Antibodies that specifically bind to the 5'→3' exonuclease domain of DNA polymerase

Abstract

To provide an antibody that inhibits the 5'→3' exonuclease activity of a DNA polymerase, or a fragment of the antibody.SOLUTION: Disclosed is an antibody that binds specifically to a 5'→3' exonuclease activity domain of a DNA polymerase, or a fragment of the antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Provided are antibodies that specifically bind to the 5' to 3' exonuclease active domain of DNA polymerase used in nucleic acid amplification methods, particularly polymerase chain reaction (hereinafter referred to as "PCR"), and related technologies. [Background technology]

[0002] Synthesis of DNA from nucleic acid templates using DNA polymerases is used and applied in various methods in the field of molecular biology, such as sequencing and nucleic acid amplification. Among these, nucleic acid amplification methods have already been put to practical use not only in research fields but also in forensic medicine, such as genetic diagnosis and paternity testing, as well as in microbial testing of food and the environment.

[0003] A typical nucleic acid amplification method is PCR. PCR involves three steps: (1) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) extension of the primer using DNA polymerase. This cycle is repeated to amplify the target nucleic acid in a sample. In some cases, (2) annealing and (3) extension are performed at the same temperature in a single step, and the two steps are counted as one cycle.

[0004] PCR has been widely used in medical and biological research and clinical diagnosis due to its sensitivity, which allows amplification from a nucleic acid sample equivalent in principle to one copy, but in reality to several copies, and its specificity, which allows amplification of only specific regions. Currently, PCR is being further developed, and various techniques exist, such as multiplex PCR, which simultaneously amplifies multiple primers, and real-time PCR, which uses fluorescent dyes or fluorescently labeled probes to monitor the generation process of amplification products over time.

[0005] These nucleic acid amplification methods are widely used in genetic analysis of large numbers of samples, such as high-throughput screening (HTS), and in food testing and environmental testing, which require processing multiple specimens. When analyzing large numbers of samples, it is expected that the nucleic acid amplification reaction solution will be left for a long period of time (e.g., several hours to several days) after preparation. However, there is a concern that leaving the reaction solution at room temperature may reduce the stability of the reaction solution. For example, in the TaqMan (registered trademark) probe method (see, for example, Non-Patent Document 1), there have been several confirmed cases where leaving the prepared reaction solution at room temperature resulted in a delay in the threshold cycle (Ct) value, or even made it impossible to detect the Ct value (Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2017-163904 [Patent Document 2] Re-tabled publication 2016 / 136324 [Non-patent literature]

[0007] [Non-Patent Document 1] Holland et al., Proc. Natl. Acad. Sci. 88, 1991, 7276-7280 Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have previously discovered the problem that nucleic acid templates, primers, probes, etc. used in nucleic acid amplification methods and the like are degraded in the presence of a DNA polymerase having a 5'→3' exonuclease activity domain.

[0009] A main objective of the present invention is to provide an antibody or a fragment thereof that specifically binds to the 5'→3' exonuclease active domain of DNA polymerase, and a method for producing the antibody or the fragment thereof. [Means for solving the problem]

[0010] As a result of extensive research aimed at solving the above problems, the present inventors have discovered an antibody or a fragment thereof that specifically binds to the 5'→3' exonuclease active domain of DNA polymerase, as well as a useful method for producing said antibody or fragment thereof. The present invention was completed as a result of further extensive research based on these findings.

[0011] Representative examples of the present invention are as follows. [Section 1] An antibody or a fragment thereof (antigen-binding fragment) that specifically binds to the 5'→3' exonuclease active domain of DNA polymerase. [Section 2] Item 2. The antibody or fragment thereof according to Item 1, wherein the DNA polymerase is selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase. [Section 3] Formula (A-1) below: GFX A3 X A4 X A5 X A6 X A7 X A8 (A-1) [In the formula, X A3 is T or S, X A4 is F, L, or I, X A5 is D, N, S, or T, X A6 is D, N, S, T, K, R, or H, X A7 is Y, F, or W, X A8 is G, S, T, W, Y or F] and a heavy chain CDR1 consisting of an amino acid sequence represented by the formula: The following formula (B-1): IX B2 X B3 X B4 X B5 X B6 X B7 X B8 (B-1) [In the formula, X B2 is G, S, T, K, R, H, D, or N, X B3 is F, Y, L, I, G, S, T, D, or N, X B4 is G, S, T, D, N, K, R, or H, X B5 is G, S, T, or A, X B6 is G, S, T, D, or N, X B7 is S, T, F, Y, D, N, K, R, or H, X B8 is S, T, V, L, I, or M] and a heavy chain CDR2 consisting of an amino acid sequence represented by the formula: The following formulas (C-1) to (C-6): VRX C1 X C2 X C3 GX C4 X C5 X C6 TGFDX C7 (C-1) VRX C1 X C2 X C3 GX C4 X C5 X C6 FDX C7 (C-2) X C8 RDGALGLAVNWFDX C7 (C-3) ATSDDYYALNI (C-4) TTAYYSRYSYYMFDX C7 (C-5) TTALRDX C7 (C-6) [In the formula, X C1 is A, S, D, K, H, or R, X C2 is G, A, D, P, K, H, or R, X C3 is S, T, L, Y, or I, X C4 is A, V, I, R, or L; X C5 is A, V, Y, or P; X C6 is A, V, S, T, or Y; X C7 is V, I, L, S, T, or N, X C8 is A or V] a heavy chain CDR3 consisting of an amino acid sequence represented by any one of the following: The following formula (D-1): X D1 X D2 X D3 X D4 X D5 X D6 (D-1) [In the formula, X D1 is E, Q, D, or N, X D2 is G, A, S, or T, X D3 is A, V, L, I, or F, X D4 is S, T, K, R, or H, X D5 is S, T, D, N, K, R, or H, X D6 is F, Y, or W] and a light chain CDR1 consisting of an amino acid sequence represented by the formula: The following formula (E-1): X E1 X E2 X E3 (E-1) [In the formula, X E1 is G, S, T, D, N, F, Y, K, R, or H, X E2is G, A, S, T, V, L or I, X E3 is K, R, H, D, N, S, or T] and a light chain CDR2 consisting of an amino acid sequence represented by the formula: The following formula (F-1) or (F-2): X F1 X F2 X F3 X F4 X F5 X F6 X F7 X F8 (F-1) X F1 X F2 X F3 X F4 X F5 X F6 X F7 X F8 X F9 (F-2) [In the formula, X F1 is L, I, E, Q, F, Y or W, X F2 is D, N, E or Q, X F3 is S, T, F, or Y, X F4 is G, S, T, F, Y, N or Q, X F5 is S, T, N, Q, L or I, X F6 is G, S, T, F, Y, or W, X F7 is S, T, P, Y or W, X F8 is L, I, P, K, R, H, Y, T, or W; X F9 is G, S, T, D or E] and a light chain CDR3 consisting of an amino acid sequence represented by An antibody or a fragment thereof (antigen-binding fragment) comprising: [Section 4] Furthermore, the following formula (E-2): X E4 X E5 X E6 X E7(E-2) [In the formula, X E4 is G, S, R, H, K, D, N, F, Y, or T, X E5 is L, I, K, H, or R; X E6 is G, A, S, T, P, F, or Y; X E7 is G, A, D, N, S, or T] Item 4. The antibody or fragment thereof according to Item 3, comprising a sequence region adjacent to the C-terminus of the light chain CDR2, which consists of the amino acid sequence represented by: [Section 5] The following formula (A-1-1): GFTFX A51 X A61 X A71 X A81 (A-1-1) X A51 is D, N, or S, X A61 is D, N, S, K, or H, X A71 is Y or W, X A81 is G, W, or Y] and a heavy chain CDR1 consisting of an amino acid sequence represented by the formula: The following formula (B-1-1): IX B21 X B31 X B41 X B51 X B61 X B71 X B81 (B-1-1) [In the formula, X B21 is G, S, T, K, or N, X B31 is Y, L, G, T, or N, X B41 is G, S, T, D, or H, X B51 is G or S, X B61 is G, S, T, or D, X B71 is S, T, Y, D, or H, X B81 is S, T, V, I, or M] and a heavy chain CDR2 consisting of an amino acid sequence represented by the formula: The following formulas (C-1-1) to (C-6-1): VRX C11 X C21 X C31 GX C41 X C51 X C61 TGFDX C71 (C-1-1) VRX C11 X C21 X C31 GX C41 X C51 X C61 FDX C71 (C-2-1) X C81 RDGALGLAVNWFDX C71 (C-3-1) ATSDDYYALNI (C-4) TTAYYSRYSYYMFDX C71 (C-5-1) TTALRDX C71 (C-6-1) [In the formula, X C11 is A or R, X C21 is P or R, X C31 is T or I, X C41 is V or L, X C51 is P or A, X C61 is T or Y, X C71 is V, T, or N, X C81 is A or V] a heavy chain CDR3 consisting of an amino acid sequence represented by any one of the following: The following formula (D-1-1): QX D21 X D31 X D41 X D51 X D61 (D-1-1) [In the formula, X D21 is G or S, X D31 is V or I, X D41 is S or K, X D51 is S, N, or K, X D61 is F or Y] and a light chain CDR1 consisting of an amino acid sequence represented by the formula: The following formula (E-1-1): X E11 X E21 X E31 (E-1-1) [In the formula, X E11 is G, T, D, Y, or R, X E21 is A, T, or V, X E31 is K, D, N, or S] and a light chain CDR2 consisting of an amino acid sequence represented by the formula: The following formula (F-1-1) or (F-2-1): X F11 QX F31 X F41 X F51 X F61 X F71 X F81 (F-1-1) X F11 QX F31 X F41 X F51 X F61 X F71 X F81 T (F-2-1) [In the formula, X F11 is L, Q, F, or Y; X F31 is S or Y, X F41 is G, N, Q, or Y; X F51 is S, N, or I, X F61 is G, S, Y, or W, XF71 is S, P, or W, X F81 is L, P, H, Y, or T] and a light chain CDR3 consisting of an amino acid sequence represented by An antibody or a fragment thereof (antigen-binding fragment) comprising: [Section 6] Furthermore, the following formula (E-2-1) or (E-2-2): SLX E61 S (E-2-1) X E42 X E52 X E62 X E72 (E-2-2) [In the formula, X E61 is A or P, X E42 is R, N, Y, or T; X E52 is L or R, X E62 is A or Y, X E72 is S or T] Item 6. The antibody or fragment thereof according to Item 5, comprising a sequence region adjacent to the C-terminus of the light chain CDR2, which consists of the amino acid sequence represented by: [Section 7] Item 7. The antibody or fragment thereof according to any one of Items 3 to 6, which specifically binds to the 5'→3' exonuclease active domain of Taq polymerase. [Section 8] Item 7. The antibody or fragment thereof according to any one of Items 3 to 6, which specifically binds to the 5'→3' exonuclease active domain of Tth polymerase. [Section 9] Item 7. The antibody or fragment thereof according to any one of Items 3 to 6, which specifically binds to the 5'→3' exonuclease active domain of Z05 polymerase. [Section 10] An antibody or a fragment thereof that specifically binds to the 5' to 3' exonuclease active domain of DNA polymerase, wherein at least one epitope is present in any of the following regions: amino acid region A selected from the region of positions 56 to 66 from the N-terminus of SEQ ID NO: 1 or the region of positions 56 to 67 from the N-terminus of SEQ ID NO: 2 or 3; amino acid region B selected from the region of positions 75 to 81 from the N-terminus of SEQ ID NO: 1 or the region of positions 76 to 82 from the N-terminus of SEQ ID NO: 2 or 3; amino acid region C selected from the region of positions 161 to 182 from the N-terminus of SEQ ID NO: 1 or the region of positions 162 to 183 from the N-terminus of SEQ ID NO: 2 or 3; or amino acid region D selected from the region of positions 269 to 285 from the N-terminus of SEQ ID NO: 1 or the region of positions 271 to 287 of SEQ ID NO: 2 or 3. [Section 11] Item 11. The antibody or fragment thereof according to Item 10, wherein the at least one epitope is present in either amino acid region A or B. [Section 12] Item 12. The antibody or fragment thereof according to Item 10 or 11, wherein the epitope in the amino acid region A is any one of SEQ ID NOs: 60 to 63, the epitope in the amino acid region B is any one of SEQ ID NOs: 64 or 65, the epitope in the amino acid region C is any one of SEQ ID NOs: 66 to 74, and the epitope in the amino acid region D is any one of SEQ ID NOs: 75 to 83. [Section 13] Item 13. The antibody or fragment thereof according to any one of Items 10 to 12, wherein the epitope in the amino acid region A is SEQ ID NO: 61 or 62, the epitope in the amino acid region B is SEQ ID NO: 64 or 65, the epitope in the amino acid region C is SEQ ID NO: 66, 67, 68, 70, or 71, and the epitope in the amino acid region D is SEQ ID NO: 77, 78, 80, or 82. [Section 14] Item 14. The antibody or fragment thereof according to any one of Items 1 to 13, which is a monoclonal antibody or a fragment thereof. [Section 15] the heavy chain CDR3 consists of the amino acid sequence shown in any one of SEQ ID NOs: 21 to 28 or an amino acid sequence in which 1 to 3 amino acids have been mutated in these amino acid sequences, Item 15. The antibody or fragment thereof according to any one of Items 1 to 14, wherein the light chain CDR3 consists of the amino acid sequence shown in any one of SEQ ID NOs: 42 to 48 or an amino acid sequence in which 1 to 3 amino acids have been mutated. [Section 16] the heavy chain CDR3 consists of an amino acid sequence shown in any one of SEQ ID NOs: 21 to 28, 16. The antibody or fragment thereof according to any one of Items 1 to 15, wherein the light chain CDR3 consists of the amino acid sequence shown in any one of SEQ ID NOs: 42 to 48. [Section 17] a heavy chain CDR1 consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 4 to 11 or an amino acid sequence in which 1 to 3 amino acids have been mutated in these amino acid sequences; a heavy chain CDR2 consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 12 to 20 or an amino acid sequence in which 1 to 3 amino acids have been mutated in these amino acid sequences; a heavy chain CDR3 consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 21 to 28 or an amino acid sequence in which 1 to 3 amino acids have been mutated in these amino acid sequences; a light chain CDR1 consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 29 to 35 or an amino acid sequence in which 1 to 3 amino acids have been mutated in these amino acid sequences; a light chain CDR2 consisting of an amino acid sequence represented by any one of YTN, YTD, YAD, YAN, DAS, GVK, RAK, GAK, and TAS; a light chain CDR3 consisting of an amino acid sequence shown in any one of SEQ ID NOS: 42 to 48 or an amino acid sequence in which 1 to 3 amino acids have been mutated in these amino acid sequences; Item 17. The antibody or fragment thereof according to any one of Items 1 to 16, comprising: [Section 18] a heavy chain CDR1 consisting of an amino acid sequence shown in any one of SEQ ID NOs: 4 to 11; a heavy chain CDR2 consisting of an amino acid sequence shown in any one of SEQ ID NOs: 12 to 20; a heavy chain CDR3 consisting of an amino acid sequence shown in any one of SEQ ID NOs: 21 to 28; a light chain CDR1 consisting of an amino acid sequence shown in any one of SEQ ID NOs: 29 to 35; a light chain CDR2 consisting of an amino acid sequence represented by any one of YTN, YTD, YAD, YAN, DAS, GVK, RAK, GAK, and TAS; a light chain CDR3 consisting of an amino acid sequence shown in any one of SEQ ID NOs: 42 to 48; Item 18. The antibody or fragment thereof according to any one of Items 1 to 17, comprising: [Section 19] Item 19. The antibody or fragment thereof according to Item 17 or 18, further comprising a sequence region adjacent to the C-terminus of the light chain CDR2, which comprises an amino acid sequence set forth in any one of SEQ ID NOs: 36 to 41 or an amino acid sequence in which 1 to 3 amino acids have been mutated in these amino acid sequences. [Section 20] 20. The antibody or fragment thereof according to any one of items 17 to 19, further comprising a sequence region adjacent to the C-terminus of the light chain CDR2 consisting of the amino acid sequence shown in any one of SEQ ID NOs: 36 to 41. [Section 21] 20. The antibody or fragment thereof of any of items 15, 17, and 19, wherein the mutation is a conservative substitution. [Section 22] A fragment of the antibody according to any one of items 1 to 21, which is an Fab, F(ab')2, or scFv fragment (antigen-binding fragment). [Section 23] A reagent comprising the antibody or fragment thereof according to any one of Items 1 to 21, or the fragment according to Item 22. [Section 24] Item 24. The reagent according to Item 23, further comprising at least one member selected from the group consisting of a DNA polymerase having a 5' to 3' exonuclease activity domain, a primer, a probe, and deoxyribonucleoside-5'-phosphate. [Section 25] Item 25. The reagent according to Item 24, wherein the DNA polymerase is selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase. [Section 26] 26. The reagent according to any one of Items 23 to 25, which is a reagent for amplifying nucleic acids. [Section 27] A method for producing an antibody or a fragment thereof (antigen-binding fragment) that specifically binds to the 5'→3' exonuclease active domain of a DNA polymerase, comprising step A of selecting an antibody having the ability to bind to the entire DNA polymerase from antibodies produced by an animal immunized with an immunogen consisting of a portion of the DNA polymerase (wherein the portion includes the 5'→3' exonuclease active domain). [Section 28] Item 28. The method of production according to Item 27, wherein the immunogen consists of the 5' to 3' exonuclease active domain of DNA polymerase. [Section 29] Item 29. The method of claim 27 or 28, wherein the DNA polymerase is selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase. [Section 30] 30. The method according to any one of Items 27 to 29, wherein the immunogen consists of the 5'→3' exonuclease active domain of Tth polymerase. [Section 31] Item 28. The production method according to Item 27, wherein step A is a step of selecting an antibody capable of binding to the entire Taq polymerase from antibodies produced by an animal immunized with an immunogen consisting of a portion of Tth polymerase (wherein the portion includes the 5'→3' exonuclease active domain). [Section 32] Item 28. The production method according to Item 27, wherein step A is a step of selecting an antibody capable of binding to the entire Taq polymerase from antibodies produced by an animal immunized with an immunogen consisting of the 5'→3' exonuclease active domain of Tth polymerase. [Section 33] A method for producing an antibody fragment (antigen-binding fragment), comprising a step of expressing a partial amino acid sequence of the antibody obtained by the production method according to any one of Items 27 to 32, by genetic engineering techniques. [Section 34] Item 22. The antibody or fragment thereof according to any one of Items 1 to 21, or the fragment according to Item 22, which exhibits 60% or more inhibitory activity against the 5' to 3' exonuclease of a DNA polymerase when coexisted with the DNA polymerase at 37°C for 24 hours. [Section 35] Item 35. The antibody or fragment thereof according to any one of Items 1 to 21 and 34, or the fragment according to Item 22, wherein the substrate DNA degradation rate is 40% or less when the substrate DNA (wherein the substrate DNA may be single-stranded or double-stranded and may function as a probe) is allowed to coexist with a DNA polymerase having a 5' to 3' exonuclease activity domain at 25°C for 24 hours. [Section 36] A reagent for stabilizing a composition comprising a DNA polymerase having a 5'→3' exonuclease active domain and at least one type of nucleic acid selected from the group consisting of a primer, a probe, and a nucleic acid template, the reagent comprising the antibody or fragment thereof according to any one of Items 1 to 21, 34, and 35, or the fragment according to Item 22. [Section 37] A method for stabilizing a composition comprising a DNA polymerase having a 5'→3' exonuclease active domain and at least one type of nucleic acid selected from the group consisting of a primer, a probe, and a nucleic acid template, the method comprising the step of adding to the composition the antibody or fragment thereof described in any one of Items 1 to 21, 34, and 35, or the fragment described in Item 22. [Effects of the Invention]

[0012] The present invention provides an antibody or a fragment thereof that specifically binds to the 5'→3' exonuclease active domain of a DNA polymerase, as well as a useful method for producing the antibody or fragment thereof. For example, adding the antibody or a fragment thereof to a reagent containing a DNA polymerase having a 5'→3' exonuclease active domain and nucleic acids such as primers and probes can suppress nucleic acid degradation and improve the stability of the reagent. Furthermore, when a target nucleic acid is amplified using the reagent, the suppression of the generation of fragments due to nucleic acid degradation can suppress nonspecific amplification of the target nucleic acid, allowing for highly efficient amplification of the target nucleic acid, thereby enabling highly sensitive detection of even very small amounts of the target nucleic acid. DETAILED DESCRIPTION OF THE INVENTION

[0013] 1.Definitions etc. As used herein, amino acids may be natural or unnatural amino acids. Examples of unnatural amino acids include, but are not limited to, citrulline, ornithine, ε-acetyl-lysine, β-alanine, aminobenzoic acid, 6-aminocaproic acid, aminobutyric acid, hydroxyproline, mercaptopropionic acid, 3-nitrotyrosine, norleucine, pyroglutamic acid, etc. Furthermore, amino acids may be, for example, L-amino acids, D-amino acids, or DL-amino acids.

[0014] As used herein, the term "identity of amino acid sequences" refers to the degree of amino acid identity when two or more amino acid sequences to be compared are optimally aligned. The identity of amino acid sequences can be calculated using commercially available analytical tools or tools available via telecommunications lines (Internet), for example, the commercially available software GENETYX (Genetyx Co., Ltd.) or the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) (http: / / www.ncbi.nlm.nih.gov / BLAST / ) using default (initial setting) parameters.

[0015] The amino acid sequences disclosed herein may have one or more (e.g., 1, 2, or 3) amino acids deleted, substituted, or modified, or one or more (e.g., 1, 2, or 3) amino acids inserted or added to the sequence, as long as the deletion, substitution, or modification does not inhibit binding to the 5' to 3' exonuclease domain of a DNA polymerase.

[0016] The amino acid substitution is preferably a substitution with another amino acid having a similar structure and / or properties (conservative substitution). Conservative substitutions include, for example, substitutions within the group shown in Table 1.

[0017] [Table 1]

[0018] Modifications of amino acids include, for example, modifications of functional groups such as amino groups, carboxyl groups, hydroxyl groups, sulfhydryl (SH) groups, etc. The modifications of functional groups may be, for example, glycosylation, methylation, esterification, amidation, PEGylation, phosphorylation, hydroxylation, binding with a protecting group such as a t-butoxycarbonyl (Boc) group or a 9-fluorenylmethyloxycarbonyl (Fmoc) group, biotinylation, binding with a fluorescent dye such as fluorescein isothiocyanate (FITC), or binding with an enzyme such as peroxidase (HRP) or alkaline phosphatase (ALP).

[0019] As used herein, the antibody may be a monoclonal or polyclonal antibody, but is preferably a monoclonal antibody. The antibody may be of any isotype, such as IgG, IgA, IgD, IgE, or IgM. Examples of the antibody include, but are not limited to, mouse antibodies, rat antibodies, guinea pig antibodies, and human antibodies. The antibody may also be a chimeric antibody, such as a guinea pig-mouse chimeric antibody or a mouse-human chimeric antibody.

[0020] Herein, the antibody fragment is not particularly limited as long as it comprises heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3, and examples include Fv, Fab, Fab', (Fab')2, scFv, scFv-Fc, diabody, triabody, tetrabody, minibody, etc. Preferably, the antibody fragment is a fragment that has antigen-binding ability (antigen-binding fragment).

[0021] Herein, heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 are identified by a homology search using IMGT / BlastSearch (http: / / www.imgt.org / blast / ).

[0022] Nucleotides such as DNA and RNA may be analogs that have been subjected to known chemical modifications, as exemplified below. For example, to prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be substituted with a chemically modified phosphate residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, -CH, -CHCHOCH, -CHCHNHC(NH)NH, -CHCONHCH, or -CHCHCN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, such as by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Furthermore, the phosphate moiety or hydroxyl moiety may be modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group, but is not limited to these.

[0023] 2. Antibodies that specifically bind to the 5'→3' exonuclease active domain of DNA polymerase (hereinafter referred to as "Domain E") The antibodies or fragments thereof of the present invention can inhibit 5'→3' exonuclease activity by specifically binding to domain E of DNA polymerase, and are useful as 5'→3' exonuclease activity inhibitors. Because the 5'→3' exonuclease activity inhibitors of the present invention are antibodies, they have high specificity and the inhibitory activity can be inactivated by heating or other methods, making them suitable for use in hot start methods. The DNA polymerase is not particularly limited as long as it contains domain E. The DNA polymerase may be a wild-type DNA polymerase, a recombinant DNA polymerase obtained by introducing a gene encoding the DNA polymerase into any host cell, or a DNA polymerase obtained by modifying the gene. For example, the DNA polymerase may be a DNA polymerase in which domain E is fused to a DNA polymerase that does not have domain E in its wild form.

[0024] In one embodiment, the DNA polymerase is preferably a thermostable DNA polymerase, where "thermostable" refers to the property of retaining preferably 50% or more of its DNA polymerase activity even after heat treatment at a high temperature, for example, at 60°C for 30 minutes. Examples of thermostable DNA polymerases include DNA polymerase derived from Thermus aquaticus (Taq polymerase), DNA polymerase derived from Thermus thermophilus HB8 (Tth polymerase), DNA polymerase derived from Thermus sp. Z05 (Z05 polymerase), DNA polymerase derived from Bacillus caldotenax (Bca polymerase), DNA polymerase derived from Bacillus stearothermophilus (Bst polymerase), DNA polymerase derived from Thermococcus kodakarensis (KOD polymerase), DNA polymerase derived from Pyrococcus furiosus (Pfu polymerase), DNA polymerase derived from Pyrococcus woesei (Pwo polymerase), DNA polymerase derived from Thermus brockianus (Tbr polymerase), DNA polymerase derived from Thermus filiformis (Tfi polymerase), and Thermus Examples of suitable polymerases include, but are not limited to, DNA polymerases derived from Thermococcus flavus (Tfl polymerase), Thermotoga maritima (Tma polymerase), Thermotoga neapolitana (Tne polymerase), Thermococcus litoralis (Vent polymerase), and Pyrococcus GB-D (DEEPVENT polymerase). The term "Taq polymerase" also includes mutants. Here, a mutant refers to a DNA polymerase that has an amino acid sequence that is 80% or more identical to the amino acid sequence of the original DNA polymerase and that maintains enzymatic properties such as polymerase activity, 5' to 3' exonuclease activity, and thermostability.The polymerase active domain of the mutant preferably consists of an amino acid sequence that is 85% or more (preferably 90% or more or 95% or more) identical to the amino acid sequence of the polymerase active domain of the original DNA polymerase. Domain E of the mutant preferably consists of an amino acid sequence that is 85% or more (preferably 90% or more or 95% or more) identical to the amino acid sequence of Domain E of the original DNA polymerase. Amino acid mutations in the mutant are preferably conservative substitutions.

[0025] In one embodiment, the DNA polymerase is preferably a DNA polymerase belonging to Family A. Examples of DNA polymerases belonging to Family A include, but are not limited to, Taq polymerase, Tth polymerase, Z05 polymerase, Tma polymerase, Bca polymerase, and Bst polymerase.

[0026] The DNA polymerase is preferably at least one selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase. In a specific embodiment, it is preferably two or more DNA polymerases selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase, and more preferably a combination of Taq polymerase and at least one selected from the group consisting of Tth polymerase and Z05 polymerase.

[0027] The polymerase activity of DNA polymerase is measured as follows: If the polymerase activity is strong, dilute the DNA polymerase solution with storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% (v / v) polyethylene glycol sorbitan monolaurate (Tween™ 20), 0.1% (v / v) octylphenyl-polyethylene glycol (Nonidet™ P40), 50% (v / v) glycerin) and then measure as follows. (1) Add 25 μl of solution A, 5 μl of solution B, 5 μl of solution C, 10 μl of sterile water, and 5 μl of DNA polymerase solution to a microtube and react at 75°C for 10 minutes. (2) Then, the mixture is cooled on ice, 50 μl of solution E and 100 μl of solution D are added, and the mixture is stirred and then cooled on ice for another 10 minutes. (3) This solution is filtered through a glass filter (Whatman GF / C filter) and thoroughly washed with 0.1N hydrochloric acid and ethanol. (4) The radioactivity of the filter is measured using a liquid scintillation counter (Packard) to determine the amount of nucleotide incorporation into the template DNA. One unit of polymerase activity is defined as the amount of DNA polymerase that incorporates 10 nmol of nucleotides into the acid-insoluble fraction (i.e., the fraction that becomes insoluble when Solution D is added) per 30 minutes under these conditions. Solution A: 40 mM Tris-HCl buffer (pH 7.5), 16 mM magnesium chloride, 15 mM dithiothreitol, 100 μg / mL BSA (bovine serum albumin) Solution B: 1.5μg / μl activated calf thymus DNA Solution C: 1.5mM dNTP(250cpm / pmol [3H]dTTP) Solution D: 20% (w / v) trichloroacetic acid (2 mM sodium pyrophosphate) Solution E: 1 mg / mL calf thymus DNA

[0028] In one embodiment, the antibody or fragment thereof of the present invention SEQ ID NO:1: The amino acid sequence shown in TIFF0007777831000002.tif32151 (the amino acid sequence of the E domain of Taq polymerase (wild type)), SEQ ID NO:2: The amino acid sequence shown in TIFF0007777831000003.tif31151 (the amino acid sequence of the E domain of Tth polymerase (wild type)), SEQ ID NO:3: The amino acid sequence shown in TIFF0007777831000004.tif32150 (the amino acid sequence of the E domain of Z05 polymerase (wild type)), and An amino acid sequence having 80% or more (preferably 85% or more, 90% or more, or 95% or more) identity to these amino acid sequences It is preferable that the E domain binds to at least one selected from the group consisting of:

[0029] In one embodiment, it is preferable that the antibody or fragment thereof of the present invention binds to (or recognizes) at least one epitope (e.g., one, two, three, four, or five) of any of the four amino acid regions enclosed in a box in the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 above. Of the four amino acid regions enclosed in the above rectangles, the amino acid region from positions 56 to 66 from the N-terminus of SEQ ID NO: 1 or the amino acid region from positions 56 to 67 from the N-terminus of SEQ ID NO: 2 or 3 is referred to as "amino acid region A"; the amino acid region from positions 75 to 81 from the N-terminus of SEQ ID NO: 1 or the amino acid region from positions 76 to 82 from the N-terminus of SEQ ID NO: 2 or 3 is referred to as "amino acid region B"; the amino acid region from positions 161 to 182 from the N-terminus of SEQ ID NO: 1 or the amino acid region from positions 162 to 183 from the N-terminus of SEQ ID NO: 2 or 3 is referred to as "amino acid region C"; and the amino acid region from positions 269 to 285 from the N-terminus of SEQ ID NO: 1 or the amino acid region from positions 271 to 287 of SEQ ID NO: 2 or 3 is referred to as "amino acid region D". The Pol I polymerase family, including Taq polymerase, is known to have multiple highly conserved regions located around the 200th amino acid from the N-terminus (Kim Y et al., Mol. Cells, Vol. 7, No. 4, pp. 468-472 (the entirety of which is incorporated herein by reference)). The antibody or fragment thereof of the present invention is particularly preferably an antibody or fragment thereof that binds to at least one epitope (e.g., one, two, three, four, or five) present in a region (e.g., amino acid regions A to C) located around the 200th amino acid from the N-terminus of Pol I polymerase. From this perspective, the antibody or fragment thereof preferably binds to part or all of amino acid region A and / or amino acid region B as at least one epitope. In a specific embodiment, the antibody or fragment thereof is preferably an antibody or fragment thereof that binds to at least part or all of amino acid region A and part or all (particularly part) of either amino acid region C or D as an epitope. In yet another embodiment, it is preferred that the antibody or fragment thereof binds to at least part or all (particularly all) of amino acid region B and part or all (particularly part) of either amino acid region C or D as an epitope.

[0030] Examples of epitopes in part or all of amino acid region A include, but are not limited to, EDGDAVIVVF (SEQ ID NO: 60), KEDGDAVIVVF (SEQ ID NO: 61), EDGYKAVFVVF (SEQ ID NO: 62), and KEDGYKAVFVVF (SEQ ID NO: 63). In one embodiment, it is preferred that part or all of the epitopes in amino acid region A comprise SEQ ID NO: 60 or 62. In a preferred embodiment, part or all of the epitopes in amino acid region A are SEQ ID NO: 60, 61, or 62, more preferably SEQ ID NO: 61 or 62. Examples of epitopes of a part or the whole of amino acid region B include, but are not limited to, HEAYGGY (SEQ ID NO: 64) and HEAYEAY (SEQ ID NO: 65). Examples of epitopes of part or all of amino acid region C include, but are not limited to, HLITPEWLW (SEQ ID NO: 66), KYGLRPEQWVDF (SEQ ID NO: 67), EKYGLRPDQWADY (SEQ ID NO: 68), KYGLRPDQWADY (SEQ ID NO: 69), GLRPEQWVDF (SEQ ID NO: 70), ITPEWLW (SEQ ID NO: 71), YLITPAWLWEKYGLRPDQWADY (SEQ ID NO: 72), HLITPEWLWEKYGLRPEQWVDF (SEQ ID NO: 73), and HLITPEWLWEKYGLKPEQWVDF (SEQ ID NO: 74). In one embodiment, the epitope of part or all of amino acid region C preferably comprises SEQ ID NO: 68, 70, or 71. In a preferred embodiment, the epitope of part or all of amino acid region C is SEQ ID NO: 66, 67, 68, 70, or 71. Examples of epitopes of part or all of amino acid region D include, but are not limited to, LERLEF (SEQ ID NO: 75), LERLEFGSLLH (SEQ ID NO: 76), LERLEFGSLLHEF (SEQ ID NO: 77), LRAFLERLERLEF (SEQ ID NO: 78), RAFLERLEF (SEQ ID NO: 79), RAFLERLEFGSLLH (SEQ ID NO: 80), LEFGSLLH (SEQ ID NO: 81), LEFGSLLHEF (SEQ ID NO: 82), and LRAFLERLERLEFGSLLHEF (SEQ ID NO: 83). In one embodiment, the epitopes of part or all of amino acid region D preferably comprise SEQ ID NO: 75, 76, 78, 79, or 81. In a preferred embodiment, the epitopes of part or all of amino acid region D are SEQ ID NO: 77, 78, 80, or 82.

[0031] The length of the epitope is not particularly limited, but is, for example, composed of 5 to 25 residues, preferably 6 to 20 residues, more preferably 6 to 15 residues, and even more preferably 7 to 14 residues.

[0032] In one embodiment, the heavy chain CDR1 of the antibody or fragment thereof of the present invention preferably consists of the following amino acid sequence: an amino acid sequence represented by formula (A-1) shown in Table 2; an amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence; or An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids of the amino acid sequence have been mutated (preferably by conservative substitution).

[0033] [Table 2]

[0034] In formula (A-1), X A3 is preferably T, and X A4 is preferably F, and X A5 is preferably D, N, or S, and X A6is preferably D, N, S, K, or H, or D, N, S, or H, and X A7 is preferably Y or W, or Y, and X A8 is preferably G, W, or Y.

[0035] The heavy chain CDR1 of the antibody or fragment thereof of the present invention preferably consists of an amino acid sequence represented by formula (A-1-1), more preferably an amino acid sequence selected from the group consisting of formulas (A-1-2) to (A-1-9), or an amino acid sequence having 90% or more identity to the amino acid sequence, preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more identity.

[0036] In one embodiment, the heavy chain CDR2 of the antibody or fragment thereof of the present invention preferably consists of the following amino acid sequence: an amino acid sequence represented by formula (B-1) shown in Table 3; an amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence; or An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids of the amino acid sequence have been mutated (preferably by conservative substitution).

[0037] [Table 3]

[0038] In formula (B-1), X B2 is preferably G, S, T, K, or N, G, S, or K, or S, and X B3 is preferably Y, L, G, T, or N, more preferably L, G, or N, L or N, or Y, G, or T; and X B4 is preferably G, S, T, D, or H, more preferably G, S, T, or D, S or T, or G, D, or H, and X B5 is preferably G or S, or G, and X B6 is preferably G, S, T, or D, G, S, or T, or G or S, and XB7 is preferably S, T, Y, D, or H, more preferably S, T, or Y, S or T, or T, Y, D, or H, and X B8 is preferably S, T, V, I, or M, more preferably S, T, or I, S or T, or T, V, I, or M.

[0039] The heavy chain CDR2 of the antibody or fragment thereof of the present invention preferably consists of an amino acid sequence represented by formula (B-1-1), more preferably an amino acid sequence selected from the group consisting of formulas (B-1-2) to (B-1-10), or an amino acid sequence having 90% or more identity to the amino acid sequence, preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more identity.

[0040] In one embodiment, the heavy chain CDR3 of the antibody or fragment thereof of the present invention preferably consists of the following amino acid sequence: an amino acid sequence represented by any one of formulas (C-1) to (C-6) shown in Table 4; an amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence; or An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids of the amino acid sequence have been mutated (preferably by conservative substitution).

[0041] [Table 4] TIFF0007777831000008.tif59152

[0042] In formula (C-1), X C1 is preferably A or R, more preferably R, and X C2 is preferably P or R, more preferably R, and X C3 is preferably T or I, more preferably T, and X C4 is preferably V or L, more preferably V, and X C5 is preferably P or A, more preferably P, and XC6 is preferably T or Y, more preferably T, and X C7 is preferably V, T, or N, more preferably V.

[0043] In formula (C-2), X C1 is preferably A or R, more preferably A, and X C2 is preferably P or R, more preferably P, and X C3 is preferably T or I, more preferably I, and X C4 is preferably V or L, and X C5 is preferably P or A, more preferably A, and X C6 is preferably T or Y, more preferably Y, and X C7 is preferably V, T, or N, more preferably V or T.

[0044] In formula (C-3), X C7 is preferably V, T, or N, more preferably N.

[0045] In formula (C-5), X C7 is preferably V, T, or N, more preferably V.

[0046] In formula (C-6), X C7 is preferably V, T, or N, more preferably V.

[0047] The heavy chain CDR3 of the antibody or fragment thereof of the present invention preferably consists of an amino acid sequence selected from the group consisting of formulas (C-1-1), (C-2-1), (C-3-1), (C-4), (C-5-1), and (C-6-1), more preferably an amino acid sequence selected from the group consisting of formulas (C-1-2), (C-2-2), (C-2-3), (C-3-2), (C-3-3), (C-4), (C-5-2), and (C-6-2), or an amino acid sequence having 90% or more identity to the amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0048] In one embodiment, the light chain CDR1 of the antibody or fragment thereof of the present invention preferably consists of the following amino acid sequence: an amino acid sequence represented by formula (D-1) shown in Table 5; an amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence; or An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids of the amino acid sequence have been mutated (preferably by conservative substitution).

[0049] [Table 5]

[0050] In formula (D-1), X D1 is preferably Q, and X D2 is preferably G or S, and X D3 is preferably V or I, and X D4 is preferably S or K, and X D5 is preferably S, N, or K, or S or N, and X D6 is preferably F or Y, or Y.

[0051] The light chain CDR1 of the antibody or fragment thereof of the present invention preferably consists of an amino acid sequence represented by formula (D-1-1), more preferably an amino acid sequence selected from the group consisting of formulas (D-1-2) to (D-1-8), or an amino acid sequence having 90% or more identity to the amino acid sequence, preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more identity.

[0052] In one embodiment, the light chain CDR2 of the antibody or fragment thereof of the present invention preferably consists of the following amino acid sequence: an amino acid sequence represented by formula (E-1) shown in Table 6A; an amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence; or An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids of the amino acid sequence have been mutated (preferably by conservative substitution).

[0053] [Table 6A]

[0054] In formula (E-1), X E1 is preferably G, T, D, Y, or R, more preferably G, Y, or R, or G, T, D, or R, even more preferably Y, or G, T, D, or R, and X E2 is preferably A, T, or V, more preferably A or T, or A or V, and X E3 is preferably K, D, N, or S, more preferably K, D, or N, or K or S, even more preferably D or N, or K or S.

[0055] The light chain CDR2 of the antibody or fragment thereof of the present invention preferably consists of an amino acid sequence represented by formula (E-1-1), more preferably an amino acid sequence represented by formula (E-1-2) or (E-1-3), and even more preferably an amino acid sequence selected from the group consisting of formulas (E-1-4) to (E-1-12), or an amino acid sequence having 90% or more identity to the amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0056] In one embodiment, the C-terminus of the light chain CDR2 of the antibody or fragment thereof of the present invention is preferably flanked by the following amino acid sequence: an amino acid sequence represented by formula (E-2) shown in Table 6B; an amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence; or An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids of the amino acid sequence have been mutated (preferably by conservative substitution).

[0057] [Table 6B]

[0058] In formula (E-2), X E4 is preferably S, R, N, Y, or T, more preferably S, or R, N, Y, or T, and X E5 is preferably L or R, or L, and X E6 is preferably A, P, or Y, more preferably A or P, or A or Y, and X E7 is preferably S or T, or S.

[0059] The C-terminus of the light chain CDR2 of the antibody or fragment thereof of the present invention is preferably flanked by an amino acid sequence represented by formula (E-2-1) or (E-2-2), more preferably an amino acid sequence selected from the group consisting of (E-2-3) to (E-2-8), or an amino acid sequence having 90% or more identity to said amino acid sequence, with the identity preferably being 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0060] The amino acid adjacent to the N-terminus of the light chain CDR2 of the antibody or fragment thereof of the present invention is not particularly limited, but is preferably Y, F, or H, and it is also preferable that these amino acids are conservatively substituted.

[0061] In one embodiment, the light chain CDR3 of the antibody or fragment thereof of the present invention preferably consists of the following amino acid sequence: an amino acid sequence represented by formula (F-1) or (F-2) shown in Table 7; an amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence; or An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids of the amino acid sequence have been mutated (preferably by conservative substitution).

[0062] [Table 7]

[0063] In formula (F-1), X F1 is preferably L, Q, F, or Y, more preferably L, Q, or Y, or Q, F, or Y, even more preferably L, Q, or Y, or Q or F, and X F2 is preferably Q, and X F3 is preferably S or Y, or Y, and X F4 is preferably G, N, Q, or Y, more preferably N, Q, or Y; G, Q, or Y; G, N, or Y; or G or Y; and X F5 is preferably S, N, or I, more preferably S or I, or S or N, and X F6 is preferably G, S, Y, or W, more preferably G, Y, or W; S, Y, or W; or G or S; and X F7 is preferably S, P, or W, more preferably P or W; S or P; or P, and X F8 is preferably L, P, H, or Y, more preferably L, H, Y, or T, or P.

[0064] The light chain CDR3 of the antibody or fragment thereof of the present invention preferably consists of the amino acid sequence represented by formula (F-1-1) or (F-2-1), more preferably the amino acid sequences represented by formulas (F-1-2) to (F-1-5) and (F-2-2) to (F-2-4), or an amino acid sequence having 90% or more identity to the amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0065] Suitable examples of combinations of heavy chain CDR1 to 3 and light chain CDR1 to 3 of the antibody or fragment thereof of the present invention are shown in Table 8A.

[0066] [Table 8A]

[0067] Suitable examples of combinations of sequences adjacent to the C-terminus of heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain CDR2 of the antibody or fragment thereof of the present invention are shown in Table 8B.

[0068] [Table 8B]

[0069] In combinations C14 to C26, it is also preferred that at least one of the amino acid sequences adjacent to the C-terminus of heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain CDR2 has one to three (preferably one or two, more preferably one) amino acid mutations (preferably conservative substitutions).

[0070] Regions other than heavy chain CDR1-3 and light chain CDR1-3 are not particularly limited and can have any amino acid sequence as long as they can be used in antibodies. Examples of constant regions that can be used include, but are not limited to, the constant regions of IgG1, IgG2, IgG3, IgA1, IgA2, and IgM. Furthermore, the constant regions may be derived from any animal, such as a mammal, such as a mouse, hamster, rat, guinea pig, rabbit, ferret, goat, monkey, or human. Examples of constant regions include the amino acid sequences shown in SEQ ID NOs: 51 and 52 (heavy chain and light chain constant regions derived from a guinea pig), the amino acid sequences shown in SEQ ID NOs: 53 and 54 (heavy chain and light chain constant regions derived from a mouse), and sequence regions consisting of amino acid sequences that share 80% or more (preferably 85% or more, 90% or more, or 95% or more) identity with these amino acid sequences.

[0071] The equilibrium dissociation constant (K D ) is, for example, 50 nM or less, preferably 10 nM or less, and for example, 1 pM or more. Dcan be measured using Biacore™ X100 (Cytiva) as in the Examples described below. Specifically, a ligand (a DNA polymerase having a 5' to 3' exonuclease active domain) is immobilized on carboxymethyl dextran of a CM5 sensor chip (Cytiva) by an amine coupling reaction using NHS (N-hydroxysuccinimide) and EDC (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride), and a flow cell on which the ligand is immobilized is prepared by blocking with 1 M ethanolamine hydrochloride solution. Then, serially diluted antibodies are added to the flow cell, and the reaction signals are analyzed, thereby determining the K. D can be calculated.

[0072] In one embodiment, when the antibody or fragment thereof of the present invention is allowed to coexist with a DNA polymerase at 37°C for 24 hours, the ability to inhibit the 5' to 3' exonuclease activity of the DNA polymerase is, for example, 50% or more, preferably 60% or more, 70% or more, 80% or more, or 90% or more. The inhibitory ability is measured by the use of the following components, as described in the Examples below: Radioisotope-labeled substrate nucleic acid λDNA; DNA polymerase alone (1 unit (U)), or DNA polymerase (1 U) and the antibody or fragment thereof of the present invention (0.005 μg / μL); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; and 1.5mM MgCl2 The radioactivity of the labeled base released when a solution containing the compound is incubated at 37°C for 24 hours is measured, and the radioactivity can be calculated using the following formula. Inhibitory effect on 5'→3' exonuclease activity (%) = (N2 - N3) ÷ (N2 - N1) × 100 N1: The amount of free nucleotides before incubating a solution not containing the antibody of the present invention or a fragment thereof at 37°C for 24 hours (or after incubating at -20°C for 24 hours), or the amount of free nucleotides after incubating a solution not containing the antibody of the present invention or a fragment thereof or DNA polymerase at 37°C for 24 hours. N2: Amount of free nucleotides after incubating a solution not containing the antibody of the present invention or a fragment thereof at 37°C for 24 hours N3: Amount of free nucleotides after incubating a solution containing the antibody of the present invention or a fragment thereof at 37°C for 24 hours

[0073] In another embodiment, when the antibody or fragment thereof of the present invention is allowed to coexist with substrate DNA (here, the substrate DNA may be single-stranded or double-stranded, and may also function as a probe) and DNA polymerase at 25°C for 24 hours, the inhibitory ability of the DNA polymerase against the 5'→3' exonuclease activity (ability to inhibit degradation of the substrate DNA) can also be quantitatively confirmed, for example, by the following method (1) or (2). (1) The antibody of the present invention or a fragment thereof is allowed to coexist with a fluorescently labeled substrate DNA (e.g., a fluorescently labeled probe) and the Ct values ​​of a reaction solution exposed to light at 25°C for 24 hours are compared with those of a control reaction solution (a reaction solution in the absence of the antibody of the present invention or a fragment thereof, after 24 hours of exposure at -20°C or before 24 hours of exposure at 25°C) in real-time PCR. A smaller difference in Ct values ​​indicates a higher inhibitory ability against the 5' to 3' exonuclease activity of DNA polymerase. (2) The antibody of the present invention or a fragment thereof is allowed to coexist with a fluorescently labeled substrate DNA (e.g., a fluorescently labeled probe) and a reaction solution exposed to light at 25°C for 24 hours is compared with a control reaction solution (a reaction solution in the absence of the antibody of the present invention or a fragment thereof, after 24 hours of exposure at -20°C, or before 24 hours of exposure at 25°C) in real-time PCR to determine the initial fluorescence intensity. The smaller the difference in fluorescence intensity, the higher the inhibitory effect on the 5'→3' exonuclease activity of DNA polymerase.

[0074] Examples of the reaction solution in (1) and (2) include: Contains the following ingredients: the antibody or fragment thereof of the present invention (0.06 μg / μL); Taq polymerase (0.05 U / μL) as set forth in SEQ ID NO: 49; anti-polymerase antibody for hot-start PCR (0.01 μg / μL); 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 0.3 mM dNTPs; cDNA derived from HeLa cell RNA (5 ng / μL); and Primer and probe solution (1 / 20 of the total liquid volume) a reaction solution comprising: Contains the following ingredients: the antibody or fragment thereof of the present invention (0.06 μg / μL); Tth polymerase set forth in SEQ ID NO: 50 or Z05 polymerase set forth in SEQ ID NO: 55 (0.05 U / μL); anti-polymerase antibody for hot-start PCR (0.01 μg / μL); 10 mM Tris-HCl (pH 8.3); 80 mM KCl; 1.5 mM MgCl2; 0.5 mg / mL BSA; 0.1% (v / v) TritonX-100; 0.1% (w / v) sodium cholate; 0.3 mM dNTPs; cDNA derived from HeLa cell RNA (5 ng / μL); and Primer and probe solution (1 / 20 of the total liquid volume) A reaction solution containing the above can be used.

[0075] As the primer and probe solutions in the reaction solution, for example, the following may be used: TaqMan® Gene Expression Assays from Thermo Fisher Scientific Co., Ltd. [gene: Interleukin 6, Assay ID: Hs00985639_m1 (hereinafter referred to as "IL6") Cyclin-dependent kinase 10, Assay ID: Hs00177586_m1 (hereafter referred to as "CDK10") APC, WNT signaling pathway regulator, Assay ID: Hs01568269_m1 (hereinafter abbreviated as "APC"); Mitogen-activated protein kinase 8, Assay ID: Hs00177083_m1 (hereafter referred to as "MAPK8") SIVA1 apoptosis inducing factor, Assay ID: Hs00276002_m1 (hereinafter abbreviated as "SIVA1") Ribosomal protein S19, Assay ID: Hs03044115_g1 (hereinafter abbreviated as "RPS19"), or serpin family B member 5, Assay ID: Hs00985285_m1 (hereinafter abbreviated as "SERPINB5")]

[0076] The reaction solution preferably satisfies one, two, or three of the following (a) to (c): (a) Ct value before 24 hours of exposure at 25°C (or after 24 hours of exposure at -20°C) / Ct value after 24 hours of exposure at 25°C ≥ 0.8 (b) Fluorescence intensity at the beginning of the cycle before 24 hours of exposure at 25°C (or after 24 hours of exposure at -20°C) / Fluorescence intensity at the beginning of the cycle after 24 hours of exposure at 25°C ≥ 0.3 (In the formula, the fluorescence intensity at the beginning of the cycle refers to the fluorescence intensity in real-time PCR until the amplification curve starts to rise, and the beginning of the cycle usually refers to the first to 30th cycles.) (c) Fluorescently labeled substrate DNA degradation rate ≦40% (wherein the formula, the rate of degradation of fluorescently labeled substrate DNA can be calculated by the following formula: Fluorescent substrate DNA degradation rate (%) = (F 13 -F11 )÷(F 12 -F 11 ) x 100 F 11 Fluorescence intensity at the beginning of the cycle before 24 hours of exposure at 25°C (or after 24 hours of exposure at -20°C) in the absence of the antibody of the present invention or a fragment thereof F 12 Fluorescence intensity at the beginning of the cycle after 24 hours of exposure at 25°C in the absence of the antibody of the present invention or a fragment thereof F 13 : Fluorescence intensity at the beginning of the cycle after 24 hours of exposure at 25°C when the antibody of the present invention or a fragment thereof is included)

[0077] The value in (a) above (Ct value ratio) is preferably 0.9 or more. The value in (b) above (fluorescence intensity ratio) is preferably 0.35 or more, more preferably 0.5 or more, and even more preferably 0.7 or more. The value in (c) above (fluorescence-labeled substrate DNA degradation rate) is preferably 30% or less, more preferably 20% or less.

[0078] In still another embodiment, when the antibody or fragment thereof of the present invention is allowed to coexist with substrate DNA (here, the substrate DNA may be single-stranded or double-stranded, and may also function as a probe) and DNA polymerase at 25°C or 37°C for 24 hours, the inhibitory ability of the DNA polymerase against the 5'→3' exonuclease activity (ability to inhibit degradation of the substrate DNA) can also be quantitatively confirmed, for example, by the method (3) or (4) below. (3) A solution in which the antibody of the present invention or a fragment thereof is coexistent with substrate DNA (e.g., double-stranded substrate DNA) and exposed at 25°C for 24 hours is compared by gel electrophoresis with a control solution (a solution after 24 hours of exposure at -20°C in the absence of the antibody of the present invention or a fragment thereof or before 24 hours of exposure at 25°C) to compare the band intensities of the substrate DNA. A smaller difference in band intensities indicates a higher inhibitory ability against the 5' to 3' exonuclease activity of DNA polymerase. (4) A reaction solution in which an antibody of the present invention or a fragment thereof is coexistent with fluorescently labeled substrate DNA (e.g., double-stranded substrate DNA in which at least one strand is fluorescently labeled) and exposed at 37°C for 24 hours is compared with a control reaction solution (a reaction solution after 24 hours of exposure at -20°C in the absence of the antibody of the present invention or a fragment thereof or before 24 hours of exposure at 37°C) in terms of fluorescence intensity at the beginning of the cycle in real-time PCR or fluorescence intensity as measured by a spectrophotometer. A smaller difference in fluorescence intensity indicates a higher inhibitory ability against the 5'→3' exonuclease activity of DNA polymerase.

[0079] Examples of the solution or reaction solution in (3) and (4) include: Contains the following ingredients: the antibody or fragment thereof of the present invention (0.06 μg / μL); Taq polymerase (0.05 U / μL) as set forth in SEQ ID NO: 49; anti-polymerase antibody for hot-start PCR (0.01 μg / μL); 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 0.3μM substrate DNA; containing, or Contains the following ingredients: the antibody or fragment thereof of the present invention (0.06 μg / μL); Tth polymerase set forth in SEQ ID NO: 50 or Z05 polymerase set forth in SEQ ID NO: 55 (0.05 U / μL); anti-polymerase antibody for hot-start PCR (0.01 μg / μL); 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 0.3μM substrate DNA; can be used that includes:

[0080] In method (3), the substrate DNA may be labeled with a fluorescent dye, a radioisotope, or the like, or may be unlabeled. Examples of double-stranded substrate DNA include, but are not limited to, double-stranded substrate DNA in which the 3' end of at least one strand overhangs the 5' end of the other strand, as exemplified by the combination of SEQ ID NO: 56 and SEQ ID NO: 57. The base length of the overhang is, for example, approximately 3 to 10 bases. Examples of gel electrophoresis include, but are not limited to, agarose gel electrophoresis and polyacrylamide gel electrophoresis. It is preferable to use an apparatus capable of quantifying the band intensity of nucleic acids. Examples of such an apparatus include, but are not limited to, a microchip electrophoresis apparatus for DNA / RNA analysis (MultiNA, Shimadzu Corporation) or a fully automated high-throughput electrophoresis system (TapeStation series, Agilent Technologies, Inc.).

[0081] In the method (4), the substrate DNA is preferably fluorescently labeled. Examples of double-stranded substrate DNA include, but are not limited to, double-stranded substrate DNAs in which the 3'-end of at least one strand overhangs the 5'-end of the other strand and at least one end of the other strand is fluorescently labeled, as represented by the combination of SEQ ID NO: 58 and SEQ ID NO: 59. The base length of the overhanging portion is, for example, about 3 to 10 bases. The change in fluorescence value can be measured, for example, using a real-time PCR device or a spectrophotometer, but is not limited to this.

[0082] The above solution or reaction liquid preferably satisfies one or two of the following conditions (d) and (e): (d) Substrate DNA degradation rate ≦40% (wherein the formula, the substrate DNA degradation rate can be calculated by the following formula: Substrate DNA degradation rate (%) = (S 11 -S 13 )÷(S 11 -S 12 ) x 100 S 11: Band intensity before 24 hours of exposure at 25°C (or after 24 hours of exposure at -20°C) in the absence of the antibody of the present invention or a fragment thereof S 12 : Band intensity after 24 hours of exposure at 25°C in the absence of the antibody of the present invention or a fragment thereof S 13 (Band intensity after 24 hours of exposure at 25°C when the antibody of the present invention or a fragment thereof is included) (e) Fluorescently labeled substrate DNA degradation rate ≦40% (wherein the formula, the rate of degradation of fluorescently labeled substrate DNA can be calculated by the following formula: Fluorescent substrate DNA degradation rate (%) = (F 23 -F 21 )÷(F 22 -F 21 )×100) F 21 Fluorescence intensity at the beginning of the cycle before 24 hours of exposure at 37°C (or after 24 hours of exposure at -20°C) in the absence of the antibody of the present invention or a fragment thereof F 22 Fluorescence intensity at the beginning of the cycle after 24 hours of exposure at 37°C in the absence of the antibody of the present invention or a fragment thereof F 23 Fluorescence intensity at the beginning of the cycle after 24 hours of exposure at 37°C when the antibody of the present invention or a fragment thereof is included Early cycle: usually cycles 1-30 Fluorescence intensity: Fluorescence intensity in real-time PCR

[0083] The rate of decomposition of the substrate DNA in (d) above is preferably 30% or less, more preferably 20% or less. The rate of decomposition of the fluorescently labeled substrate DNA in (e) above is preferably 30% or less, more preferably 20% or less.

[0084] The antibody of the present invention or a fragment thereof can suppress nucleic acid degradation caused by the 5' to 3' exonuclease activity of DNA polymerase, even when coexisting with DNA polymerase and nucleic acids such as nucleic acid templates, primers, and probes, for example, for 24 hours, 48 ​​hours, or 72 hours at 25° C., or for 24 hours at 37° C. Therefore, the antibody of the present invention or a fragment thereof can be suitably used to improve the stability of nucleic acid amplification reagents and the like.

[0085] The antibody or a fragment thereof of the present invention can be obtained, for example, by immunizing an animal with a portion or all of a DNA polymerase containing domain E as an immunogen. The immunogen is preferably a portion containing domain E of a DNA polymerase, more preferably a portion containing domain E of at least one DNA polymerase selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase, and even more preferably a portion containing domain E of Tth polymerase. Examples of animals include, but are not limited to, mammals such as mice, hamsters, rats, guinea pigs, rabbits, ferrets, goats, monkeys, and humans.

[0086] The antibodies of the present invention can be obtained by screening antibodies produced by animals immunized with the above-mentioned immunogens. For example, when a portion containing domain E of DNA polymerase is used as the immunogen, screening may be performed using the binding ability to the whole DNA polymerase as an index. When the whole DNA polymerase is used as the immunogen, screening may be performed using the binding ability to the portion containing domain E of DNA polymerase, or the difference between the binding ability to the whole DNA polymerase and the binding ability to the DNA polymerase other than the portion containing domain E as an index. In one embodiment, screening is preferably performed using a portion containing domain E of DNA polymerase as the immunogen and the binding ability to the whole DNA polymerase as an index. It is even more preferable to screen using a portion containing domain E of Tth polymerase as the immunogen and the binding ability to the whole DNA polymerase as an index.

[0087] Specific screening methods may include, for example, the hybridoma method in which mammalian spleen cells are fused with myeloma cells, or the phage display method in which antibodies having affinity for a target molecule are selected from an antibody phage library. Alternatively, a method may be used in which antigen-specific plasma cells are selected from an immunized animal, antibody genes (full-length or part of the variable region, etc.) are isolated, and recombinant antibodies with high affinity for the antigen are obtained.

[0088] Methods for selecting antigen-specific plasma cells include, for example, the method described in U.S. Patent Application Publication No. 2014 / 031528 (incorporated by reference in its entirety) and the method described in U.S. Patent Application Publication No. 2018 / 292407 (incorporated by reference in its entirety). In the former method, antigen-specific plasma cells can be identified by treating a cell suspension prepared from an immunized animal with a fluorescently labeled antigen and an endoplasmic reticulum-affinity fluorescent dye, fluorescently labeling antibodies expressed on the cell surface. In the latter method, antigen-specific plasma cells can be identified by fixing a cell population containing antibody-producing cells with a crosslinking agent and lysing the cell membrane with a surfactant, allowing antibodies expressed inside the cells to bind to the fluorescently labeled antigen. In this method, single-cell analysis using a cell sorter can be performed to isolate at least one plasma cell bound to the target antigen. Furthermore, this method can use a fluorescent probe that has high staining selectivity for the endoplasmic reticulum of cells and can distinguish plasma cells and plasmablasts from other cells. As the fluorescent probe, for example, those described in US Patent Application Publication No. 2013 / 029325 (the entire specification is incorporated by reference) can be used.

[0089] Methods for obtaining antibody genes from antigen-specific plasma cells include, but are not limited to, the hybridoma method and antibody gene cloning. Examples of the latter method include a method for obtaining antibody genes by extracting mRNA from antigen-specific plasma cells, performing reverse transcription, and synthesizing cDNA, and this may be the method described in U.S. Patent Application Publication No. 2011 / 020879 (incorporated by reference in its entirety). This method involves extracting mRNA from antigen-specific plasma cells using magnetic beads and obtaining antibody genes by RT-PCR, and utilizes a reaction device capable of performing multiple sequential reactions in parallel, such as cDNA synthesis from mRNA and DNA amplification, optionally including a washing step.

[0090] A method for obtaining a recombinant antibody from an antibody gene may include, for example, constructing an antibody expression vector containing the antibody gene and expressing the antibody from the antibody expression vector. Examples of such methods include those described in U.S. Patent Application Publication No. 2013 / 023009 (incorporated herein by reference in its entirety) and U.S. Patent Application Publication No. 2011 / 117609 (incorporated herein by reference in its entirety). The former method involves ligating one or more double-stranded DNA fragments to a PCR amplification product containing a target gene sequence, thereby specifically producing a ligated DNA fragment containing a sequence derived from the target gene of interest without purifying the PCR amplification product. The latter method involves retaining, in the homologous recombination regions at both ends of a linearized vector, sequences inside the amplification primer sequence that are present only in the amplification primer sequence and the target gene, allowing selective homologous recombination of the target DNA fragment to construct a vector.

[0091] The antibodies or fragments thereof of the present invention can also be obtained by genetic engineering techniques based on the amino acid sequence information of the antibodies or fragments thereof obtained by the above-mentioned methods. For example, they can be obtained by expressing in any host cell known in the art an expression vector incorporating an antibody gene designed so that the light chain CDR1 to 3 and, optionally, the C-terminal flanking region of the light chain CDR2 each have 80% or more identity to the amino acid sequences of the light chain CDR1 to 3 and, optionally, the C-terminal flanking region of the light chain CDR2 of the antibody obtained by the above-mentioned methods, and the heavy chain CDR1 to 3 each have 80% or more identity to the amino acid sequences of the heavy chain CDR1 to 3 of the antibody obtained by the above-mentioned methods.

[0092] 3. Polynucleotides Preferably, the polynucleotide of the present invention comprises a coding sequence for the antibody or fragment thereof described in 2 above.

[0093] In one embodiment, the polynucleotide of the present invention preferably comprises an expression cassette for the antibody or fragment thereof described in 2 above. The expression cassette is not particularly limited as long as it enables expression in a host cell, and includes, for example, a promoter and a coding sequence placed under the control of the promoter.

[0094] The promoter is not particularly limited and can be selected appropriately depending on the type of host cell. For example, various Pol II promoters can be used. Pol II promoters are not particularly limited, but examples include the CMV promoter, EF1 promoter, SV40 promoter, and MSCV promoter. Other examples of promoters include tryptophan promoters such as trc and tac; lac promoters; T7 promoters; T5 promoters; T3 promoters; SP6 promoters; arabinose-inducible promoters; cold shock promoters; and tetracycline-inducible promoters.

[0095] The expression cassette may contain other elements as necessary. Examples of other elements include a multiple cloning site (MCS), a drug resistance gene, a replication origin, an enhancer sequence, a repressor sequence, an insulator sequence, a reporter protein coding sequence, and a drug resistance gene coding sequence. These elements may be used alone or in combination of two or more.

[0096] The polynucleotide of the present invention may be in the form of, for example, a vector. An appropriate vector is selected depending on the intended use, the type of host cell, and the like. For example, examples of vectors using E. coli as a host include M13 phage or modified forms thereof, λ phage or modified forms thereof, and pBR322 or modified forms thereof (e.g., pB325, pAT153, pUC8); examples of vectors using yeast as a host include pYepSec1, pMFa, pYES2, and pPIC3.5K; examples of vectors using insect cells as a host include pAc and pVL; and examples of vectors using mammalian cells as a host include pcDNA, pCDM8, and pMT2PC.

[0097] 4.Cells The cells of the present invention preferably contain the polynucleotide described in 3 above. Examples of the cells include Escherichia coli such as Escherichia coli K12, Bacillus bacteria such as Bacillus subtilis MI114, yeast such as Saccharomyces cerevisiae AH22, the Sf cell line derived from Spodoptera frugiperda or the HighFive cell line derived from Trichoplusia ni, insect cells such as olfactory nerve cells, and animal cells. Examples of the animal cells include preferably cultured cells derived from mammals, specifically COS7 cells, CHO cells, HEK293 cells, Expi293 cells, 293F cells, 293T cells, 293FT cells, Hela cells, PC12 cells, N1E-115 cells, and SH-SY5Y cells.

[0098] In one embodiment, the cell of the present invention preferably expresses an antibody or a fragment thereof that specifically binds to domain E of DNA polymerase.

[0099] In one embodiment, the cells of the present invention preferably secrete or have on their cell surface an antibody or fragment thereof that specifically binds to domain E of DNA polymerase.

[0100] 5. Reagents The reagent of the present invention preferably comprises the antibody or fragment thereof described in 2 above, the polynucleotide described in 3 above, or the cell described in 4 above. The reagent of the present invention preferably further comprises an excipient or carrier and / or an additive.

[0101] Examples of the excipient or carrier include starch, lactose, crystalline cellulose, sorbitol, calcium hydrogen phosphate, water, ethanol, (poly)ethylene glycol, (poly)propylene glycol, glycerol, vegetable oil, etc. These may be used alone or in combination of two or more.

[0102] Examples of the additives include buffering agents, isotonicity agents, thickening agents, chelating agents, emulsifying agents, coloring agents, preservatives, etc. These may be used alone or in combination of two or more.

[0103] The reagent of the present invention is preferably a reagent for nucleic acid amplification.

[0104] In one embodiment, the reagent of the present invention preferably comprises a DNA polymerase having domain E, and an antibody or a fragment thereof that specifically binds to domain E of the DNA polymerase. The molar ratio of the antibody or fragment thereof to the DNA polymerase is not limited as long as the effects of the present invention are achieved, but is preferably about 1:1 to about 500:1. The reagent may further comprise a DNA polymerase that does not have domain E. The reagent is preferably a reagent for nucleic acid amplification.

[0105] In one embodiment, the reagent of the present invention preferably comprises at least one selected from the group consisting of a DNA polymerase having domain E, a primer, a probe, and deoxyribonucleoside-5'-phosphate, and an antibody or a fragment thereof that specifically binds to domain E of the DNA polymerase (preferably an antibody that binds to at least one (e.g., one or two) epitope present in any of amino acid regions A to D in domain E). The reagent may further comprise a metal salt such as manganese or magnesium, a buffer, etc., in order to improve DNA polymerase activity. The reagent is preferably a reagent for nucleic acid amplification.

[0106] When the reagent of the present invention contains a DNA polymerase having domain E, examples of the DNA polymerase include those described in 2 above.

[0107] When the reagent of the present invention contains primers, the primers may be at least two types of primers. The at least two types of primers may be oligonucleotides substantially complementary to the nucleic acid sequence to be amplified, defining both ends of the nucleic acid sequence to be amplified, and functioning as templates for further synthesis when the extension products synthesized from each primer are separated from their complements. The primers may be appropriately selected and designed depending on the target nucleic acid and are not particularly limited. Furthermore, if the target nucleic acid is expected to be a subtype, degenerate primers may be used. Generally, the primers may be oligonucleotides having 12 to 60 nucleotides. The primers may be synthesized using a DNA synthesizer or isolated from a biological source.

[0108] When the reagent of the present invention contains a probe, the probe may be a hybridization probe labeled with at least one type of labeling substance. By using such a probe, analysis of nucleic acid amplification products can be monitored by monitoring fluorescent signals rather than by conventional electrophoresis, thereby reducing analytical labor. Furthermore, there is no need to open the reaction vessel, further reducing the risk of contamination. For example, it is possible to identify the subtype of a target nucleic acid by labeling each hybridization probe corresponding to a subtype of the nucleic acid sequence to be detected with a different fluorescent dye. Examples of hybridization probes include TaqMan hydrolysis probes [U.S. Pat. Nos. 5,210,015, 5,538,848, 5,487,972, and 5,804,375 (incorporated herein by reference in their entireties)], molecular beacons [U.S. Pat. No. 5,118,801 (incorporated herein by reference in their entireties)], and FRET hybridization probes [WO 97 / 46707, WO 97 / 46712, and WO 97 / 46714 (incorporated herein by reference in their entireties)].

[0109] The reagent of the present invention may contain a double-stranded DNA-binding fluorescent compound instead of a probe, including, but not limited to, SYBR® Green I, SYBR® Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), EvaGreen® (Biotium), LCGreen (Idaho), and LightCycler® 480 ResoLight (Roche Applied Science).

[0110] When the reagent of the present invention contains a deoxyribonucleoside-5'-phosphate, the deoxyribonucleoside-5'-phosphate is, for example, dATP, dCTP, dTTP, dGTP, or a mixture thereof. Note that the terms dATP and the like also include chemically modified deoxyribonucleoside-5'-phosphates.

[0111] When the reagent of the present invention is a reagent for nucleic acid amplification, examples of nucleic acid amplification methods include, but are not limited to, PCR, Loop-Mediated Isothermal Amplification (LAMP), Transcription Reverse Transcription Concerted Reaction (TRC), and Nucleic Acid Sequence-Based Amplification (NASBA). The nucleic acid amplification method is preferably PCR. Among PCR methods, a preferred example is hot-start PCR, which inhibits primer annealing up to a predetermined temperature using a monoclonal antibody specific to DNA polymerase. The hot-start PCR reagent of the present invention can more effectively suppress nonspecific reactions by containing a combination of an antibody that specifically binds to the polymerase activity domain of DNA polymerase and an antibody that specifically binds to domain E of DNA polymerase. The hot-start PCR reagent of the present invention preferably contains a primer, deoxyribonucleoside-5'-phosphate, DNA polymerase, an antibody that specifically binds to the polymerase activity domain of DNA polymerase, and an antibody that specifically binds to domain E of DNA polymerase. The reagent is mixed with a reagent containing the target nucleic acid, and the resulting mixture is heated to 60°C or higher (e.g., 95°C for 20 seconds or more) to inactivate both antibodies and allow the formation of primer extension products. [Example]

[0112] The present invention will be specifically explained below with reference to test examples, although the present invention is not limited to the following test examples.

[0113] Test Example 1. Preparation of antigen When whole DNA polymerase was used as the antigen, Taq polymerase having the amino acid sequence of SEQ ID NO: 49 (TAP-201, Toyobo Co., Ltd., hereinafter referred to as "whole Taq") and Tth polymerase having the amino acid sequence of SEQ ID NO: 50 (TTH-301, Toyobo Co., Ltd., hereinafter referred to as "whole Tth") were used. The sequence identity between whole Taq and whole Tth is approximately 87%.

[0114] When domain E of DNA polymerase was used as the antigen, a polypeptide having the amino acid sequence of SEQ ID NO: 1 (from the N-terminus to the 290th amino acid of whole Taq) (hereafter referred to as "Taq exo") and a polypeptide having the amino acid sequence of SEQ ID NO: 2 (from the N-terminus to the 292nd amino acid of whole Tth) (hereafter referred to as "Tth exo") were expressed in E. coli JM109 strain and purified using heparin-Sepharose chromatography. Both antigens were dissolved in phosphate buffer.

[0115] Test Example 2. Guinea pig immunization Slc:Hartley guinea pigs (7-week-old males) were injected subcutaneously (in the lumbar region) on the back with 0.8 mL of an antigen preparation containing 400 μg of antigen. The antigen preparation used was an emulsion prepared by mixing the antigen solution prepared in Test Example 1, in which the antigen was dissolved in phosphate buffer, with the adjuvant TiterMAX Gold (TiterMAX) at a 1:1 (volume ratio). Three weeks later, a booster immunization was performed by injecting 0.8 mL of the antigen preparation containing 400 μg of antigen. Three weeks later, a booster immunization was performed by injecting 0.4 mL of the antigen solution containing 400 μg of antigen. Lymph node swelling in the guinea pigs after immunization increased in the following order: whole Taq, Taq exo, whole Tth, and Tth exo.

[0116] Test Example 3. Preparation of fluorescently labeled proteins The entire DNA polymerase and the DNA polymerase lacking domain E were each fluorescently labeled. The DNA polymerase lacking domain E was obtained by expressing Taq polymerase (hereinafter referred to as "ΔTaq") in which the 289th amino acid from the N-terminus of SEQ ID NO: 49 was deleted, and Tth polymerase (hereinafter referred to as "ΔTth") in which the 291st amino acid from the N-terminus of SEQ ID NO: 50 was deleted, using the E. coli JM109 strain, and purifying them using heparin Sepharose chromatography.

[0117] Whole Taq and whole Tth were fluorescently labeled using DyLight™ 488 NHS Ester (Thermo Fisher Scientific), while ΔTaq and ΔTth were fluorescently labeled using DyLight™ 550 NHS Ester (Thermo Fisher Scientific).

[0118] Test Example 4. Isolation of domain E-specific plasma cells and construction of antibody expression vector Using the methods described in U.S. Patent Application Publication Nos. 2014 / 031528, 2018 / 292407, and 2013 / 029325, cell suspensions were prepared from the iliac lymph nodes of the guinea pigs immunized in Test Example 2, and domain E-specific plasma cells were selected using a flow cytometer. Domain E-specific plasma cells were selected using the following five methods, varying the combination of the antigen used for immunization and the fluorescently labeled protein prepared in Test Example 3.

[0119] [Method 1] From cells immunized with whole Taq, domain E-specific plasma cells were selected by subtraction using DyLight488-labeled whole Taq and DyLight550-labeled ΔTaq. That is, plasma cells that showed fluorescence corresponding to DyLight488 but not DyLight550 were selected.

[0120] [Method 2] From cells immunized with Taq exo, domain E-specific plasma cells were selected using DyLight488-labeled whole Taq.

[0121] [Method 3] From cells immunized with Tth exo, domain E-specific plasma cells were selected using DyLight488-labeled whole Taq.

[0122] [Method 4] From cells immunized with whole Tth, domain E-specific plasma cells were selected by subtraction using DyLight488-labeled whole Tth and DyLight550-labeled ΔTth. That is, plasma cells that showed fluorescence corresponding to DyLight488 but not DyLight550 were selected.

[0123] [Method 5] From cells immunized with Tth exo, domain E-specific plasma cells were selected using DyLight488-labeled whole Tth.

[0124] The number of plasma cells selected using domain E of Taq polymerase as a target was higher in method 3 than in methods 1 and 2, reaching 288 in method 3. The number of plasma cells selected using domain E of Tth polymerase as a target was 192 in method 4 and 240 in method 5.

[0125] Using the plasma cells selected by methods 3 to 5, antibody expression vectors were constructed according to the methods described in U.S. Patent Application Publication Nos. 2011 / 020879, 2013 / 023009, and 2011 / 117609. The guinea pig heavy and light chain constant regions used herein were the amino acid sequences set forth in SEQ ID NOs: 51 and 52, respectively. 22 antibody expression vectors were obtained from method 3, 9 from method 4, and 66 from method 5.

[0126] Methods 3 to 5 resulted in greater lymph node swelling and a greater number of isolated plasma cells in guinea pigs than methods 1 and 2, suggesting that Tth polymerase elicited a stronger immune response as an antigen than Taq polymerase. Therefore, it was demonstrated that domain E-specific plasma cells can be efficiently selected for both Taq and Tth polymerase by using Tth polymerase as an antigen. Furthermore, method 5 yielded a greater number of isolated plasma cells and antibody expression vectors than method 4. Therefore, it was demonstrated that immunization with domain E alone can more efficiently obtain antibodies that specifically bind to domain E (anti-domain E antibodies) than immunization with the entire DNA polymerase.

[0127] In the following test examples, antibodies obtained by expressing the antibody expression vectors obtained by methods 3 to 5 were used.

[0128] Test Example 5. Evaluation of antibody binding ability to domain E Antibody expression vectors were introduced into 293FT cells using the method described in U.S. Patent Application Publication No. 2018 / 292407, and the culture supernatant containing the secreted antibody was collected. A commercially available hot-start antibody (TCP-101, Toyobo Co., Ltd.) was immobilized on an ELISA plate (Sumitomo Bakelite Co., Ltd., MS-8896F) using carbonate buffer. After washing each well, blocking was performed using 1x TBS (Nacalai Tesque) containing 1% (w / v) bovine serum albumin (globulin-free, Nacalai Tesque). After washing each well, antigens (whole Taq, whole Tth) diluted with 1x TBS-T (Nacalai Tesque) were added to each well. After washing each well, the culture supernatant was added to each well. After washing each well, Goat Anti-Guinea pig IgG H&L (HRP) (Abcam) diluted 50,000 times was added. After washing each well, TMB solution (TMBW-1000-01, SURMODICS) was added to develop color, and the reaction was stopped by adding 1N sulfuric acid (Nacalai Tesque). Wavelengths of 450 to 620 nm were measured using a plate reader. In this binding assay, the DNA polymerase activity domain is occupied by the immobilized antibody, so the binding ability of the antibody to domain E is evaluated.

[0129] Of the 22 antibodies obtained by Method 3, 20 antibodies bound to whole Taq (hit rate 91%), and 19 antibodies bound to both whole Taq and whole Tth (hit rate 86%).

[0130] Of the nine antibodies obtained by Method 4, one antibody bound to whole Tth (hit rate 11%), and this antibody did not show binding to whole Taq.

[0131] Of the 66 antibodies obtained by Method 5, 32 antibodies bound to whole Tth (hit rate: 48%), and 12 antibodies bound to both whole Tth and whole Taq (hit rate: 18%).

[0132] Compared with Methods 4 and 5, Method 3 showed a 2- to 8-fold higher hit rate for each DNA polymerase and for both DNA polymerases. These results suggest that the method of using fluorescently labeled whole Taq and Tth Exo, which strongly induces an immune response, as an antigen to select domain E-specific plasma cells is an efficient method for obtaining antibodies that specifically bind to domain E of Taq polymerase. Furthermore, the method of using fluorescently labeled whole Tth and Tth Exo as an antigen to select domain E-specific plasma cells produced unexpected results, demonstrating a high probability of isolating domain E-specific antibodies.

[0133] Test Example 6. Degradation of probe by DNA polymerase containing domain E It was confirmed that the probe was decomposed when a PCR reaction mixture containing a DNA polymerase having domain E was exposed to 25°C for 24 hours.

[0134] (1) Components of PCR reaction solution [PCR mix] PCR mix 1 having the composition shown below was prepared. PCR Mix 1: Taq polymerase (0.05 U / μL, TAP-201, Toyobo Co., Ltd.); Anti-polymerase antibody for hot start PCR (0.01 μg / μL, TCP-101, Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; and 0.3 mM dNTPs.

[0135] [Primers / Probes] TaqMan® Gene Expression Assays (Thermo Fisher Scientific) was used as a 20x primer / probe mixture, and the genes amplified / detected by the primers / probes in this mixture were IL6, CDK10, APC, MAPK8, SIVA1, RPS19, or SERPINB5.

[0136] [Nucleic acid template] cDNA was prepared from HeLa cell RNA (derived from human cervical cancer). RNA was extracted and cDNA was synthesized using Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep™ II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) according to the manufacturer's instructions.

[0137] (2) Reaction A 20 μL PCR reaction mixture was prepared by mixing PCR Mix 1 with each primer / probe in a 1:20 ratio and the nucleic acid template in a 1:20 ratio. The PCR reaction mixture was stored at -20°C or 25°C for 24 hours. The reaction was then carried out using a real-time PCR device (Applied Biosystems 7500 Fast Real-Time PCR System) with the following temperature cycle: Fluorescence readings were taken at 60°C with a 60-second extension step. (Temperature cycle) Step 1: 95℃ 1 minute Step 2: 95°C 15 seconds - 60°C 60 seconds, 50 cycles (PCR)

[0138] (3) Results In this reaction system, each gene is detected in the FAM channel. Table 9 shows the Ct values ​​when each gene (IL6, CDK10, APC, MAPK8, SIVA1, RPS19, SERPINB5) in HeLa cDNA was detected using real-time PCR, and the fluorescence value at the 10th cycle in the multicomponent data.

[0139] When the PCR reaction solution was exposed to 25°C for 24 hours, a delay of 2 or more in the Ct values ​​was observed for the IL6, CDK10, and SIVA1 genes compared to when it was exposed to -20°C for 24 hours, and no Ct value was calculated for the RPS19 gene (indicated by "-" in the results table). The reason that a Ct value was not calculated for the RPS19 gene is presumably because all of the probe was degraded, preventing an increase in the fluorescence value corresponding to the amplified product.

[0140] Furthermore, when the PCR reaction solution was exposed to 25°C for 24 hours, the fluorescence value at the 10th cycle for all seven genes increased compared to when it was exposed to -20°C for 24 hours. The reason for the increase in fluorescence value is presumed to be that the fluorescently labeled probe was decomposed before the start of the cycles during 24 hours of exposure at 25°C, liberating the fluorescent label, which resulted in the release of fluorescence due to the release of quenching by the quencher. Therefore, we found that all of the fluorescently labeled probes detecting the seven genes were decomposed when exposed to 25°C for 24 hours.

[0141] [Table 9]

[0142] Test Example 7. Inhibition of probe degradation by anti-domain E antibody When a PCR reaction mixture containing a DNA polymerase having domain E and an anti-domain E antibody was exposed to light at 25°C for 24 hours, it was confirmed whether the anti-domain E antibody would suppress degradation of the probe.

[0143] (1) Preparation of anti-domain E antibody Using the method described in U.S. Patent Application Publication No. 2018 / 292407, 293FT cells were transfected with the antibody expression vectors obtained by the methods described in Test Examples 1 to 4, and the culture supernatant containing the secreted antibody was collected. The culture supernatant was passed through a HiTrap Protein A HP column (Cytiva) using AKTA pure 25 (Cytiva) to adsorb the antibody. The column was washed with wash buffer (20 mM phosphate buffer, pH 7.4) and then eluted with elution buffer (0.1 M citric acid-NaOH, pH 3.5). The antibody was concentrated using an Amicon Ultra-15 (Merck) and quantified using a Nanodrop One (Thermo Fisher Scientific). Clone numbers Anti-TAQ1 to Anti-TTH1 to Anti-TTH5 are anti-domain E antibodies obtained by Method 3 of Test Example 4, and clone numbers Anti-TTH1 to Anti-TTH5 are anti-domain E antibodies obtained by Method 5 of Test Example 4.

[0144] (2) Components of PCR reaction solution [PCR mix] The same PCR mix 1 as used in Test Example 6 was used. In addition, the following two types of PCR mixes 2 and 3 were prepared and used. PCR Mix 2: Tth polymerase (0.05 U / μL, TTH-301, Toyobo Co., Ltd.); Anti-polymerase antibody for hot-start PCR (0.01 μg / μL, TCP-101, Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.3); 80 mM KCl; 1.5 mM MgCl2; 0.5 mg / mL BSA; 0.1% (v / v) TritonX-100; 0.1% (w / v) sodium cholate; and 0.3 mM dNTPs. PCR Mix 3: Tth polymerase (mutant) described in WO 2018 / 096961 (0.05 U / μL); Anti-polymerase antibody for hot-start PCR (0.01 μg / μL, TCP-101, Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.3); 80 mM KCl; 1.5 mM MgCl2; 0.5 mg / mL BSA; 0.1% (v / v) TritonX-100; 0.1% (w / v) sodium cholate; and 0.3 mM dNTPs.

[0145] [Primers / Probes] TaqMan® Gene Expression Assays (Thermo Fisher Scientific) was used as a 20x primer / probe mixture, and the gene amplified / detected by the primers / probe in this mixture was RPS19.

[0146] [Nucleic acid template] cDNA was prepared from HeLa cell RNA (derived from human cervical cancer). RNA was extracted and cDNA was synthesized using Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep™ II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) according to the manufacturer's instructions.

[0147] (3) Reaction (Reaction solution 1) A 19 μL mixture was prepared by mixing PCR Mix 1 with primers / probes at a ratio of 1:20 and nucleic acid template at a ratio of 1:20. As a control, 1 μL of 20 mM Tris-HCl (pH 7.5) was added to the mixture and exposed to temperatures of -20°C or 25°C for 24 hours. Similarly, as a control, 1 μL of Platinum Taq Monoclonal Antibody (10965-028, Thermo Fisher Scientific) was added to the mixture and exposed to temperatures of 25°C for 24 hours. For the anti-domain E antibody, 1 μL of a 0.8 mg / mL solution was added to the mixture (introduced amount: 0.8 μg) and exposed to temperatures of 25°C for 24 hours. (Reaction solution 2) A 19 μL mixture was prepared by mixing PCR mix 2 with primers / probes at a ratio of 1:20 and nucleic acid template at a ratio of 1:20. As a control, 1 μL of 20 mM Tris-HCl (pH 7.5) was added to the mixture, and the mixture was exposed to temperatures of -20°C or 25°C for 24 hours. For the anti-domain E antibody, 1 μL of a 1.2 mg / mL solution was added to the mixture (amount carried over: 1.2 μg), and the mixture was exposed to temperatures of 25°C for 24 hours. (Reaction solution 3) A 19 μL mixture was prepared by mixing PCR mix 3 with primers / probes at a ratio of 1:20 and nucleic acid template at a ratio of 1:20. As a control, 1 μL of 20 mM Tris-HCl (pH 7.5) was added to the mixture, and the mixture was exposed to temperatures of -20°C or 25°C for 24 hours. For the anti-domain E antibody, 1 μL of a 1.2 mg / mL solution was added to the mixture (introduced amount: 1.2 μg) and the mixture was exposed to temperatures of 25°C for 24 hours. (reaction) Reaction solutions 1 to 3 were subjected to the following temperature cycle using a real-time PCR device (Applied Biosystems 7500 Fast Real-Time PCR System). Fluorescence readings were taken at 60°C for a 60-second extension step. (Temperature Cycle) Step 1: 95℃ 1 minute Step 2: 95°C 15 seconds - 60°C 60 seconds, 50 cycles (PCR)

[0148] (4) Results The Ct values ​​when detecting the RPS19 gene and the fluorescence values ​​at the 10th cycle in the multicomponent data for reaction solutions 1, 2, and 3 are shown in Tables 10, 11, and 12, respectively. Table 13 shows the sequences of heavy chain (H chain) complementarity-determining regions (CDRs) 1 to 3, light chain (L chain) complementarity-determining regions (CDRs) 1 to 3, and the sequence adjacent to the C-terminus of L chain CDR2 for Anti-TAQ1 to 5 and Anti-TTH1 to 5.

[0149] When reaction solution 1 containing Tris-HCl was exposed to 25°C for 24 hours, the Ct value of the RPS19 gene was delayed by about 5-6 hours compared to when it was exposed to -20°C for 24 hours, indicating that the detection sensitivity was 2. 5 ~2 6 In contrast, in reaction solution 1 to which anti-domain E antibody was added, all Ct values ​​were below 30. Furthermore, all clones of anti-domain E antibody satisfied the probe degradation inhibition indexes (a) to (c): (a) Ct value ratio [Ct value measured after 24 hours of exposure at -20°C (equivalent to before 24 hours of exposure at 25°C) / Ct value measured after 24 hours of exposure at 25°C] ≥ 0.8 (b) Fluorescence intensity ratio [fluorescence intensity at the beginning of the cycle measured after 24 hours of exposure at -20°C (corresponding to before 24 hours of exposure at 25°C) / fluorescence intensity at the beginning of the cycle measured after 24 hours of exposure at 25°C] ≥ 0.3 (c) Probe decomposition rate [(F 33 -F 31 )÷(F 32 -F 31 )×100]≦40% F 31 : Fluorescence intensity at the beginning of the cycle measured after 24 hours of exposure at -20°C in the absence of anti-domain E antibody (equivalent to before 24 hours of exposure at 25°C) F 32 : Fluorescence intensity at the beginning of the cycle measured after 24 hours of exposure at 25°C in the absence of anti-domain E antibody F 33 : Fluorescence intensity at the beginning of the cycle measured after 24 hours of exposure at 25°C in the presence of anti-domain E antibody From this, it was found that all of the anti-domain E antibody clones have the effect of inhibiting probe degradation.

[0150] When reaction solution 2 containing Tris-HCl was exposed to 25°C for 24 hours, the RPS19 gene could not be detected. In contrast, the RPS19 gene was detected in all of reaction solution 2 containing anti-domain E antibodies. Furthermore, all of the anti-domain E antibody clones satisfied the probe degradation inhibition indexes (a) to (c), demonstrating the probe degradation inhibition effect. Anti-TTH2 and 3 were also found to be effective in reaction solution 1 containing Taq.

[0151] When reaction solution 3 containing Tris-HCl was exposed to 25°C for 24 hours, the RPS19 gene could not be detected. In contrast, the RPS19 gene was detected in all reaction solutions containing anti-domain E antibodies. All anti-domain E antibody clones satisfied the probe degradation inhibition indices (a) to (c), demonstrating their effectiveness in inhibiting probe degradation. When anti-domain E antibodies were added to reaction solution 3, the probe degradation inhibition indices (b) and (c) significantly exceeded 100%. This is likely due to the reaction solution reaching room temperature during preparation of control reagents or loading into the real-time PCR instrument, resulting in degradation of the fluorescently labeled probe. Therefore, this antibody can inhibit probe degradation in reaction solutions not only during long-term storage of reaction solutions, but also during preparation of standard nucleic acid amplification reagents.

[0152] [Table 10]

[0153] [Table 11]

[0154] [Table 12]

[0155] [Table 13]

[0156] Test Example 8: Effect of exposure time of PCR reaction solution at 25°C The exposure time of the PCR reaction solution at 25°C was varied to confirm the effect of the anti-domain E antibody in inhibiting probe degradation. (1) Components of PCR reaction solution [PCR mix] The same PCR mix 1 as used in Test Example 6 was used.

[0157] [Primers / Probes] TaqMan® Gene Expression Assays (Thermo Fisher Scientific) was used as a 20x primer / probe mixture, and the genes amplified / detected by the primers / probes in this mixture were IL6, CDK10, and RPS19.

[0158] [Nucleic acid template] cDNA was prepared from HeLa cell RNA (derived from human cervical cancer). RNA was extracted and cDNA was synthesized using Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep™ II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) according to the manufacturer's instructions.

[0159] (2) Reaction (Reaction solution) A 19 μL mixture was prepared by mixing each primer / probe and nucleic acid template in a 1:20 ratio with PCR Mix 1. As a control, 1 μL of 20 mM Tris-HCl (pH 7.5) was added to the mixture and exposed to temperatures of -20°C or 25°C for 24 hours. For the anti-domain E antibody, 1 μL of a 0.1 mg / mL solution was added to the mixture (introduced amount: 0.1 μg) and exposed to temperatures of 25°C for 24 hours. The reaction was then performed using a real-time PCR device (Applied Biosystems 7500 Fast Real-Time PCR System) with the following temperature cycle: Fluorescence readings were taken at 60°C for a 60-second extension step. (Temperature cycle) Step 1: 95℃ 1 minute Step 2: 95°C 15 seconds - 60°C 60 seconds, 50 cycles (PCR)

[0160] (3) Results Table 14 shows the Ct values ​​obtained when detecting each gene (IL6, CDK10, RPS19). When the reaction solution containing Tris-HCl was exposed to 25°C for 24 hours, three genes were undetectable. On the other hand, when 0.1 μg of each anti-domain E antibody was added to the reaction solution, no delay in Ct value was observed, and all genes were detectable. Furthermore, no delay in Ct value was observed even when the reaction solution was exposed to 25°C for 72 hours. Furthermore, no increase in fluorescence value was observed, indicating that the addition of the anti-domain E antibody suppressed probe degradation. Therefore, it was confirmed that the use of this antibody makes it possible to store PCR reaction solutions at 25°C for 72 hours.

[0161] [Table 14]

[0162] Test Example 9. Inhibition of probe degradation by a small amount of anti-domain E antibody It was confirmed whether probe degradation was suppressed when a PCR reaction solution containing 0.1 μg of anti-domain E antibody was exposed to 25° C. for 24 hours.

[0163] (1) Components of the reaction solution [PCR mix] The same PCR mix 2 as used in Test Example 7 was used.

[0164] [Primers / Probes] TaqMan® Gene Expression Assays (Thermo Fisher Scientific) was used as a 20x primer / probe mixture, and the genes amplified / detected by the primers / probes in this mixture were IL6, CDK10, SIVA1, and RPS19.

[0165] [Nucleic acid template] cDNA was prepared from HeLa cell RNA (derived from human cervical cancer). RNA was extracted and cDNA was synthesized using Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep™ II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) according to the manufacturer's instructions.

[0166] (2) Reaction Each primer / probe and nucleic acid template were mixed in a 1 / 20 ratio with PCR mix 2 to prepare 15 μL of a mixed solution. As a control, 5 μL of 20 mM Tris-HCl (pH 7.5) was added to the mixed solution, and the mixture was exposed to temperatures of -20°C or 25°C for 24 hours. For the anti-domain E antibody, 1 μL of a 0.1 mg / mL solution was added to the mixed solution (introduced amount: 0.1 μg), and the mixture was exposed to temperatures of 25°C for 24 hours. Each reaction mixture was analyzed using a real-time PCR instrument (Applied Biosystems The reactions were run using a 7500 Fast Real-Time PCR System with the following temperature cycle: 60°C, 60 seconds extension step, and fluorescence readings were taken. (Temperature cycle) Step 1: 95℃ 1 minute Step 2: 95°C 15 seconds - 60°C 60 seconds, 50 cycles (PCR)

[0167] (3) Results Table 15 shows the Ct values ​​when each gene (IL6, CDK10, SIVA1, RPS19) was detected.

[0168] When reaction solution 4 containing Tris-HCl was exposed for 24 hours at 25°C, a delay in Ct values ​​was observed for each gene. On the other hand, when reaction solution 4 containing 0.1 μg of each anti-domain E antibody was exposed for 24 hours at 25°C, no delay in Ct values ​​was observed.

[0169] When the control reaction solution containing Tris-HCl was exposed for 24 hours at 25°C, a delay in Ct values ​​was observed for each gene. On the other hand, when reaction solution 5 containing 0.1 μg of anti-domain E antibody was exposed for 24 hours at 25°C, no delay in Ct values ​​was observed.

[0170] [Table 15]

[0171] Test Example 10. Expression of chimeric anti-domain E antibody (1) Preparation of antibody expression plasmid An antibody sequence containing the CDRs of anti-TTH4 was designed, and oligonucleotides were synthesized. Using the Mammalian PowerExpress System™ (MPH-102 and MPL-202, Toyobo Co., Ltd.), antibody expression plasmids containing the mouse-derived heavy and light chain constant regions set forth in SEQ ID NOs: 53 and 54 were prepared according to the accompanying instructions.

[0172] (2) Antibody Expression by ExpiCHO-S™ Cells Antibody expression was performed using the ExpiCHO™ Expression System (Thermo Fisher Scientific). Culture conditions were 37°C, 5% (v / v) CO2, and shaking at 80 rpm. ExpiCHO-S™ cells were resuscitated according to the accompanying instructions, and a viable cell count of 2.0 x 10 cells was obtained. 5 The cells were cultured with shaking at 6.0 × 10 cells / mL. Subculture was continued until the viability reached 95%. 6 A culture medium was prepared at 100 cells / mL. 1.0 μg of antibody expression plasmid and 80 μL of ExpiFectamine™ CHO Reagent, diluted with 2 mL of OptiPro SFM™, were added to 25 mL of culture medium, and the cells were cultured at 37°C, 5% (v / v) CO2, and 80 rpm with shaking. After 24 hours, 150 μL of ExpiCHO™ Enhancer and 6 mL of ExpiCHO™ Feed were added, and the cells were cultured at 37°C, 5% (v / v) CO2, and 80 rpm with shaking until the viability reached 50%.

[0173] (3) Purification of antibodies using a Protein A column The culture supernatant of ExpiCHO-S™ cells was collected by centrifugation. The culture supernatant was passed through a HiTrap Protein A HP column (Cytiva) using AKTA pure 25 (Cytiva) to adsorb the antibody. The column was washed with wash buffer (20 mM phosphate buffer, pH 7.4) and then eluted with elution buffer (0.1 M citric acid-NaOH, pH 3.5). The antibody was concentrated using an Amicon Ultra-15 (Merck) and quantified using a Nanodrop One (Thermo Fisher Scientific).

[0174] In the following test examples, a chimeric anti-domain E antibody having a mouse-derived constant region obtained by the above method was used.

[0175] Test Example 11. Inhibition of probe degradation by chimeric anti-domain E antibody When a PCR reaction solution containing the chimeric anti-domain E antibody was exposed to light at 25°C for 24 hours, it was confirmed whether the chimeric anti-domain E antibody would suppress degradation of the probe.

[0176] (1) Components of the reaction solution [PCR mix] The same PCR mix 1 as used in Test Example 6 and PCR mix 2 as used in Test Example 7 were used.

[0177] [Primers / Probes] TaqMan® Gene Expression Assays (Thermo Fisher Scientific) was used as a 20x primer / probe mixture, and the gene amplified / detected by the primers / probe in this mixture was RPS19.

[0178] [Nucleic acid template] cDNA was prepared from HeLa cell RNA (derived from human cervical cancer). RNA was extracted and cDNA was synthesized using Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep™ II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) according to the manufacturer's instructions.

[0179] (2) Reaction (Reaction solutions 4 and 5) The primers and probes were mixed at a ratio of 1:20 with PCR mix 1 and 2, respectively, to prepare 18 μL of mixed solution (corresponding to reaction solutions 4 and 5, respectively). TMNucleic acid templates quantified using Thermo Fisher Scientific One were diluted to 100, 10, 1, or 0.1 ng / μL, and 1 μL of each was added to the mixture (intake amounts: 100, 10, 1, or 0.1 ng). As a control, 1 μL of 20 mM Tris-HCl (pH 7.5) was added to the mixture and exposed for 24 hours at -20°C or 25°C. For the chimeric anti-domain E antibody, 1 μL of a 0.1 mg / mL solution was added to the mixture (intake amount: 0.1 μg) and exposed for 24 hours at 25°C. The reaction was then performed using a real-time PCR device (Applied Biosystems 7500 Fast Real-Time PCR System) with the following temperature cycle: Fluorescence readings were taken at 60°C for a 60-second extension step. (Temperature cycle) Step 1: 95℃ 1 minute Step 2: 95°C 15 seconds - 60°C 60 seconds, 50 cycles (PCR)

[0180] (3) Results Table 16 for reaction solution 4 and Table 17 for reaction solution 5 show the Ct values ​​when the RPS19 gene was detected and the fluorescence values ​​at the 10th cycle in the multicomponent data.

[0181] For reaction solutions 4 and 5 containing Tris-HCl, the RPS19 gene could not be detected when exposed for 24 hours at 25° C., whereas for reaction solutions 4 and 5 containing chimeric anti-domain E antibody, the RPS19 gene was detectable in 100, 10, 1, and 0.1 ng of HeLa cDNA with Ct values ​​equivalent to those obtained when exposed for 24 hours at −20° C. Furthermore, the probe degradation rates were estimated using the same method as in Test Example 7(c) to be 4.4% for Anti-TAQ2 and 3.3% for Anti-TTH4.

[0182] [Table 16]

[0183] [Table 17]

[0184] Test Example 12. Inhibition of probe degradation when Taq polymerase (mutant) or Z05 polymerase is coexistent with anti-domain E antibody When a PCR reaction solution containing an anti-domain E antibody and Taq polymerase (mutant) or Z05 polymerase was exposed at 25°C for 24 hours, it was confirmed whether the anti-domain E antibody would inhibit degradation of the probe.

[0185] (1) Components of the reaction solution [PCR mix] The following three types of PCR mixes 4 to 6 were prepared and used. PCR Mix 4: QuantiNova Probe RT-PCR Kit (QIAGEN, 208352) containing Taq polymerase (mutant) PCR Mix 5: TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, 4444556) containing Taq polymerase (mutant) PCR Mix 6: Z05 polymerase (0.05 U / μL, Roche Diagnostics, HawkZ05, SEQ ID NO: 55); Anti-polymerase antibody for hot-start PCR (0.01 μg / μL, TCP-101, Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.3); 80 mM KCl; 1.5 mM MgCl2; 0.5 mg / mL BSA; 0.1% (v / v) TritonX-100; 0.1% (w / v) sodium cholate; and 0.3 mM dNTPs.

[0186] [Primers / Probes] TaqMan® Gene Expression Assays (Thermo Fisher Scientific) was used as a 20x primer / probe mixture, and the gene amplified / detected by the primers / probe in this mixture was RPS19.

[0187] [Nucleic acid template] cDNA was prepared from HeLa cell RNA (derived from human cervical cancer). RNA was extracted and cDNA was synthesized using Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep™ II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) according to the manufacturer's instructions.

[0188] (2) Reaction (Reaction solutions 6 to 8) PCR mixes 4 to 6 were each mixed with a 1:20 primer / probe and a 1:20 nucleic acid template to prepare 19 μL mixtures (corresponding to reactions 6 to 8, respectively). As a control, 1 μL of 20 mM Tris-HCl (pH 7.5) was added to the mixture and the mixture was exposed to temperatures of -20°C or 25°C for 24 hours. For the anti-domain E antibody, 1 μL of a 0.1 mg / mL solution was added to the mixture (introduced amount: 0.1 μg) and the mixture was exposed to temperatures of 25°C for 24 hours. The reaction was then performed using a real-time PCR device (Applied Biosystems 7500 Fast Real-Time PCR System) with the following temperature cycle: Fluorescence readings were taken at the 60°C extension step. (Temperature cycle of reaction solution 6 containing PCR mix 4) Step 1: 95℃ 2 minutes Step 2: 95°C for 5 seconds, 60°C for 24 seconds, 50 cycles (PCR) (Temperature cycle of reaction solution 7 containing PCR mix 5) Step 1: 95℃ 20 seconds Step 2: 95°C for 3 seconds, 60°C for 30 seconds, 50 cycles (PCR) (Temperature cycle of reaction solution 8 containing PCR mix 6) Process 1: 95℃ 60 seconds Step 2: 95°C 15 seconds - 60°C 45 seconds, 50 cycles (PCR)

[0189] (3) Results The Ct values ​​when the RPS19 gene was detected and the fluorescence values ​​at the 10th cycle in the multicomponent data are shown in Tables 18 and 19, respectively.

[0190] When reactions 8–10 containing Tris-HCl were exposed to 25°C for 24 hours, a delay in the Ct value at which the RPS19 gene could be detected was observed, and some genes became undetectable. On the other hand, reactions 8–10 containing anti-domain E antibody were able to detect each gene at Ct values ​​equivalent to those at -20°C after 24 hours at 25°C. Therefore, it was demonstrated that anti-domain E antibody significantly improved the stability of PCR reactions containing Taq mutants. It was also demonstrated that anti-domain E antibody significantly improved the stability of PCR reactions containing Z05 polymerase.

[0191] [Table 18]

[0192] [Table 19]

[0193] Test Example 13: Inhibitory effect of anti-domain E antibodies on 5'→3' exonuclease activity The obtained anti-domain E antibodies were examined for their ability to inhibit the 5' to 3' exonuclease activity of Taq polymerase or Tth polymerase.

[0194] (1) Reaction (Reaction Solution 9) A Taq polymerase solution containing 1 unit of Taq polymerase (TAP-201, Toyobo Co., Ltd.) and 0.2 μg of the commercially available hot-start antibody Anti-Taq high (TCP-101, Toyobo Co., Ltd.) was prepared. A mixture of Taq polymerase solution and 0.1 μg of Anti-TAQ2 was added to a reaction solution containing substrate DNA (9000 cpm, count rate 80%) radioactively labeled with 32P at the 5' end (final concentration: 10 mM Tris-HCl (pH 8.6), 50 mM KCl, 1.5 mM MgCl2) to prepare a 20 μL solution (Sample 3). As controls, Sample 1, which contained neither Taq polymerase solution nor Anti-TAQ2, and Sample 2, which contained only Taq polymerase solution, were prepared. Each sample was incubated at 37°C for 24 hours. Then, 100 μL of 10% (w / v) TCA was added to each sample to precipitate the substrate DNA, and the radioactivity of the free 32 P-labeled base remaining in the supernatant was measured.

[0195] (Reaction solution 10) A Tth polymerase enzyme solution containing 1 unit of Tth polymerase (TTH-301, Toyobo Co., Ltd.) and 0.6 μg of the commercially available hot-start antibody Anti-Taq high (TCP-101, Toyobo Co., Ltd.) was prepared. A mixture of Tth polymerase enzyme solution and 0.1 μg of Anti-TTH4 was added to a reaction solution (final concentrations: 10 mM Tris-HCl (pH 8.6), 50 mM KCl, 1.5 mM MgCl2) containing substrate DNA radioactively labeled with 32P at the 5' end (9000 cpm, count rate 80%) to prepare a 20 μL reaction solution (Sample 3). As controls, Sample 1, which contained neither Tth polymerase enzyme solution nor Anti-TTH4, and Sample 2, which contained only Tth polymerase enzyme solution, were prepared. Each sample was incubated at 37°C for 24 hours. Then, 100 μL of 10% (w / v) TCA was added to each sample to precipitate the substrate DNA, and the radioactivity of the free 32 P-labeled base remaining in the supernatant was measured.

[0196] [Substrate DNA] A reaction mixture was prepared by mixing 10 μg of λDNA and 30 units of ScaI (Toyobo Co., Ltd.) according to the manufacturer's instructions and incubated at 37°C for 24 hours. The pellet obtained by phenol / chloroform / isoamyl alcohol (25:24:1 volume ratio) treatment and ethanol precipitation was dissolved in 100 μL of TE buffer. To 80 μL of this solution, 5 μL of P-32 Adenosine 5'-triphosphate, [γ-32P]- (PerkinElmer, NEG002), 5 μL of T4 Polynucleotide Kinase (Toyobo, PNK-111), and 10 μL of 10× Blunt End Kinase Buffer (Toyobo, PNK-111 accessory) were added and incubated at 37°C for 1 hour. The pellet obtained by phenol / chloroform / isoamyl alcohol (25:24:1 volume ratio) treatment and ethanol precipitation was dissolved in 100 μL of TE buffer.

[0197] (2) Results For reaction solution 9, Sample 1, which did not contain either Taq polymerase enzyme solution or Anti-TAQ2, showed no substrate DNA degradation and was therefore set to 100%, while Sample 2, which contained only Taq polymerase enzyme solution and showed the most substrate DNA degradation and was therefore set to 0%. The remaining rate of substrate DNA in Sample 3 was calculated as the 5'→3' exonuclease activity inhibitory capacity, and the results are shown in Table 20. The Anti-TAQ2 activity inhibitory capacity was calculated to be 91%.

[0198] For reaction solution 10, sample 1, which did not contain either the Tth polymerase enzyme solution or anti-TTH4, showed no substrate DNA degradation and was therefore set to 100%, while sample 2, which contained only the Tth polymerase enzyme solution and showed the most substrate DNA degradation and was therefore set to 0%, and the remaining rate of substrate DNA in sample 3 was calculated as the 5'→3' exonuclease activity inhibitory ability, and the results are shown in Table 21. The anti-TTH4 activity inhibitory ability was calculated to be 98%.

[0199] [Table 20]

[0200] [Table 21]

[0201] Test Example 14: Inhibitory effect of anti-domain E antibody on 5'→3' exonuclease activity when the amount of added antibody is changed The amount of the anti-domain E antibody added was varied to confirm its ability to inhibit the 5'→3' exonuclease activity of Taq polymerase.

[0202] (1) Reaction (Reaction solution) A Taq polymerase enzyme solution containing 1 unit of Taq polymerase (TAP-201, Toyobo Co., Ltd.) and 0.2 μg of the commercially available hot-start antibody Anti-Taq high (TCP-101, Toyobo Co., Ltd.) was prepared. A mixture of Taq polymerase enzyme solution and Anti-TAQ2 (0.05, 0.1, 0.2, or 0.4 μg) was added to a reaction solution (final concentration: 10 mM Tris-HCl (pH 8.6), 50 mM KCl, 1.5 mM MgCl2) containing substrate DNA radioactively labeled with 32P at the 5' end (9000 cpm, count rate 80%) to prepare a 20 μL reaction solution (Samples 3–6). As controls, Sample 1 contained neither Taq polymerase enzyme solution nor Anti-TAQ2, and Sample 2 contained only Taq polymerase enzyme solution. Each sample was incubated at 37°C for 24 hours. Then, 100 μL of 10% (w / v) TCA was added to each sample to precipitate the substrate DNA, and the radioactivity of the free 32 P-labeled base remaining in the supernatant was measured.

[0203] [Substrate DNA] The same substrate DNA as in Test Example 13 was used.

[0204] (2) Results In Sample 1, which does not contain Taq polymerase enzyme solution or Anti-TAQ2, the substrate DNA is not degraded and is therefore set to 100%, while in Sample 2, which contains only Taq polymerase enzyme solution, the substrate DNA is degraded the most and is therefore set to 0%.The residual rate of substrate DNA in Samples 3 to 6 was calculated as the 5'→3' exonuclease activity inhibitory ability, and the results are shown in Table 22.

[0205] [Table 22]

[0206] Test Example 15: Inhibition of probe degradation by Taq polymerase by an antibody that specifically binds to domain E of Taq polymerase When a PCR reaction mixture containing an antibody that specifically binds to domain E of Taq polymerase and Tth polymerase was exposed at 25° C. for 24 hours, it was confirmed whether the antibody inhibited probe degradation by Tth polymerase.

[0207] (1) Components of PCR reaction solution [PCR mix] The same PCR mix 1 as used in Test Example 6 and PCR mix 2 as used in Test Example 7 were used.

[0208] [Primers / Probes] TaqMan® Gene Expression Assays (Thermo Fisher Scientific) was used as a 20x primer / probe mixture, and the genes amplified / detected by the primers / probes in this mixture were IL6, CDK10, SIVA1, RPS19, and SERPINB5.

[0209] [Nucleic acid template] cDNA was prepared from HeLa cell RNA (derived from human cervical cancer). RNA was extracted and cDNA was synthesized using Human HeLa Cell Total RNA (product code: 636543, Takara Bio Inc.) and SuperPrep™ II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) according to the manufacturer's instructions.

[0210] (2) Reaction Each primer / probe was mixed with PCR Mix 1 or 2 at a 1:20 ratio to prepare 18 μL of the mixture. Nucleic acid template quantified using a Nanodrop™ One (Thermo Fisher Scientific) was diluted to 100 ng / μL and added to the mixture in an amount of 100 ng. As a control, 1 μL of 20 mM Tris-HCl (pH 7.5) was added and the mixture was exposed to -20°C or 25°C for 24 hours. For Anti-TAQ2, 4 μL of a 0.1 mg / mL solution was added (0.4 μg) and the mixture was exposed to 25°C for 24 hours. The reaction was then run using a real-time PCR instrument (Applied Biosystems 7500 Fast Real-Time PCR System) with the following temperature cycle: Fluorescence readings were taken at 60°C with a 45-second extension step. (Temperature cycle) Step 1: 95℃ 1 minute Step 2: 95°C 15 seconds - 60°C 45 seconds, 50 cycles (PCR)

[0211] (3) Results Table 23 shows the Ct values ​​for each gene detected and the fluorescence values ​​at the 10th cycle of the multicomponent data. When the reaction mixture containing Tris-HCl was exposed to 25°C for 24 hours, either no genes were detected or the Ct values ​​were significantly delayed, indicating a decrease in detection sensitivity. On the other hand, when the reaction mixture containing Anti-TAQ2 was exposed to 25°C for 24 hours, each gene in HeLa cDNA was detected with Ct values ​​equivalent to those observed when the reaction mixture containing Tris-HCl was exposed to -20°C for 24 hours. Furthermore, it was confirmed that Anti-TAQ2 inhibited probe degradation against both Taq and Tth polymerases. Therefore, it was confirmed that antibodies with neutralizing activity against both Taq and Tth could be obtained by Method 3, which screened for antibodies against whole Taq using the Tth exo as an immunogen.

[0212] [Table 23]

[0213] Test Example 16: Inhibitory effect of anti-domain E antibody on 5'→3' exonuclease activity when substrate DNA is changed Double-stranded substrate DNAs derived from λDNA shown in SEQ ID NOs: 56 and 57 were designed, and the ability of anti-domain E antibodies to inhibit the 5' to 3' exonuclease activity of Taq polymerase or Tth polymerase was confirmed.

[0214] (1) Sample preparation The following two types of activity measurement mixes 1 and 2 were prepared and used. Activity Assay Mix 1: Taq polymerase (0.05 U / μL, TAP-201, Toyobo Co., Ltd.); Anti-polymerase antibody for hot-start PCR (0.01 μg / μL, TCP-101, Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; 1.5 mM MgCl2; 0.3μM double-stranded substrate DNA; Activity Assay Mix 2: Tth polymerase (0.05 U / μL, TTH-301, Toyobo Co., Ltd.); Anti-polymerase antibody for hot-start PCR (0.01 μg / μL, TCP-101, Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; 1.5 mM MgCl2; 0.3μM double-stranded substrate DNA;

[0215] [Double-stranded substrate DNA] A double-stranded substrate DNA was designed from λDNA having the oligonucleotides shown in SEQ ID NOs: 56 and 57. The oligonucleotides shown in SEQ ID NOs: 56 and 57 were synthesized separately and mixed in equal amounts for use.

[0216] As a control, 1 μL of 20 mM Tris-HCl (pH 7.5) was added to 19 μL of each activity measurement mix and exposed for 24 hours at -20°C or 25°C. For the anti-domain E antibody, 1 μL of a 0.1 mg / mL solution (introduced amount: 0.1 μg) was added to 19 μL of activity measurement mix 1, and 1 μL of a 0.4 mg / mL solution (introduced amount: 0.4 μg) was added to 19 μL of activity measurement mix 2, and exposed for 24 hours at 25°C. Each sample was then analyzed using a microchip electrophoresis system for DNA / RNA analysis (MultiNA, Shimadzu Corporation) and a DNA-500 kit (S292-27910-91, Shimadzu Corporation).

[0217] (2) Results The quantitative band values ​​obtained when each sample was analyzed are shown in Table 24. For both Activity Measurement Mix 1 and 2, the quantitative band values ​​were significantly lower when Tris-HCl was added and the sample was exposed for 24 hours at 25°C than when it was exposed for 24 hours at -20°C, confirming degradation of double-stranded substrate DNA. On the other hand, when Anti-TAQ2 was added and the sample was exposed for 24 hours at 25°C, the quantitative band values ​​were equivalent to those when Tris-HCl was added and the sample was exposed for 24 hours at -20°C, and degradation of double-stranded substrate DNA was not confirmed. Furthermore, the ability to inhibit 5' to 3' exonuclease activity was determined by calculating the rate of degradation of double-stranded substrate DNA (d) below. (d) Double-stranded substrate DNA degradation rate (%) [(S 21 -S 23 )÷(S 21 -S 22 )×100] S 21 : Band intensity after 24 hours of exposure at -20°C without anti-domain E antibody (equivalent to before 24 hours of exposure at 25°C) S 22 : Band intensity after 24 hours of exposure at 25°C without anti-domain E antibody S 23 : Band intensity after 24 hours of exposure at 25°C when anti-domain E antibody is included In samples exposed to Anti-TAQ2 at 25°C for 24 hours, the (d) double-stranded DNA degradation rate (%) by Taq polymerase and Tth polymerase was calculated to be ≦10%. Therefore, it was confirmed that Anti-TAQ2 exhibits sufficient inhibitory activity against double-stranded DNA degradation by both Taq polymerase and Tth polymerase.

[0218] [Table 24]

[0219] Test Example 17: Inhibition of DNA polymerase degradation of fluorescently labeled double-stranded substrate DNA (probe) by an antibody that specifically binds to domain E of Taq polymerase We investigated whether a PCR reaction mixture containing an antibody that specifically binds to domain E of Taq polymerase and a DNA polymerase (Taq polymerase or Tth polymerase) inhibits the degradation of fluorescently labeled double-stranded substrate DNA by the polymerase when the reaction mixture was exposed to 37°C for 24 hours.

[0220] (1) Components of the reaction solution [PCR mix] The following two types of PCR mixes 7 and 8 were prepared and used. PCR Mix 7: Taq polymerase (0.05 U / μL, TAP-201, Toyobo Co., Ltd.); Anti-polymerase antibody for hot-start PCR (0.01 μg / μL, TCP-101, Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; 1.5 mM MgCl2; and 0.3 μM fluorescently labeled double-stranded substrate DNA. PCR Mix 8: Tth polymerase (0.05 U / μL, TTH-301, Toyobo Co., Ltd.); Anti-polymerase antibody for hot-start PCR (0.01 μg / μL, TCP-101, Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; 1.5 mM MgCl2; and 0.3 μM fluorescently labeled double-stranded substrate DNA.

[0221] [Fluorescently labeled double-stranded substrate DNA] A fluorescently labeled double-stranded substrate DNA derived from λDNA was designed, having the oligonucleotides set forth in SEQ ID NOs: 58 and 59 (here, the 5' end of SEQ ID NO: 58 was labeled with FAM and the 5' end with BHQ1). The oligonucleotides set forth in SEQ ID NOs: 58 and 59 were synthesized separately and mixed in equal amounts for use.

[0222] (2) Reaction As a control, 1 μL of 20 mM Tris-HCl (pH 7.5) was added to 19 μL of PCR mix, and the reaction solution was exposed to -20°C or 37°C for 24 hours. 1 μL of 0.4 mg / mL anti-domain E antibody solution (0.4 μg added) was added to 19 μL of PCR mix, and the reaction solution was exposed to 37°C for 24 hours. The reaction was then performed using a real-time PCR device (Applied Biosystems 7500 Fast Real-Time PCR System) with the following temperature cycle: Fluorescence readings were taken at 60°C with a 45-second extension step. (Temperature cycle) Step 1: 95℃ 1 minute Step 2: 95°C 15 seconds - 60°C 45 seconds, 50 cycles (PCR)

[0223] (3) Results The fluorescence values ​​at the 10th cycle for the multicomponent data are shown in Table 25. When the reaction solution containing Tris-HCl was exposed to 37°C for 24 hours, an increase in fluorescence value was confirmed compared to when it was exposed to -20°C for 24 hours. On the other hand, when the reaction solution containing Anti-TAQ2 was exposed to 37°C for 24 hours, the fluorescence value was equivalent to when the reaction solution containing Tris-HCl was exposed to -20°C for 24 hours, and no increase in fluorescence value was confirmed for either Taq polymerase or Tth polymerase. Specifically, (e) the rate of degradation of fluorescently labeled double-stranded substrate DNA can be calculated using the following formula. (e) Fluorescently labeled double-stranded substrate DNA degradation rate [(F 43 -F 41 )÷(F 42 -F 41 )×100)] F 41 : Fluorescence intensity at the 10th cycle after 24 hours of exposure at -20°C (equivalent to before 24 hours of exposure at 37°C) without anti-domain E antibody F 42 : Fluorescence intensity at the 10th cycle after 24 hours of exposure at 37°C without anti-domain E antibody F 43 : Fluorescence intensity at the 10th cycle after 24 hours of exposure at 37°C when anti-domain E antibody is included In samples exposed to Anti-TAQ2 at 37°C for 24 hours, the calculated (e) degradation rate (%) of fluorescently labeled double-stranded substrate DNA was ≦10% for both Taq and Tth polymerases. Therefore, it was confirmed that Anti-TAQ2 exhibits sufficient inhibitory activity against degradation of fluorescently labeled double-stranded substrate DNA for both Taq and Tth polymerases.

[0224] [Table 25]

[0225] Test Example 18. Antibody binding rate constant ka value, dissociation rate constant kd value, and equilibrium dissociation constant KD Measurement of values The affinity of the antibody for Tth polymerase was determined using surface plasmon resonance (SPR). The measurement device used was a Biacore X100 instrument (Cytiva). The running buffer used was 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.05% (v / v) Surfactant P 20 (Cytiva). (1) Immobilization by amine coupling The ligand (Tth polymerase) was immobilized on a CM5 sensor chip (Cytiva) using EDC and NHS, followed by blocking with 1 M ethanolamine hydrochloride solution, resulting in immobilization of Tth polymerase at a density of 200-500 RU on flow cells 1-4. (2) Interaction measurement The antibody was serially diluted in the range of 0.222 to 81 nM and applied to the flow cell. The obtained sensorgram was fitted to the bivalent analyte model in the Biacore X100 evaluation software to obtain the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K D ) was decided. (3) Results The results of analyzing the interactions of anti-domain E antibodies, Anti-TTH2, Anti-TTH4, and Anti-TTH5, are shown in Table 24. All of the anti-domain E antibodies had a K of 10 nM or less. D showed. [Table 26]

[0226] Test Example 19. Antibody epitope mapping Epitope mapping was performed using conformational epitope mapping from PEPperPRINT's PEPperMAP™ Peptide Microarray analysis service. From the amino acid sequence of Taq exo (SEQ ID NO: 1) (from the N-terminus of whole Taq to amino acid position 290) and the amino acid sequence of Tth exo (SEQ ID NO: 2) (from the N-terminus of whole Tth to amino acid position 292), peptides consisting of 7, 10, and 13 amino acids were synthesized on a peptide array, with overlaps of 6, 9, and 12 amino acids, shifted by one amino acid. Subsequently, detection signals indicating binding of Anti-TAQ2 and Anti-TTH4 to each peptide array were measured, and the epitopes interacting with the antibodies were identified.

[0227] (result) Anti-TAQ2 was confirmed to bind to at least two regions of the amino acid sequences KEDGDAVIVVF (SEQ ID NO: 61) and LERLEFGSLLHEF (SEQ ID NO: 77) in Taq exo (SEQ ID NO: 1), and to at least four regions of the amino acid sequences EDGYKAVFVVF (SEQ ID NO: 62), HLITPEWLW (SEQ ID NO: 66), KYGLRPEQWVDF (SEQ ID NO: 67), and LRAFLERLEF (SEQ ID NO: 78) in Tth exo (SEQ ID NO: 2). Anti-TTH4 was confirmed to bind to at least three regions of the amino acid sequences HEAYGGY (SEQ ID NO: 64), EKYGLRPDQWADY (SEQ ID NO: 68), and RAFLERLEFGSLLH (SEQ ID NO: 80) in Taq exo (SEQ ID NO: 1), and to at least five regions of the amino acid sequences HEAYEAY (SEQ ID NO: 65), GLRPEQWVDF (SEQ ID NO: 70), ITPEWLW (SEQ ID NO: 71), LRAFLERLEF (SEQ ID NO: 78), and LEFGSLLHEF (SEQ ID NO: 82) in Tth exo (SEQ ID NO: 2).

[0228] Anti-TAQ2 was an antibody that recognized and bound to an epitope containing a sequence (EDGDAVIVVF (sequence number 60) or EDGYKAVFVVF (sequence number 62)) in amino acid region A that was common or similar between Taq exo and Tth exo, and an epitope containing a common sequence (LERLEF (sequence number 75)) in amino acid region D. Anti-TTH4 was an antibody that recognized and bound to an epitope containing a sequence in amino acid region B (HEAYGGY (sequence number 64) or HEAYEAY (sequence number 65)) common or similar between Taq exo and Tth exo, an epitope containing a consensus sequence in amino acid region C (EKYGLRPDQWADY (sequence number 68), GLRPEQWVDF (sequence number 70), or ITPEWLW (sequence number 71)), and an epitope containing a consensus sequence in amino acid region D (RAFLERLEF (sequence number 79) or LEFGSLLH (sequence number 81)).

[0229] Furthermore, it was shown that both Anti-TAQ2 and Anti-TTH4 bind to an epitope containing the consensus sequence (LERLEFGSLLH (SEQ ID NO: 76)) in amino acid region D of the Taq exon, and recognize and bind to an epitope containing the consensus sequence (GLRPEQWVDF (SEQ ID NO: 70) or ITPEWLW (SEQ ID NO: 71)) in amino acid region C of the Tth exon, and an epitope containing the consensus sequence (LRAFLERLEF (SEQ ID NO: 78)) in binding region D of the Tth exon. It was also confirmed that Anti-TAQ2 and Anti-TTH4 bind to an epitope containing the consensus sequence (LERLEF (SEQ ID NO: 75)) in amino acid region D of the Taq exon and the Tth exon.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the 5' to 3' exonuclease active domain of at least one DNA polymerase selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase, wherein at least one epitope to which the antibody or antigen-binding fragment thereof binds is located in any of the following regions: amino acid region A selected from the region at positions 56 to 66 from the N-terminus of SEQ ID NO: 1 or the region at positions 56 to 67 from the N-terminus of SEQ ID NO: 2 or 3; amino acid region B selected from the region at positions 75 to 81 from the N-terminus of SEQ ID NO: 1 or the region at positions 76 to 82 from the N-terminus of SEQ ID NO: 2 or 3; amino acid region C selected from the region at positions 161 to 182 from the N-terminus of SEQ ID NO: 1 or the region at positions 162 to 183 from the N-terminus of SEQ ID NO: 2 or 3; or amino acid region D selected from the region at positions 269 to 285 from the N-terminus of SEQ ID NO: 1 or the region at positions 271 to 287 of SEQ ID NO: 2 or 3.

2. The antibody or antigen-binding fragment thereof according to claim 1 , wherein the at least one epitope is present in either amino acid region A or B.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the epitope in the amino acid region A is any one of SEQ ID NOs: 60 to 63, the epitope in the amino acid region B is any one of SEQ ID NOs: 64 or 65, the epitope in the amino acid region C is any one of SEQ ID NOs: 66 to 74, and the epitope in the amino acid region D is any one of SEQ ID NOs: 75 to 83.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the epitope in the amino acid region A is SEQ ID NO: 61 or 62, the epitope in the amino acid region B is SEQ ID NO: 64 or 65, the epitope in the amino acid region C is SEQ ID NO: 66, 67, 68, 70, or 71, and the epitope in the amino acid region D is SEQ ID NO: 77, 78, 80, or 82.

5. The antibody or antigen-binding fragment thereof described in claim 1, which binds to two or more epitopes selected from the epitopes of SEQ ID NOs: 60 to 63 in the amino acid region A, the epitopes of SEQ ID NOs: 64 and 65 in the amino acid region B, the epitopes of SEQ ID NOs: 66 to 74 in the amino acid region C, and the epitopes of SEQ ID NOs: 75 to 83 in the amino acid region D.

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