Synthesis of cysteine-containing polypeptides) (1) Add DCM to HMPB-ChemMatrix resin (0.49 mmol / g) (612 mg, 300 μmol) and allow to swell at room temperature for at least 1 hour. Next, add Fmoc-D-Ser(tBu)-OH (575 mg, 1.5 mmol), MSNT (444 mg, 1.5 mmol), and 1-methylimidazole (120 μL, 1.5 mmol) to DCM (9 mL) and mix well. Add this mixed solution to the swollen resin and stir overnight at room temperature. Remove the reaction solution and thoroughly wash the resin with DCM and DMF. Next, add acetic anhydride (1.5 mL, 15 mmol) and pyridine (1.2 mL, 15 mmol) to DMF (9 mL), mix with the resin, and stir at room temperature for 30 minutes. After that, thoroughly wash the resin with DMF.

[0103] (2) Using a microwave-assisted automated peptide synthesizer, a resin (100 μmol) formed by condensing Fmoc-D-Ser(tBu)-OH was extended to 68 residues at 72C-139S. The amounts of each reagent used in the condensation reaction were Fmoc-D-amino acid (500 μmol), DIC (500 μmol), Oxyma Pure™ (500 μmol), and DIPEA (50 μmol). DMF was used for the condensation reaction and resin washing, and 20% piperidine / DMF was used for the removal of the Fmoc group. Microwave irradiation was used to accelerate the reaction during the condensation and Fmoc group removal reactions.

[0104] (3) A 68-residue polypeptide to which the resin was bound was added (100 μmol) to a mixture of TFA:H2O:TIPS:EDT (92.5:2.5:2.5, v / v / v / v) (10 mL) and stirred at room temperature. After 3 hours, the reaction mixture was added to ice-cold diethyl ether (80 mL), and the resulting precipitate was air-dried to obtain the deprotected crude peptide (396 mg).

[0105] (4) The peptide was purified by RP-HPLC (Triart-C18, 20 × 250 mm, Linear gradient of B: 30-45, 9.9 mL / min, 30 min, 60°C, 220 nm, A: aqueous solution containing 0.1% TFA, B: 90% MeCN, 10% H2O containing 0.09% TFA, manufactured by YMC) to obtain a 68-residue peptide segment (72C-139S) (37.2 mg, 5.03 μmol).

[0106] (Ligate of polypeptides) To 546 μL of 0.2 M phosphate buffer (containing 8 M guanidine hydrochloride, 40 mM 4-mercaptoacetic acid, 40 mM sodium ascorbate, and 40 mM tris(2-carboxyethyl)phosphine (TCEP), pH 7.1), peptide thioester (13A-71T) (6.0 mg, 0.933 μmol) and peptide segment (72C-139S) (9.3 mg, 1.26 μmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted 2-fold with 0.2 M phosphate buffer (containing 8 M guanidine hydrochloride and 0.2 M sodium 2-mercaptoethanesulfonate, pH 7.0) and stirred at room temperature. After stirring for more than 1 hour, the mixture was purified by RP-HPLC (Proteonavi C1, 4.6 × 250 mm, Linear gradient of B: 15-35, 1.0 mL / min, 30 min, 60℃, 220 nm, A: aqueous solution containing 0.1% TFA, B: 90% MeCN, 10% H2O containing 0.09% TFA, manufactured by Shiseido) to obtain a full-length 127-residue [Cys60]-(1-127) peptide (6.7 mg, 0.504 μmol).

[0107] (Desulfurization reaction) [Cys60]-(1-127) peptide (4.7 mg, 0.354 μmol) was dissolved in 707 μL of 0.2 M phosphate buffer (pH 7.1) containing 8 M guanidine hydrochloride and 0.25 M TCEP. Tert-butanethiol (40 μL) and VA-044 / H2O (250 mg / mL, 27 μL) were added sequentially, and the mixture was stirred at room temperature while protected from light. After 4 hours, 1.4 mL of 0.2 M phosphate buffer (pH 7.1) containing 8 M guanidine hydrochloride and 0.25 M TCEP was added and mixed well. Diethyl ether (1.7 mL) was added to this mixture, mixed well, and then allowed to stand, and the aqueous phase was collected. This process was repeated three times, and after thoroughly removing the diethyl ether from the recovered aqueous phase, the solution was purified by RP-HPLC (Proteonavi C1, 4.6 × 250 mm, Linear gradient of B: 15-45, 1.0 mL / min, 30 min, 60℃, 220 nm, A: aqueous solution containing 0.1% TFA, B: 90% MeCN, 10% H2O containing 0.09% TFA, manufactured by Shiseido) to obtain a polypeptide with a total length of 127 residues (2.6 mg, 0.196 μmol).

[0108] (Preparation of tetramers) A 127-residue polypeptide was dissolved in denaturation buffer (50 mM Tris-HCl, 6 M guanidine-HCl, 1 mM EDTA, 200 mM NaCl, pH 8.0 (4 °C)) to a concentration of 1 mg / mL and heated at 85 °C for 45 minutes. 5 mL of the heated solution was dialyzed at 4 °C for 12 hours with buffer 1 containing 3 M guanidine HCl (50 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, pH 8.0). Next, it was dialyzed at 4 °C for 36 hours with buffer 1 containing 2 M guanidine HCl. Dialysis was performed at 4 °C for 12 hours with buffer 1 containing 0.4 M L-arginine HCl and 1 M guanidine HCl. Dialysis was performed at 4 °C for 36 hours with buffer 1 containing 0.4 M L-arginine HCl and 0.5 M guanidine HCl. Finally, 4-hour dialysis at 4 °C was repeated twice with buffer 1, and dialysis was performed overnight at room temperature with buffer 1.

[0109] (Gel filtration chromatography analysis) After dialysis, polypeptides were concentrated using an Amicon Ultra-15 10K centrifugal filter device (Merck KGaA). 500 μL of each concentrate was then passed through a Superdex200 10 / 300GL (Cytiva) using an AKTA Prime system (Cytiva). Buffer 1 was flowed at a rate of 0.4 mL / min, and gel filtration chromatography was performed, using the change in absorbance at 280 nm over time as an indicator of elution. Furthermore, to estimate the molecular weight, commercially available tetramerized natural core streptavidin (Roche) was also subjected to similar gel filtration chromatography.

[0110] 1-2.Results Figure 3 shows the time course of absorbance at a wavelength of 280 nm in gel filtration chromatography of the polypeptide prepared above. Fraction 8 is the peak of aggregates. Fractions 14 to 18 are presumed to be the peaks of the tetrameric polypeptide, as similar peaks were observed in commercially available tetrameric core streptavidin. Fractions 14 to 18 were collected and concentrated using an Amicon Ultra-15 10K centrifugal filter device (Merck KGaA) to prepare tetrameric D-streptavidin composed of D-amino acid residues (hereinafter, for convenience, simply referred to as "D-streptavidin" in this example).

[0111] 2. Example 2: Evaluation of biotin binding ability of D-streptavidin by SPR analysis 2-1. Method The binding ability of D-streptavidin obtained in Example 1 to the natural form of D-biotin and L-biotin, an optical isomer of D-biotin, was evaluated using an intermolecular interaction analyzer (Biacore T200) that uses SPR as its measurement principle.

[0112] L-biotin was synthesized as a mixture of D-biotin and L-biotin using a method described in non-patent literature (Journal of the American Chemical Society 1978, 100, 1558-1563), and then prepared by optical resolution using liquid chromatography, which was commissioned to Daicel Corporation. A CHIRALPAK IG column (Daicel, Φ46×50 mm) was used, and optical resolution was performed under conditions of methanol:acetic acid mixed solvent (100:0.1 (v / v)) as the mobile phase, flow rate of 1.0 mL / min, column temperature of 40°C, and detection wavelength of 205 nm.

[0113] (1) Preparation of biotin-labeled albumin To 2.8 mL of 50 mg / mL bovine serum albumin / 0.1 M phosphate buffer (pH 7.5), 16.3 μL of N,N-dimethylformamide solution containing 10 mg / mL D-biotin-AC5-OSu (Dojin Chemical Co., Ltd.) was added, and after stirring, the mixture was allowed to stand at 35°C for 1 hour. Subsequently, desalting was performed using PD-10 (Cytiva, 17085101) equilibrated with 0.1 M phosphate buffer (pH 7.5), and D-biotin-labeled albumin was recovered. The number of D-biotin labels per albumin molecule by the HABA method was 0.4. Similarly, L-biotin-labeled albumin was prepared using L-biotin-AC5-OSu [see (Amidation of L-biotin) described in Example 3 above].

[0114] (2) Fixing to the sensor chip Bovine serum albumin was immobilized at a target level of 400 RU on flow cells 1 and 3 of the CM5 sensor chip (Cytiva) of the Biacore T200 system using an amine coupling kit (Cytiva). Similarly, L-biotin-labeled albumin and D-biotin-labeled albumin were immobilized at a target level of 400 RU on flow cells 2 and 4 using the same amine coupling method. All flow cells were blocked with 1 M ethanolamine solution, pH 8.5 (Cytiva).

[0115] (Measurement of interaction with biotin) HBS-EP+ (manufactured by Cytiva) was used as the running buffer, and D-streptavidin prepared in Example 1 was flowed into each flow cell at a flow rate of 30 μL / min in the range of 10 pM to 100 nM using a single-cycle method.

[0116] 2-2.Results Figure 4A shows the sensorgram of the intermolecular interaction between D-streptavidin and L-biotin-labeled albumin, and Figure 4B shows the sensorgram of the intermolecular interaction between D-streptavidin and D-biotin-labeled albumin. Figure 5 shows the sensorgram of the intermolecular interaction between natural core streptavidin and D-biotin-labeled albumin.

[0117] A comparison of the sensorgrams of natural core streptavidin and D-biotin-labeled albumin with those of D-streptavidin and L-biotin-labeled albumin revealed that the D-streptavidin and L-biotin-labeled albumin in this embodiment bind very strongly, similar to the combination of natural core streptavidin and D-biotin-labeled albumin.

[0118] In Figure 4, no specific sensogram changes were observed between D-streptavidin and D-biotin-labeled albumin due to the binding of the analyte D-streptavidin. The dissociation constant Kd = 3.48 × 10⁻¹⁰ between the streptavidin mutant shown in Patent Document 1 and the natural type of D-biotin-labeled albumin. -7 Considering this, the binding affinity between D-streptavidin and D-biotin in this embodiment is extremely low, and it can be said that they do not bind at all.

[0119] 3. Example 3: Evaluation of the effect on the D-biotin measurement system in ELISA. 3-1. Method (Preparation of streptavidin-bound solid phase) A 96-well ELISA plate (Thermo Fisher Scientific) was washed three times with PBS, and then D-streptavidin (1 μg / mL, PBS, 100 μL) prepared in Example 1 was added and left to stand overnight at 4°C. After washing three times with PBS, PBS buffer (200 μL) containing 2% bovine serum albumin was added and the mixture was shaken at 25°C and a rotation speed of 600 rpm for 2 hours to prepare a D-streptavidin-immobilized plate. The same procedure was performed for L-streptavidin (Roche) to prepare an L-streptavidin-immobilized plate.

[0120] (Amidation of L-biotin) Biotin (43 mg) and N-hydroxysuccinimide (NHS) (24.3 mg) were dissolved in DMF (1.2 mL), and ethyl (dimethylaminopropyl) carboxydiimide (EDC) (40.5 mg) was added and the mixture was stirred at room temperature for 23 hours. The residue obtained by distilling off the solvent was recrystallized from ethanol:acetic acid:water (95:5:1) to obtain 6-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide)hexanoic acid 6-aminohexanoic acid (compound 2) (63.9 mg). [ka]

[0121] 6-aminohexanoic acid (24.2 mg) was dissolved in 0.4 mL of 0.25 M aqueous sodium carbonate solution. This solution was added to a 1 mL solution of compound 2 in DMF and stirred at room temperature for 23 hours. After removing the solvent by distillation, the residue was washed with water. The solution was acidified with 4 M hydrochloric acid, and the resulting solid was collected by filtration to obtain 6-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide)hexanoic acid (compound 3) (68.5 mg). [ka]

[0122] Compound 3 (68.5 mg) and NHS (40.5 mg) were dissolved in DMF (3.3 mL), EDC (67.5 mg) was added at room temperature, and the mixture was stirred at 35°C for 21 hours. The residue obtained by distilling off the solvent was subjected to silica gel column chromatography, and 2,5-dioxopyrrolidine-1-yl5-((3aR, 4R, 6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate (compound 4) (47.6 mg) was obtained from the DCM-methanol elution portion. [ka]

[0123] (Preparation of biotin-labeled antibodies) 5.1 mg / mL thyroid-stimulating hormone antibody (TSH antibody) (T2-194) (Kitayama Labess) (25 μL) was mixed with DMSO solution (1.3 μL) containing 16.7 mg / mL L-biotin-AC5-OSu, and after stirring, it was allowed to stand at 35°C for 1 hour. Then, it was subjected to PD-10 (Cytiva, 17085101) equilibrated with 0.1 M phosphate buffer (pH 7.5), and 500 μL fractions were taken. The absorption peak fraction at 280 nm wavelength was collected to prepare L-biotin-labeled TSH antibody. The same procedure was performed on D-biotin-AC5-OSu (Dojin Chemical) to prepare D-biotin-labeled TSH antibody.

[0124] (Evaluation of the effect on the measurement system in the presence of D-biotin) (1) 400 μL of HISCL® TSH calibrator C3 (Sysmex Corporation) and 400 μL of HISCL® TSH R1 reagent (Sysmex Corporation) were added to a 1.5 mL tube as a sample, and the mixture was reacted at 37°C and 600 rpm for 30 minutes. In addition, to evaluate the effect of D-biotin on the sample, 0 ng / mL, 1 ng / mL, 10 ng / mL, or 100 ng / mL of D-biotin were added to the sample and reacted with the R1 reagent.

[0125] (2) The D-streptavidin-immobilized plates after blocking were washed three times with HISCL® wash solution (Sysmex Corporation), and 60 μL of the reaction solution in a 1.5 mL tube was added to each well. The reaction was carried out at 37°C and 600 rpm for 20 minutes. The L-biotin-labeled TSH antibody prepared above was added, and the reaction was carried out at 37°C and 600 rpm for 30 minutes.

[0126] (3) After the reaction, the plate was washed five times with HISCL® wash solution (Sysmex Corporation), and 50 μL of HISCL® R4 reagent (Sysmex Corporation) was added to each plate, followed by 100 μL of HISCL® R5 reagent (Sysmex Corporation), and the reaction was carried out for 30 minutes at 37°C and 600 rpm. After the reaction, luminescence detection was performed using a microplate reader (TECAN, Infinite 200 PRO). The same measurements were also performed on the L-streptavidin-immobilized plate and the D-biotin-labeled TSH antibody after blocking.

[0127] 3-2.Results Figure 6 shows the detection results of TSH by ELISA in the presence of D-biotin. Figure 6 shows the percentage of the signal measurement value for each sample, with the signal measurement value when a sample with a D-biotin concentration of 0 ng / mL was measured being set to 100% in each measurement system. When D-biotin was present in the sample, the signal decreased in the D-biotin / L-streptavidin measurement system depending on the concentration of D-biotin added. On the other hand, the L-biotin / D-streptavidin measurement system showed almost no effect from the addition of D-biotin. The D-streptavidin / L-biotin measurement system was virtually unaffected by the endogenous D-biotin present in the sample.

[0128] 4. Example 4: Confirmation of the quantitative accuracy of the measurement system 4-1. Method (1) HISCL® TSH calibrator C0~C5 (Sysmex Corporation) was added as a sample to a 1.5 mL tube to obtain concentrations of 0 μIU / mL, 2 μIU / mL, 10 μIU / mL, 50 μIU / mL, 120 μIU / mL, or 200 μIU / mL. Then, 400 μL of HISCL® TSH R1 reagent (Sysmex Corporation), to which HISCL® TSH calibrator C0~C5 (Sysmex Corporation) had been added, was added, and the mixture was reacted at 37°C and 600 rpm for 30 minutes.

[0129] (2) The D-streptavidin-immobilized plates after blocking, prepared in the same manner as in Example 3, were washed three times with HISCL® wash solution (Sysmex Corporation), and 60 μL of the reacted solution in a 1.5 mL tube was added to each well. The reaction was carried out for 20 minutes at 37°C and 600 rpm. The L-biotin-labeled TSH antibody prepared above was added, and the reaction was carried out for 30 minutes at 37°C and 600 rpm.

[0130] (3) After the reaction, the plate was washed five times with HISCL® wash solution (Sysmex Corporation), and 50 μL of HISCL® R4 reagent (Sysmex Corporation) was added in stages, followed by 100 μL of HISCL® R5 reagent (Sysmex Corporation), and the reaction was carried out for 30 minutes at 37°C and 600 rpm. After the reaction, luminescence detection was performed using a microplate reader (Infinite 200 PRO).

[0131] 4-2.Results The results measured in Example 4 are shown in Table 1 and Figure 7. Figure 7 is a graph showing the count values ​​for TSH concentrations from 0 to 120 μIU / mL. As shown in Figure 7, the L-biotin / D-streptavidin system is R 2 A good correlation of 0.998 was observed. Since the reference range for TSH is 0.34–4.22 μIU / mL, it was found that the system exhibits good linearity within the measurement range.

[0132] [Table 1]

[0133] In addition, five samples with a TSH concentration of 50 μIU / mL were prepared, and the TSH concentration was measured for each. The results are shown in Table 2. The coefficient of variation was approximately 5%. The measurement system in this example was found to be reproducible.

[0134] [Table 2]

[0135] 5. Example 5: L-amino acid residue substitution of amino acid residues involved in structural maintenance 5-1. Method We investigated the effect on the three-dimensional structure of D-streptavidin, shown in Sequence ID No. 1, by changing some amino acid residues to the L-isomer. In silico analysis was performed using Discovery Studio (Dassault Aviation).

[0136] First, the crystal structure of the natural core streptavidin tetramer (PDB ID: 3RY2) was obtained from the Protein Data Bank (PDB), and a monomer structure was extracted from the tetramer. Based on the extracted structure, the monomer structure of D-streptavidin was constructed by converting all amino acid residues to D-amino acid residues in Discovery Studio. Subsequently, the solvent contact degree of the amino acid residues constituting the D-streptavidin monomer structure was confirmed. Among the amino acid residues of the D-streptavidin core sequence, amino acid residues other than glycine with a solvent contact degree of 25% or less were identified. As a result, the amino acid residues in question were, in order of increasing solvent contact, the 36 amino acid residues at positions 21, 29, 77, 104, 130, 54, 56, 39, 43, 27, 75, 31, 92, 90, 128, 102, 33, 79, 71, 73, 106, 23, 60, 96, 81, 122, 28, 86, 88, 50, 45, 108, 38, 110, 132, and 42 of SEQ ID NO: 1. These amino acid residues are thought to be particularly involved in maintaining the internal structure of the D-streptavidin monomer. When one of the above amino acid residues was substituted with an L-amino acid residue, the structure of the compound was optimized using Clean Geometry with respect to the substituted amino acid residue, the amino acid residue adjacent to the N side of the substituted amino acid residue, and the amino acid residue adjacent to the C side of the substituted amino acid residue. Subsequently, the presence or absence of interatomic collisions within D-streptavidin was confirmed from the three-dimensional structure.

[0137] 5-2.Results The results of Example 5 showed that for all 36 amino acid residues, the interatomic collisions that occurred when substituting D-amino acid residues with L-amino acid residues were resolved by performing structural optimization using the Clean Geometry function. As an example, Figures 8A and 8C show how the interatomic collisions that occur when the 21st amino acid residue, a D-tryptophan residue, is substituted with an L-tryptophan residue are resolved. Figure 8A shows the structure before substituting the 21st D-tryptophan residue with the L-isomer. Figure 8B shows how interatomic collisions occur within D-streptavidin when the 21st D-tryptophan residue is substituted with an L-tryptophan residue (the cylinders highlighted with ellipses represent interatomic collisions). Figure 8C shows how the interatomic collisions within D-streptavidin are resolved by performing structural optimization using the Clean Geometry function.

[0138] In silico analysis, it was found that for any of the amino acid residues involved in maintaining the structure, the interatomic collisions that occur when a D-amino acid residue is replaced with an L-amino acid residue are resolved. It is thought that even in vitro, when some of the amino acid residues involved in maintaining the structure are replaced with L-amino acid residues, there exists a structure in which atoms within D-streptavidin move and interatomic collisions are resolved. Therefore, even if some of the amino acid residues involved in maintaining the monomer structure are replaced with L-amino acid residues, it is thought that the structure capable of binding to biotin will be maintained.

[0139] 6. Example 6: Synthesis of avidin-like protein derived from Pleurotus cornucopiae, consisting of D amino acids. An avidin-like protein derived from Pleurotus cornucopiae, consisting of D amino acids (hereinafter referred to as D-Tamavidin® 2), was synthesized. The polypeptide having amino acid sequences 2 to 141 of SEQ ID NO: 4 was divided into five peptide segments, and each was synthesized using an automated peptide synthesizer (Prelude, Protein Technologies, Inc.). These five segments were segment 1 containing sequences 2 to 23 of SEQ ID NO: 4, segment 2 containing sequences 24 to 49, segment 3 containing sequences 50 to 76, segment 4 containing sequences 77 to 105, and segment 5 containing sequences 106 to 141. Next, segments 1 and 2 were linked by chemical ligation to prepare segment 1-2, segments 3 and 4 were linked by chemical ligation to prepare segment 3-4, segment 5 was linked to segment 3-4 by chemical ligation to prepare segment 3-4-5, and by chemical ligation of segments 1-2 and 3-4-5, D-tamavidin (trademark) 2 having the amino acid sequence from the 2nd to the 141st amino acid of SEQ ID NO: 4 was produced. (Preparation of tetramers)

[0140] D-Tamavidin® 2 was dissolved in denaturation buffer (80 mM Tris-HCl, 6 M guanidine-HCl, 1 mM DTT, pH 8.0) to a concentration of 20 mg / mL and heated at 85°C for 45 minutes. Five times the volume of buffer (80 mM Tris-HCl, 1 mM DTT, pH 8.0) was added to the heated solution, and it was allowed to stand at room temperature for 30 minutes. Next, the solution was diluted 50-fold with buffer (80 mM Tris-HCl, 1 mM DTT, pH 8.0) and allowed to stand at 4°C overnight. After that, the solution was concentrated with Amicon Ultra-4 (30k) (Merck Millipore) and recovered as D-Tamavidin® 2 protein solution.

[0141] 7. Example 7: Evaluation of biotin binding ability of D-Tamavidin™ 2 by SPR analysis 7-1. Method (1) Preparation of biotin-labeled albumin To 2.8 mL of 50 mg / mL bovine serum albumin / 0.1 M phosphate buffer (pH 7.5), 16.3 μL of N,N-dimethylformamide solution containing 10 mg / mL D-biotin-AC5-OSu (Dojin Chemical Co., Ltd.) was added, and after stirring, the mixture was allowed to stand at 35°C for 1 hour. Subsequently, desalting was performed using PD-10 (Cytiva, 17085101) equilibrated with 0.1 M phosphate buffer (pH 7.5), and D-biotin-labeled albumin was recovered. The number of D-biotin labels per albumin molecule by the HABA method was 0.4. Similarly, L-biotin-labeled albumin was prepared using L-biotin-AC5-OSu [see (Amidation of L-biotin) described in Example 3 above].

[0142] (2) Fixing to the sensor chip Bovine serum albumin was immobilized at a target level of 400 RU on flow cells 1 and 3 of the CM5 sensor chip (Cytiva) of the Biacore T200 system using an amine coupling kit (Cytiva). Similarly, L-biotin-labeled albumin and D-biotin-labeled albumin were immobilized at a target level of 400 RU on flow cells 2 and 4 using the same amine coupling method. All flow cells were blocked with 1 M ethanolamine solution, pH 8.5 (Cytiva).

[0143] (3) Measurement of interaction with biotin Using HBS-EP+ (Cytiva) as the running buffer, the prepared D-Tamavidin™ 2 was flowed through each flow cell at a flow rate of 30 μL / min in the range of 100 pM to 100 nM using a single-cycle method, and its binding and dissociation to D-biotin or L-biotin were evaluated using Biacore T200 Evaluation Software (Cytiva).

[0144] 7-2.Results Figure 9A shows the change in the SPR signal of the intermolecular interaction between D-Tamavidin® 2 and L-biotin-labeled albumin. An increase in the SPR signal was observed in proportion to the amount of D-Tamavidin® 2 added.

[0145] Figure 9B shows the SPR signal changes in the intermolecular interaction between D-Tamavidin® 2 and D-biotin-labeled albumin. No increase in the SPR signal dependent on the addition of D-Tamavidin® 2 was observed. These results indicate that D-Tamavidin™ 2 does not substantially bind to D-biotin-labeled albumin, but does bind to L-biotin-labeled albumin.

[0146] 8. Example 8: Evaluation of the effect of D-tamavidin™ immobilized plate on the D-biotin measurement system in ELISA. 8-1. Method (1) 200 μL of HISCL® TSH calibrator C3 (Sysmex Corporation) and 200 μL of HISCL® TSH R1 reagent (Sysmex Corporation) were added to a 1.5 mL tube as a sample, and the mixture was reacted at 37°C and 600 rpm for 30 minutes. In addition, to evaluate the effect of D-biotin on the sample, 0 ng / mL, 1 ng / mL, 10 ng / mL, or 100 ng / mL of D-biotin were added to the sample and reacted with the R1 reagent.

[0147] D-Tamavidin® 2 (1 μg / mL, PBS, 50 μL) was immobilized on a 96-well ELISA plate (Thermo Fisher) by allowing it to stand overnight at 4°C, and blocking was performed with 2% BSA / PBS.

[0148] (2) The D-Tamavidin® 2 immobilized plate prepared in (1) after blocking was washed three times with HISCL® wash solution (Sysmex Corporation), and 60 μL of the reacted solution in a 1.5 mL tube was added to each well. The reaction was carried out for 20 minutes at 37°C and 600 rpm. The L-biotin-labeled TSH antibody prepared in (see Preparation of Biotin-Labeled Antibody) described in Example 3 was added, and the reaction was carried out for 30 minutes at 37°C and 600 rpm.

[0149] (3) After the reaction, the plate was washed five times with HISCL® wash solution (Sysmex Corporation), and 50 μL of HISCL® R4 reagent (Sysmex Corporation) was added in stages, followed by 100 μL of HISCL® R5 reagent (Sysmex Corporation), and the reaction was carried out for 30 minutes at 37°C and 600 rpm. After the reaction, luminescence detection was performed using a microplate reader (SpectraMAXiD3, Molecular Devices Corporation).

[0150] 8-2.Results Figure 10 shows the detection results of TSH by ELISA in the presence of D-biotin. Figure 10 shows the percentage of the signal measurement value for each sample, with the signal measurement value when a sample with a D-biotin concentration of 0 ng / mL was measured in each measurement system being set to 100%. In the L-biotin / D-tamavidin™ 2 measurement system, there was almost no effect from the addition of D-biotin.

[0151] The measurement system for D-Tamavidin™ 2 / L-Biotin was suggested to be virtually unaffected by endogenous D-biotin present in the sample.

[0152] 9. Example 9: Confirmation of quantitative accuracy of a measurement system using a D-Tamavidin™ immobilized plate. 9-1. Method (1) 200 μL of HISCL® TSH calibrator C0~C5 (Sysmex Corporation) was added to a 1.5 mL tube as a sample to achieve concentrations of 0 μIU / mL, 2 μIU / mL, 10 μIU / mL, 50 μIU / mL, 120 μIU / mL, or 200 μIU / mL. Then, 200 μL of HISCL® TSH R1 reagent (Sysmex Corporation), to which HISCL® TSH calibrator C0~C5 (Sysmex Corporation) had been added, was added, and the mixture was reacted at 37°C and 600 rpm for 30 minutes.

[0153] (2) The D-tamavidin™ 2 immobilized plate prepared in Example 8(1) after blocking was washed three times with HISCL™ wash solution (Sysmex Corporation), and 60 μL of the reacted solution in a 1.5 mL tube was added to each well. The reaction was carried out for 20 minutes at 37°C and 600 rpm. The L-biotin-labeled TSH antibody prepared above was added, and the reaction was carried out for 30 minutes at 37°C and 600 rpm.

[0154] (3) After the reaction, the plate was washed five times with HISCL® wash solution (Sysmex Corporation), and 50 μL of HISCL® R4 reagent (Sysmex Corporation) was added in stages, followed by 100 μL of HISCL® R5 reagent (Sysmex Corporation), and the reaction was carried out for 30 minutes at 37°C and 600 rpm. After the reaction, luminescence detection was performed using a microplate reader (SpectraMAXiD3, Molecular Devices Corporation).

[0155] 9-2.Results The measurement results are shown in Table 3 and Figure 11. Concentration-dependent signal changes were observed.

[0156] [Table 3]

[0157] In addition, five samples with a TSH concentration of 50 μIU / mL were prepared, and the TSH concentration was measured for each. The results are shown in Table 4. The coefficient of variation was approximately 5%. It was clear that the measurement system in this example was also problem-free in terms of reproducibility.

[0158] [Table 4]

[0159] 10. Example 10: In silico analysis The binding affinity of D-bradavidin and D-avidin to L-biotin or D-biotin was investigated by in silico analysis. 10-1. Method

[0160] The structure of L-biotin used in the analysis is shown in Figure 12A, and the structure of D-biotin is shown in Figure 12B. Discovery Studio 2018 was used to prepare and calculate the structures of L-biotin or D-biotin. For the analysis, ligands other than D-biotin were removed from the tetramerized crystal structure data of the natural streptavidin-D-biotin complex (PDB ID: 3RY2 Biological Assembly1), the natural bladavidin-D-biotin complex (PDB ID: 4BBO Biological Assembly1), or the natural avidin-D-biotin complex (PDB ID: 2AVI Biological Assembly1). Then, hydrogen atoms were added using the Prepare protein command to remove D-biotin from the structure. For each structure, the L / D Conversion command was used to generate the structures of the optical isomer proteins composed of D-amino acids. A binding site with a radius of 12 Å was set at coordinates corresponding to the biotin binding site in the natural organism, and this was used to predict ligand docking of D-biotin and L-biotin.

[0161] The ligand docking structure was predicted using the CDOCKER function in Discovery Studio, and an interaction score (CDOCKER ENERGY) was obtained. The interaction score between natural streptavidin and the optical isomers of each biotin serves as a criterion for binding affinity.

[0162] 10-2.Results The results are as follows. All units are in kcal / mol. aD-Streptavidin L-biotin -33.5867 D-biotin -29.2717 bD-Bradavidine L-biotin -30.6423 D-biotin -25.9508 cD-avidin L-biotin -31.5848 D-biotin -27.6185

[0163] Both optical isomers showed scores approximately 3.9–4.7 kcal / mol lower for binding to L-biotin compared to D-biotin. This suggests that D-bladavidin and D-avidin, like D-streptavidin, do not bind substantially to D-biotin but strongly bind to L-biotin.

Claims

1. A method for measuring a test substance using a solid phase on which a polypeptide belonging to the avidin-streptavidin family or a polymer with the polypeptide as a monomer unit is immobilized, and a capture body that binds to the test substance in a sample, The polypeptide comprises either the sequence from position 19 to 133 of the amino acid sequence shown in SEQ ID NO: 1 or the sequence from position 4 to 127 of the amino acid sequence shown in SEQ ID NO: 4, and contains D-amino acid residues in 90% or more of the amino acid residues other than glycine in the polypeptide, has the ability to bind to L-biotin, and is a polypeptide in which no specific sensorgram change due to D-biotin binding is observed when measured using Biacore T200. The aforementioned capture body has L-biotin attached to it. The L-biotin and the polypeptide or polymer on the solid phase bind to each other, so that when the test substance is detected, the capture body is fixed to the solid phase. Method for measuring the test substance.

2. The measurement method according to claim 1, wherein the polymer is a tetramer.

3. The measurement method according to claim 1 or 2, wherein the polypeptide comprises either the sequence from the 19th to the 133rd amino acid sequence shown in SEQ ID NO: 1 or the sequence from the 4th to the 127th amino acid sequence shown in SEQ ID NO: 4, and the amino acid residues of the polypeptide other than glycine consist only of D-amino acid residues.

4. The measurement method according to any one of claims 1 to 3, wherein the polypeptide comprises amino acid residues from the 19th to the 133rd amino acid residues of the amino acid sequence shown in SEQ ID NO:

1.

5. The measurement method according to any one of claims 1 to 4, wherein the polypeptide comprises amino acid residues from the 13th to the 139th amino acid residues of the amino acid sequence shown in SEQ ID NO:

1.

6. The measurement method according to any one of claims 1 to 5, wherein the capture body comprises an antibody, antigen, lectin, nucleic acid, enzyme, or substrate.

7. A step of bringing the solid phase, the capture body, the detection body bound to the test substance, and the test substance into contact to form a composite including the solid phase, the capture body, the detection body, and the test substance, and The process includes measuring the test substance based on the detection agent contained in the complex, The measurement method according to any one of claims 1 to 6.

8. The measurement method according to claim 7, wherein the detection body comprises a fluorescent substance or an enzyme.

9. The measurement method according to any one of claims 1 to 8, wherein the sample is serum, plasma, blood, cerebrospinal fluid, or lymph fluid.

10. The dissociation constant (Kd) between the polypeptide and D-biotin is 10 -4 A measurement method according to any one of claims 1 to 9, wherein the value is M or greater.

11. The dissociation constant (Kd) between the polypeptide and D-biotin is 10 -2 The measurement method according to any one of claims 1 to 10, wherein M is greater than or equal to M.

12. A reagent kit for measuring a test substance, comprising a solid phase on which a polypeptide belonging to the avidin-streptavidin family or a polymer with the polypeptide as a monomer unit is immobilized, and a capture body to which L-biotin is attached, The polypeptide comprises either the sequence from position 19 to 133 of the amino acid sequence shown in SEQ ID NO: 1 or the sequence from position 4 to 127 of the amino acid sequence shown in SEQ ID NO: 4, and contains D-amino acid residues in 90% or more of the amino acid residues other than glycine, has the ability to bind to L-biotin, and is a polypeptide in which no specific sensorgram change due to D-biotin binding is observed when measured using Biacore T200. Reagent kit.

13. The reagent kit according to claim 12, wherein the polymer is a tetramer.

14. The reagent kit according to claim 12 or 13, wherein the polypeptide comprises either the sequence from the 19th to the 133rd amino acid sequence shown in SEQ ID NO: 1 or the sequence from the 4th to the 127th amino acid sequence shown in SEQ ID NO: 4, and the amino acid residues of the polypeptide other than glycine consist only of D-amino acid residues.

15. The reagent kit according to any one of claims 12 to 14, wherein the polypeptide comprises amino acid residues from the 19th to the 133rd amino acid residues of the amino acid sequence shown in SEQ ID NO:

1.

16. The reagent kit according to any one of claims 12 to 15, wherein the polypeptide comprises amino acid residues from the 13th to the 139th amino acid residues of the amino acid sequence shown in SEQ ID NO:

1.

17. A reagent kit according to any one of claims 12 to 16, used in the measurement method according to any one of claims 1 to 11.

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