CN116583600A

CN116583600A - Antibodies that specifically bind to the 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase

Info

Publication number: CN116583600A
Application number: CN202180082185.3A
Authority: CN
Inventors: 吉兼峻史; 黑泽信幸; 矶部正治
Original assignee: Toyobo Co Ltd; University of Toyama NUC
Current assignee: Toyobo Co Ltd; University of Toyama NUC
Priority date: 2020-12-11
Filing date: 2021-12-10
Publication date: 2023-08-11

Abstract

Antibodies or fragments thereof are provided that inhibit 5 '. Fwdarw.3' exonuclease activity of a DNA polymerase. An antibody or fragment thereof that specifically binds to the 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase is disclosed.

Description

Antibodies that specifically bind to the 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase

Technical Field

Antibodies and related techniques thereof that specifically bind to the 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase used in a nucleic acid amplification method, particularly in a polymerase chain reaction (Polymerase Chain Reaction, hereinafter referred to as "PCR"), and the like, are provided.

Background

In the field of molecular biology, the synthesis of DNA from a nucleic acid template using a DNA polymerase has been utilized and applied to various methods such as sequencing methods and nucleic acid amplification methods. Among them, the nucleic acid amplification method has been put into practical use not only in the research field but also in the forensic field such as genetic diagnosis and paternity test, and in microbiological examination in foods and environments.

A representative nucleic acid amplification method is PCR. The PCR method comprises the following steps: the target nucleic acid in the sample is amplified by repeating 1 cycle of (1) DNA denaturation (dissociation of double-stranded DNA into single-stranded DNA from double-stranded DNA), (2) annealing of the primer to the template single-stranded DNA, and (3) extension of the primer using a DNA polymerase. Sometimes (2) annealing and (3) elongation are also performed at the same temperature and in the same step, taking 2 steps as 1 cycle.

PCR is widely used in medical and biological research, clinical diagnosis, and the like because it has characteristics such as sensitivity that it is possible to amplify 1 copy of a nucleic acid sample, in reality, a nucleic acid sample corresponding to several copies, and specificity that only a specific portion is amplified. At present, PCR has been further developed, and there are various techniques such as a multiplex PCR method in which a plurality of primers are amplified simultaneously, and a real-time PCR method in which the progress of the amplification product generation is monitored with time by using a fluorescent dye or a fluorescent-labeled probe.

These nucleic acid amplification methods are also widely used for genetic analysis of a large number of samples such as HTS (High Throughput Screening ), food inspection requiring treatment of a plurality of subjects, environmental inspection, and the like. In the case of analyzing a large number of samples, it is expected that the nucleic acid amplification reaction solution will be left for a long period of time (for example, several hours to several days) after preparation. However, the stability of the reaction solution may be lowered by allowing the reaction solution to stand at normal temperature. For example, in the case of the TaqMan (registered trademark) probe method (for example, refer to non-patent document 1), it has been confirmed many times that the Ct (Threshold cycle) value is delayed or the Ct value cannot be detected due to the reaction solution after preparation being left at normal temperature (patent documents 1 and 2).

Prior art literature

Patent literature

Patent document 1 Japanese patent application laid-open No. 2017-163904

Patent document 2 Japanese re-watch 2016/136324 publication

Non-patent literature

Non-patent document 1 Holland et al Proc. Natl. Acad. Sci. Vol.88, 1991, pages 7276-7280)

Disclosure of Invention

Problems to be solved by the invention

The present inventors have found the following problems: a nucleic acid template, a primer, a probe, or the like used in a nucleic acid amplification method or the like is decomposed in the presence of a DNA polymerase having a 5 '. Fwdarw.3' exonuclease active domain.

The main object of the present invention is to provide an antibody or a fragment thereof directed against (specifically binding to) the 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase, and a method for producing the antibody or the fragment thereof.

Solution for solving the problem

As a result of intensive studies to solve the above problems, the present inventors have found an antibody or a fragment thereof directed against (specifically binding to) the 5 '. Fwdarw.3' exonuclease active domain of DNA polymerase and a practical method for producing the antibody or the fragment thereof. The present invention has been completed based on these findings and further repeated intensive studies.

Representative inventions are described below.

[ 1] an antibody or a fragment thereof (antigen-binding fragment) that specifically binds to the 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase.

[ 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.

[ 3] an antibody or a fragment thereof (antigen-binding fragment), comprising:

a heavy chain CDR1 comprising an amino acid sequence represented by the following formula (A-1),

GFX ^A3 X ^A4 X ^A5 X ^A6 X ^A7 X ^A8 (A-1)

[ in the above-mentioned, a method for producing a semiconductor device,

X ^A3 is a T or S, and the number of the T or S,

X ^A4 is a compound of formula F, L or I,

X ^A5 is a compound of D, N, S or T,

X ^A6 is a compound which is D, N, S, T, K, R or H,

X ^A7 is a metal-containing alloy of Y, F or W,

X ^A8 g, S, T, W, Y or F]；

A heavy chain CDR2 comprising an amino acid sequence represented by the following formula (B-1),

IX ^B2 X ^B3 X ^B4 X ^B5 X ^B6 X ^B7 X ^B8 (B-1)

[ in the above-mentioned, a method for producing a semiconductor device,

X ^B2 is a compound of G, S, T, K, R, H, D or N,

X ^B3 is a compound of F, Y, L, I, G, S, T, D or N,

X ^B4 is a compound which is G, S, T, D, N, K, R or H,

X ^B5 is a compound of G, S, T or A,

X ^B6 is a compound of G, S, T, D or N,

X ^B7 is a compound which is S, T, F, Y, D, N, K, R or H,

X ^B8 s, T, V, L, I or M]；

Heavy chain CDR3, VRX comprising an amino acid sequence represented by any one of the following formulas (C-1) to (C-6) ^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 above-mentioned, a method for producing a semiconductor device,

X ^C1 is a compound of formula A, S, D, K, H or R,

X ^C2 is a compound of formula G, A, D, P, K, H or R,

X ^C3 is a compound of formula S, T, L, Y or I,

X ^C4 is a compound which is A, V, I, R or L,

X ^C5 Is a compound of A, V, Y or P,

X ^C6 is a compound which is A, V, S, T or Y,

X ^C7 is a compound of V, I, L, S, T or N,

X ^C8 is A or V]；

A light chain CDR1 comprising an amino acid sequence represented by the following formula (D-1),

X ^D1 X ^D2 X ^D3 X ^D4 X ^D5 X ^D6 (D-1)

[ in the above-mentioned, a method for producing a semiconductor device,

X ^D1 is a compound of E, Q, D or N,

X ^D2 is a compound of G, A, S or T,

X ^D3 is a compound of A, V, L, I or F,

X ^D4 is a compound which is S, T, K, R or H,

X ^D5 is a compound which is S, T, D, N, K, R or H,

X ^D6 f, Y or W]；

A light chain CDR2 comprising an amino acid sequence represented by the following formula (E-1),

X ^E1 X ^E2 X ^E3 (E-1)

[ in the above-mentioned, a method for producing a semiconductor device,

X ^E1 is a compound which is G, S, T, D, N, F, Y, K, R or H,

X ^E2 is a compound of formula G, A, S, T, V, L or I,

X ^E3 k, R, H, D, N, S or T]The method comprises the steps of carrying out a first treatment on the surface of the And

light chain CDR3, X comprising the amino acid sequence of the following formula (F-1) or (F-2) ^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 above-mentioned, a method for producing a semiconductor device,

X ^F1 is a metal-containing alloy of L, I, E, Q, F, Y or W,

X ^F2 is D, N, E or Q, and the total number of the components is equal to or less than the total number of the components,

X ^F3 is a compound which is S, T, F or Y,

X ^F4 is G, S, T, F, Y, N or Q, and the total number of the components is equal to or less than the total number of the components,

X ^F5 is a compound of formula S, T, N, Q, L or I,

X ^F6 is a metal-containing alloy of G, S, T, F, Y or W,

X ^F7 is a metal-containing alloy of S, T, P, Y or W,

X ^F8 is a metal-containing alloy of L, I, P, K, R, H, Y, T or W,

X ^F9 g, S, T, D or E]。

[ 4] the antibody or fragment thereof according to item 3, further comprising a sequence region adjacent to the C-terminus of the light chain CDR2, which is composed of an amino acid sequence represented by the following formula (E-2),

X ^E4 X ^E5 X ^E6 X ^E7 (E-2)

[ in the above-mentioned, a method for producing a semiconductor device,

X ^E4 is a compound of G, S, R, H, K, D, N, F, Y or T,

X ^E5 is a compound of formula L, I, K, H or R,

X ^E6 Is a compound which is G, A, S, T, P, F or Y,

X ^E7 g, A, D, N, S or T]。

[ 5] an antibody or a fragment thereof (antigen-binding fragment), comprising:

a heavy chain CDR1 comprising an amino acid sequence represented by the following formula (A-1-1),

GFTFX ^A51 X ^A61 X ^A71 X ^A81 (A-1-1)

X ^A51 is D, N or S, and the number of the components is D, N or S,

X ^A61 is a compound which is D, N, S, K or H,

X ^A71 in the form of Y or W, the ratio of the total number of the components to the total number of the components,

X ^A81 g, W or Y]；

A heavy chain CDR2 comprising an amino acid sequence represented by 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 above-mentioned, a method for producing a semiconductor device,

X ^B21 is a compound of G, S, T, K or N,

X ^B31 is a compound of Y, L, G, T or N,

X ^B41 is a compound which is G, S, T, D or H,

X ^B51 in the form of G or S,

X ^B61 is a compound of G, S, T or D, and the compound is a compound of G, S, T or D,

X ^B71 is a compound which is S, T, Y, D or H,

X ^B81 s, T, V, I or M]；

A heavy chain CDR3 comprising an amino acid sequence represented by any one of 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 above-mentioned, a method for producing a semiconductor device,

X ^C11 is a group of A or R,

X ^C21 is a group of the formula P or R,

X ^C31 is a group of T or I,

X ^C41 is a group of V or L,

X ^C51 is a compound of the formula P or A,

X ^C61 is a group of T or Y, and the total number of the groups is the same,

X ^C71 is a compound of V, T or N,

X ^C81 is A or V]；

A light chain CDR1 comprising an amino acid sequence represented by the following formula (D-1-1),

QX ^D21 X ^D31 X ^D41 X ^D51 X ^D61 (D-1-1)

[ in the above-mentioned, a method for producing a semiconductor device,

X ^D21 in the form of G or S,

X ^D31 is a group of V or I,

X ^D41 in the form of S or K,

X ^D51 is a compound of S, N or K,

X ^D61 is F or Y]；

A light chain CDR2 comprising an amino acid sequence represented by the following formula (E-1-1),

X ^E11 X ^E21 X ^E31 (E-1-1)

[ in the above-mentioned, a method for producing a semiconductor device,

X ^E11 is a compound of formula G, T, D, Y or R,

X ^E21 is a compound of A, T or V,

X ^E31 k, D, N or S]The method comprises the steps of carrying out a first treatment on the surface of the And

a light chain CDR3 consisting of an amino acid sequence represented by 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 above-mentioned, a method for producing a semiconductor device,

X ^F11 is a compound which is L, Q, F or Y,

X ^F31 in the form of S or Y,

X ^F41 is a compound which is G, N, Q or Y,

X ^F51 is a compound of formula S, N or I,

X ^F61 is a metal-containing alloy of G, S, Y or W,

X ^F71 is a metal-containing alloy of S, P or W,

X ^F81 l, P, H, Y or T]。

[ 6] the antibody or the fragment thereof according to item 5, which further comprises a sequence region adjacent to the C-terminus of the light chain CDR2, which is composed of an amino acid sequence represented by 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 above-mentioned, a method for producing a semiconductor device,

X ^E61 is a group A or P, and the group A is a group B,

X ^E42 is a compound of R, N, Y or T,

X ^E52 is a group of L or R,

X ^E62 is a group of A or Y, and the group of B is a group of C,

X ^E72 is S or T]。

[ 7] the antibody or fragment thereof according to any one of items 3 to 6, which specifically binds to a 5 '. Fwdarw.3' exonuclease active domain of Taq polymerase.

[ 8] the antibody or the fragment thereof according to any one of items 3 to 6, which specifically binds to a 5 '. Fwdarw.3' exonuclease active domain of Tth polymerase.

The antibody or fragment thereof according to any one of items 3 to 6, which specifically binds to a 5 '. Fwdarw.3' exonuclease active domain of Z05 polymerase.

[ 10] an antibody or a fragment thereof (antigen-binding fragment) which specifically binds to a 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase, wherein at least 1 epitope of the antibody or the fragment thereof is present in any one of the following regions A to D: an amino acid region A selected from the region from 56 to 66 th from the N-terminus of SEQ ID NO. 1 and the region from 56 to 67 th from the N-terminus of SEQ ID NO. 2 or 3; an amino acid region B selected from the region from 75 to 81 th from the N-terminus of SEQ ID NO. 1 and the region from 76 to 82 th from the N-terminus of SEQ ID NO. 2 or 3; an amino acid region C selected from the region 161 to 182 from the N-terminus of SEQ ID NO. 1 and the region 162 to 183 from the N-terminus of SEQ ID NO. 2 or 3; selected from the region from 269 to 285 from the N-terminus of SEQ ID NO. 1 or the amino acid region D in the region from 271 to 287 of SEQ ID NO. 2 or 3.

The antibody or fragment thereof according to item 10, wherein the at least 1 epitope is present in either of the amino acid regions A or B.

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 SEQ ID NO. 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.

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.

The antibody or fragment thereof according to any one of items 1 to 13, which is a monoclonal antibody or fragment thereof.

[ 15] the antibody or fragment thereof according to any one of items 1 to 14, wherein the heavy chain CDR3 is composed of an amino acid sequence represented by any one of SEQ ID Nos. 21 to 28 or an amino acid sequence in which 1 to 3 amino acids in these amino acid sequences have been mutated,

The light chain CDR3 is composed of an amino acid sequence represented by any one of SEQ ID Nos. 42 to 48 or an amino acid sequence in which 1 to 3 amino acids in these amino acid sequences have been mutated.

[ 16] the antibody or fragment thereof according to any one of items 1 to 15, wherein the heavy chain CDR3 is composed of an amino acid sequence shown in any one of SEQ ID NOS 21 to 28,

the light chain CDR3 is composed of an amino acid sequence represented by any one of SEQ ID NOS 42 to 48.

The antibody or fragment thereof according to any one of items 1 to 16, which comprises:

a heavy chain CDR1 comprising an amino acid sequence represented by any one of SEQ ID Nos. 4 to 11 or an amino acid sequence in which 1 to 3 amino acids in the amino acid sequence have been mutated;

a heavy chain CDR2 comprising an amino acid sequence represented by any one of SEQ ID Nos. 12 to 20 or an amino acid sequence in which 1 to 3 amino acids in the amino acid sequence have been mutated;

a heavy chain CDR3 comprising an amino acid sequence represented by any one of SEQ ID NOS.21 to 28 or an amino acid sequence having a mutation of 1 to 3 amino acids in the amino acid sequence;

a light chain CDR1 comprising an amino acid sequence represented by any one of SEQ ID Nos. 29 to 35 or an amino acid sequence in which 1 to 3 amino acids in the amino acid sequence have been mutated;

A light chain CDR2 consisting of an amino acid sequence as set forth in either YTN, YTD, YAD, YAN, DAS, GVK, RAK, GAK or TAS; and

a light chain CDR3 comprising an amino acid sequence represented by any one of SEQ ID NOS.42 to 48 or an amino acid sequence having a mutation of 1 to 3 amino acids in the amino acid sequence.

The antibody or fragment thereof according to any one of items 1 to 17, which comprises:

a heavy chain CDR1 comprising an amino acid sequence represented by any one of SEQ ID NOS.4 to 11;

a heavy chain CDR2 comprising an amino acid sequence represented by any one of SEQ ID NOS 12 to 20;

a heavy chain CDR3 consisting of an amino acid sequence represented by any one of seq id nos 21 to 28;

a light chain CDR1 comprising an amino acid sequence of any one of SEQ ID NOS 29 to 35;

a light chain CDR3 comprising the amino acid sequence depicted in any one of seq id nos 42 to 48.

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 the amino acid sequence shown in any one of SEQ ID NOS 36 to 41 or an amino acid sequence having a mutation of 1 to 3 amino acids in these amino acid sequences.

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, which is composed of an amino acid sequence shown in any one of SEQ ID NOS 36 to 41.

The antibody or fragment thereof according to any one of items 15, 17 and 19, wherein the variation is a conservative substitution.

[ 22] a fragment (antigen-binding fragment) which is a fragment of the antibody of any one of claims 1 to 21, wherein the fragment is Fab, F (ab') 2 or scFv.

[ 23] an agent comprising the antibody or fragment thereof according to any one of items 1 to 21, or the fragment according to item 22.

[ 24] the reagent according to item 23, which further comprises at least one selected from the group consisting of a DNA polymerase having a 5 '. Fwdarw.3 ' exonuclease activity domain, a primer, a probe, and deoxyribonucleoside-5 ' -phosphate.

The reagent according to item 24, wherein the DNA polymerase is selected from the group consisting of Taq polymerase, tth polymerase and Z05 polymerase.

The reagent according to any one of items 23 to 25, which is a reagent for nucleic acid amplification.

[ 27] A method for producing an antibody or a fragment thereof (antigen-binding fragment) which specifically binds to a 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase, comprising the following step A: an antibody having binding ability to the whole DNA polymerase is selected from antibodies produced by animals immunized with an immunogen comprising a portion of the DNA polymerase (herein, the portion comprises a 5 '. Fwdarw.3' exonuclease active domain).

[ 28] the production method according to item 27, wherein the immunogen is composed of a 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase.

[ 29] the production method according to item 27 or 28, wherein the DNA polymerase is selected from the group consisting of Taq polymerase, tth polymerase and Z05 polymerase.

The production method according to any one of items 27 to 29, wherein the immunogen is constituted of a 5 '. Fwdarw.3' exonuclease active domain of Tth polymerase.

[ 31] the production method according to claim 27, wherein the step A is a step of: from among antibodies produced by animals immunized with an immunogen comprising a portion of Tth polymerase (herein, the portion comprising a 5 '. Fwdarw.3' exonuclease active domain), an antibody having binding ability to Taq polymerase as a whole is selected.

[ 32] the production method according to claim 27, wherein the step A is a step of: from among antibodies produced by animals immunized with an immunogen composed of the 5 '. Fwdarw.3' exonuclease active domain of Tth polymerase, an antibody having binding ability to Taq polymerase as a whole is selected.

[ 33] A method for producing an antibody fragment (antigen-binding fragment), comprising the steps of: based on the amino acid sequence of the antibody obtained by the production method according to any one of claims 27 to 32, a part of the amino acid sequence thereof is expressed by a genetic engineering method.

[ 34] the antibody or fragment thereof according to any one of items 1 to 21, or the fragment according to item 22, wherein the 5 '. Fwdarw.3' exonuclease inhibitory activity of a DNA polymerase is 60% or more when it coexists with the DNA polymerase at 37℃for 24 hours.

[ 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 (herein, the substrate DNA is optionally single-stranded or double-stranded, the substrate DNA is optionally functional as a probe) is allowed to coexist with the DNA polymerase having a 5 '. Fwdarw.3' exonuclease activity domain for 24 hours at 25℃and the substrate DNA degradation rate is 40% or less.

[ 36] an agent for stabilizing a composition comprising a DNA polymerase having a 5 '. Fwdarw.3' exonuclease activity domain and at least one nucleic acid selected from the group consisting of a primer, a probe and a nucleic acid template, the agent 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.

[ 37] A method for stabilizing a composition comprising a DNA polymerase having a 5 '. Fwdarw.3' exonuclease activity domain and at least one nucleic acid selected from the group consisting of a primer, a probe and a nucleic acid template, which comprises the step of adding the antibody or fragment thereof according to any one of items 1 to 21, 34 and 35, or the fragment according to item 22 to the composition.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, an antibody or a fragment thereof specifically binding to the 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase and a practical method for producing the antibody or the fragment thereof can be provided. For example, by adding the antibody or a fragment thereof to a reagent containing a DNA polymerase having a 5 '. Fwdarw.3' exonuclease active domain and containing a nucleic acid such as a primer or a probe, degradation of the nucleic acid can be suppressed and the stability of the reagent can be improved. In addition, when the target nucleic acid is amplified using the above-described reagent, the generation of fragments due to nucleic acid degradation can be suppressed, whereby non-specific amplification of the target nucleic acid can be suppressed, and the target nucleic acid can be efficiently amplified, so that even a very small amount of target nucleic acid can be detected with high sensitivity.

Detailed Description

1. Definition etc

In the present specification, the amino acid may be a natural amino acid or an unnatural amino acid. Examples of the unnatural amino acid include citrulline, ornithine, epsilon-acetyl lysine, beta-alanine, aminobenzoic acid, 6-aminocaproic acid, aminobutyric acid, hydroxyproline, mercaptopropionic acid, 3-nitrotyrosine, norleucine, pyroglutamic acid, and the like, but are not limited thereto. In addition, the amino acid may be, for example, an L-amino acid, a D-amino acid or a DL-amino acid.

In the present specification, the identity of amino acid sequences means: the amino acid sequences of 2 or more amino acid sequences to be compared are aligned optimally. Amino acid sequence identity may be calculated using commercially available analytical tools or available via telecommunications lines (Internet), for example, using commercially available software GENETYX (Genetyx Corporation) or using default (initial set) parameters in the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) (http:// www.ncbi.nlm.nih.gov/BLAST /).

The amino acid sequences disclosed in the present specification may have 1 or more (for example, 1, 2 or 3) amino acids deleted, substituted or modified, or 1 or more (for example, 1, 2 or 3) amino acids inserted or added, as long as the amino acid sequences do not inhibit the binding property to the 5 '. Fwdarw.3' exonuclease domain of DNA polymerase.

Substitutions of amino acids are preferably made by other amino acids of similar structure and/or nature (conservative substitutions). Conservative substitutions are exemplified by those within the group shown in Table 1.

TABLE 1

[ Table 1]

Examples of the modification of the amino acid include modification of a functional group such as an amino group, a carboxyl group, a hydroxyl group, and a mercapto group (SH). Modification of the above functional group may be, for example, glycosylation; methylation; esterification; amidation; PEGylation; phosphorylation; hydroxylation; is combined with protecting groups such as tert-butyloxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); biotinylation; binding with Fluorescein Isothiocyanate (FITC) and other fluorescent pigments; binding to enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP).

In the present specification, the antibody may be a monoclonal antibody, a polyclonal antibody, or preferably a monoclonal antibody. The antibody may be of any isotype, for example IgG, igA, igD, igE, igM and the like. Examples of the antibody include, but are not limited to, a mouse antibody, a rat antibody, a guinea pig antibody, and a human antibody. The antibody may be a chimeric antibody such as a guinea pig-mouse chimeric antibody or a mouse-human chimeric antibody.

In the present specification, the fragment of the antibody is not particularly limited as long as it contains heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3, and examples thereof include Fv, fab, fab ', (Fab') ₂ scFv, scFv-Fc, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, minibodies, and the like. The fragment of the antibody is preferably a fragment having an antigen-binding ability (antigen-binding fragment).

In the present specification, the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 are identified by homology search using IMGT/BlastSearch (http:// www.imgt.org/blast /).

The nucleotide such as DNA and RNA may be an analogue obtained by subjecting the nucleotide to known chemical modification as exemplified below. For example, in order to prevent decomposition by hydrolytic enzymes such as nucleases, the phosphate residue (phosphate) of each nucleotide may be replaced with a chemically modified phosphate residue such as Phosphorothioate (PS), methylphosphonate, phosphorodithioate, or the like. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be replaced by-OR (R represents, for example, -CH) ₃ 、-CH ₂ CH ₂ OCH ₃ 、-CH ₂ CH ₂ NHC(NH)NH ₂ 、-CH ₂ CONHCH ₃ 、-CH ₂ CH ₂ CN, etc.). Further, the base moiety (pyrimidine, purine) may be chemically modified, and examples thereof include introduction of a methyl group or a cationic functional group into the 5-position of a pyrimidine base, substitution of a carbonyl group at the 2-position with a thiocarbonyl group, and the like. Further, examples of the modified phosphoric acid moiety and hydroxyl moiety include biotin, amino, lower alkylamino, acetyl and the likeBut are not limited to these.

2. Specific binding to the 5 '. Fwdarw.3' exonuclease active domain of DNA polymerase (hereinafter referred to as "domain E") Synthetic antibodies

The antibody or fragment thereof of the present invention can inhibit 5 '. Fwdarw.3' exonuclease activity by specifically binding to domain E of DNA polymerase, and is useful as an inhibitor of 5 '. Fwdarw.3' exonuclease activity. The 5 '. Fwdarw.3' exonuclease activity inhibitor of the present invention has the following advantages: the specificity is high because of being an antibody; in addition, the inhibition activity can be deactivated by heating or the like, and thus is also easily used in the hot start method. The DNA polymerase is not particularly limited as long as it has the 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 an arbitrary host cell, or a DNA polymerase obtained by modifying the gene. For example, the DNA polymerase may be a DNA polymerase obtained by fusing the domain E to a DNA polymerase having no domain E in the wild type.

In one embodiment, the DNA polymerase is preferably a thermostable DNA polymerase. Here, "heat resistance" means, for example: even if the heat treatment is performed at a high temperature, for example, 60℃for 30 minutes, the DNA polymerase activity is maintained at preferably 50% or more. Examples of thermostable DNA polymerases include a DNA polymerase (Taq polymerase) derived from Thermus aquaticus (Thermus aquaticus), a DNA polymerase (Tth polymerase) derived from Thermus thermophilus (Thermus thermophilus) HB8, a DNA polymerase (Z05 polymerase) derived from Thermus (Thermus sp) Z05, a DNA polymerase (Bca polymerase) derived from Bacillus stearothermophilus (Bacillus caldotenax), a DNA polymerase (Bst polymerase) derived from Bacillus stearothermophilus (Bacillus stearothermophilus), a DNA polymerase (KOD polymerase) derived from Pyrococcus islandii (Thermococcus kodakarensis), a DNA polymerase (Ppu polymerase) derived from Pyrococcus furiosus (Pyrococcus furiosus), a DNA polymerase (Pwopolymerase) derived from UltraUltraThermococcus (Pyrococcus woesei), a DNA polymerase (Tbr polymerase) derived from Brucella (Thermus brockianus), a DNA polymerase (Tfi polymerase) derived from Thermus filifolius (Thermus filiformis), a DNA polymerase (Tfl polymerase) derived from Thermus flavum, a DNA polymerase (TmTmIn polymerase) derived from Pyrococcus polymerase (TmInd polymerase), a DNA polymerase (TmBt polymerase) derived from Pyrococcus polymerase (TmInd polymerase) and a DNA polymerase (TmBt polymerase) derived from Pyrococcus sp) and a DNA polymerase (Bt polymerase) derived from Pyrococcus sp (Bt polymerase) of Thermus (Bt polymerase) from Pbheat polymerase (Bt polymerase) and a DNA polymerase (Bt polymerase) derived from Thermus sp) of Bt polymerase (Bt polymerase) and Bt polymerase (Bt polymerase) derived from Bt polymerase (Bt polymerase), but are not limited to, it should be noted that, variants are also included in the terms of Taq polymerase, etc. Herein, a variant refers to: the DNA polymerase is composed of an amino acid sequence having an identity of 80% or more to the amino acid sequence of the original DNA polymerase, and maintains the enzymatic properties such as polymerase activity, 5 '. Fwdarw.3' exonuclease activity and heat resistance. The polymerase active domain of the variant preferably consists of an amino acid sequence having an identity of 85% or more (preferably 90% or more or 95% or more) with respect to the amino acid sequence of the polymerase active domain of the original DNA polymerase. The domain E of the variant is preferably composed of an amino acid sequence having an identity of 85% or more (preferably 90% or more or 95% or more) with respect to the amino acid sequence of the domain E of the original DNA polymerase. Amino acid variations in the variants are preferably conservative substitutions.

In one embodiment, the DNA polymerase is a DNA polymerase belonging to family A. Examples of the DNA polymerase belonging to family A include Taq polymerase, tth polymerase, Z05 polymerase, tma polymerase, bca polymerase, bst polymerase, and the like, but are not limited thereto.

The DNA polymerase is preferably at least 1 selected from the group consisting of Taq polymerase, tth polymerase and Z05 polymerase. In a specific embodiment, it is preferably 2 or more DNA polymerases selected from the group consisting of Taq polymerase, tth polymerase and Z05 polymerase, more preferably a combination of Taq polymerase and at least 1 selected from the group consisting of Tth polymerase and Z05 polymerase.

The polymerase activity of the DNA polymerase can be determined as follows. In the case where the polymerase activity was strong, the DNA polymerase solution was diluted with a preservation buffer (50 mM Tris-HCl (pH 8.0), 50mM KCl, 1mM dithiothreitol, 0.1% (v/v) polyethylene glycol sorbitan monolaurate (Tween (trademark) 20), 0.1% (v/v) octylphenyl-polyethylene glycol (Nonidet (trademark) P40), and 50% (v/v) glycerol) and then measured as follows.

(1) Mu.l of the following solution A, 5. Mu.l of the following solution B, 5. Mu.l of the following solution C, 10. Mu.l of sterilized water and 5. Mu.l of the DNA polymerase solution were added to the microtubes, and reacted at 75℃for 10 minutes.

(2) Then, the mixture was cooled with ice, 50. Mu.l of E solution and 100. Mu.l of D solution were added thereto, and the mixture was stirred and cooled with ice for another 10 minutes.

(3) The liquid was filtered through a glass filter (GF/C filter, manufactured by Whatman), and thoroughly washed with 0.1N hydrochloric acid and ethanol.

(4) The radioactivity of the filters was measured by a liquid scintillation counter (Packard), whereby the nucleotide uptake of the template DNA was measured. 1 unit (unit) of polymerase activity was set as: an amount of DNA polymerase that can ingest 10nmol of nucleotides into the acid insoluble fraction (i.e., the fraction insoluble when the solution D is added) every 30 minutes under this condition.

And (3) solution A: 40mM Tris-HCl buffer (pH 7.5) 16mM magnesium chloride 15mM dithiothreitol 100. Mu.g/mL BSA (bovine serum albumin)

And (2) liquid B: 1.5. Mu.g/. Mu.l activated calf thymus DNA

And C, liquid: 1.5mM dNTP (250 cpm/pmol [3H ] dTTP)

And D, liquid: 20% (w/v) trichloroacetic acid (2 mM sodium pyrophosphate)

E, liquid: calf thymus DNA 1mg/mL

In one embodiment, the antibody or fragment thereof of the invention preferably binds to at least one E domain selected from the group consisting of:

sequence number 1:

the amino acid sequence shown (amino acid sequence of the E domain of Taq polymerase (wild-type)), SEQ ID NO. 2:

the amino acid sequence shown (amino acid sequence of the E domain of Tth polymerase (wild-type)), SEQ ID NO. 3:

The amino acid sequences shown (the amino acid sequences of the E domain of the Z05 polymerase (wild-type)) and amino acid sequences having an identity of 80% or more (preferably 85% or more, 90% or more or 95% or more) with respect to these amino acid sequences.

In one embodiment, the antibody or fragment thereof of the present invention preferably binds to (or recognizes) at least 1 (e.g., 1, 2, 3, 4 or 5) epitope(s) of any one of the 4 amino acid regions enclosed by a rectangle in the amino acid sequence represented by any one of the above sequence numbers 1 to 3. Among the above-mentioned 4 amino acid regions surrounded by a rectangle, the amino acid region from the 56 th to 66 th positions of the N-terminus of SEQ ID NO. 1 or the amino acid region from the 56 th to 67 th positions of the N-terminus of SEQ ID NO. 2 or 3 is referred to as "amino acid region A"; the amino acid region from the 75 th to 81 th amino acid region from the N-terminus of SEQ ID NO. 1 or the amino acid region from the 76 th to 82 th amino acid region from the N-terminus of SEQ ID NO. 2 or 3 is referred to as "amino acid region B"; the amino acid region from position 161 to 182 from the N-terminus of SEQ ID NO. 1 or the amino acid region from position 162 to 183 from the N-terminus of SEQ ID NO. 2 or 3 is referred to as "amino acid region C"; the amino acid region from 269 to 285 th from the N-terminus of SEQ ID NO. 1 or the amino acid region from 271 to 287 th from SEQ ID NO. 2 or 3 is referred to as "amino acid region D". It is known that the polI polymerase family represented by Taq polymerase has a plurality of regions of particularly high conservation from the N-terminus to the vicinity of the 200 th position (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 which binds to at least 1 (e.g., 1, 2, 3, 4 or 5) epitopes present in a region from the N-terminus to the vicinity of the 200 th position (e.g., amino acid regions A to C) of the polI polymerase. From such a viewpoint, the antibody or a fragment thereof of the present invention is preferably one which can bind to a part or all of the amino acid region a and/or the amino acid region B as at least 1 epitope. In a specific embodiment, antibodies or fragments thereof that bind at least a portion or all of amino acid region a and a portion or all (particularly a portion) of either amino acid region C or D as an epitope are preferred. In yet another embodiment, antibodies or fragments thereof that bind at least a portion or all (particularly all) of amino acid region B and a portion or all (particularly a portion) of either amino acid region C or D as an epitope are preferred.

Examples of epitopes that are part or all of the amino acid region a include EDGDAVIVVF (SEQ ID NO. 60), KEDGDAVIVVF (SEQ ID NO. 61), EDGYKAVFVVF (SEQ ID NO. 62), KEDGYKAVFVVF (SEQ ID NO. 63), but are not limited thereto. In one embodiment, the epitope of a portion or all of amino acid region a preferably comprises sequence number 60 or 62. In a preferred embodiment, part or all of the amino acid region A has an epitope of SEQ ID NO. 60, 61 or 62, more preferably SEQ ID NO. 61 or 62.

Examples of the epitope of part or all of the amino acid region B include HEAYGGY (SEQ ID NO: 64) and HEAYEAY (SEQ ID NO: 65), but are not limited thereto.

Examples of epitopes of part or all of the amino acid region C include 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), but are not limited thereto. In one embodiment, a portion or all of the epitope of amino acid region C preferably comprises sequence number 68, 70 or 71. In a preferred embodiment, a portion or all of the epitope of amino acid region C is sequence number 66, 67, 68, 70 or 71.

Examples of the epitope of a part or the whole of the 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), LRAFLERLEF (SEQ ID NO: 78), RAFLERLEF (SEQ ID NO: 79), RAFLERLEFGSLLH (SEQ ID NO: 80), lefgllh (SEQ ID NO: 81), LEFGSLLHEF (SEQ ID NO: 82), and LRAFLERLEFGSLLHEF (SEQ ID NO: 83). In one embodiment, the epitope of a portion or all of amino acid region D preferably comprises sequence numbers 75, 76, 78, 79 or 81. In a preferred embodiment, a portion or all of the epitope of amino acid region D is sequence number 77, 78, 80 or 82.

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

In one embodiment, the heavy chain CDR1 of the antibody or fragment thereof of the present invention preferably consists of the amino acid sequence:

an amino acid sequence represented by the formula (A-1) shown in Table 2,

An amino acid sequence having an identity of 90% or more (preferably 95% or more) 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 conservatively substituted).

TABLE 2

In the formula (A-1), X ^A3 Preferably T, X ^A4 Preferably F, X ^A5 Preferably D, N or S, X ^A6 Preferably D, N, S, K or H, or D, N, S or H, X ^A7 Preferably Y or W, or Y, X ^A8 Preferably G, W or Y.

The heavy chain CDR1 of the antibody or fragment thereof of the present invention is preferably composed of an amino acid sequence represented by the formula (A-1-1), more preferably an amino acid sequence selected from the group consisting of the formulae (A-1-2) to (A-1-9) 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.

In one embodiment, the heavy chain CDR2 of the antibody or fragment thereof of the present invention preferably consists of the amino acid sequence:

an amino acid sequence represented by the formula (B-1) shown in Table 3,

TABLE 3

In the formula (B-1), X ^B2 Preferably G, S, T, K or N, G, S or K, or S, X ^B3 Preferably Y, L, G, T or N, further preferably L, G or N, L or N, or Y, G or T, X ^B4 Preferably G, S, T, D or H, more preferably G, S, T or D, S or T, or G, D or H, X ^B5 Preferably G or S, or G, X ^B6 Preferably G, S, T or D, G, S or T, or G or S, X ^B7 Preferably S, T, Y, D or H, more preferably S, T or Y, S or T, or T, Y, D or H, X ^B8 Preferably S, T, V, I or M, more preferably S, T or I, S or T, or T, V, I or M.

The heavy chain CDR2 of the antibody or fragment thereof of the present invention is preferably composed of an amino acid sequence represented by the formula (B-1-1), more preferably an amino acid sequence selected from the group consisting of the formulae (B-1-2) to (B-1-10) 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.

In one embodiment, the heavy chain CDR3 of an antibody or fragment thereof of the present invention preferably consists of the amino acid sequence:

an amino acid sequence represented by any one of the formulae (C-1) to (C-6) shown in Table 4,

TABLE 4

In the formula (C-1), X ^C1 Preferably A or R, more preferably R, X ^C2 Preferably P or R, more preferably R, X ^C3 Preferably T or I, more preferably T, X ^C4 Preferably V or L, more preferably V, X ^C5 Preferably P or A, more preferably P, X ^C6 Preferably T or Y, more preferably T, X ^C7 Preferably V, T or N, and more preferably V.

In the formula (C-2), X ^C1 Preferably A or R, more preferably A, X ^C2 Preferably P or R, more preferably P, X ^C3 Preferably T or I, more preferably I, X ^C4 Preferably V or L, X ^C5 Preferably P or A, more preferably A, X ^C6 Preferably T or Y, more preferably Y, X ^C7 Preferably V, T or N, more preferably V or T.

In the formula (C-3), X ^C7 Preferably V, T or N, more preferably N.

In the formula (C-5), X ^C7 Preferably V, T or N, and more preferably V.

In the formula (C-6), X ^C7 Preferably V, T or N, and more preferably V.

The heavy chain CDR3 of the antibody or fragment thereof of the present invention is preferably composed of an amino acid sequence selected from the group consisting of the formulae (C-1-1), (C-2-1), (C-3-1), (C-4), (C-5-1) and (C-6-1), more preferably composed of an amino acid sequence selected from the group consisting of (C-1-2), (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 with respect 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.

In one embodiment, the light chain CDR1 of the antibody or fragment thereof of the present invention preferably consists of the amino acid sequence:

an amino acid sequence represented by the formula (D-1) shown in Table 5,

TABLE 5

In the formula (D-1), X ^D1 Preferably Q, X ^D2 Preferably G or S, X ^D3 Preferably V or I, X ^D4 Preferably S or K, X ^D5 Preferably S, N or K, or S or N, X ^D6 Preferably F or Y, or Y.

The light chain CDR1 of the antibody or fragment thereof of the present invention is preferably composed of an amino acid sequence represented by the formula (D-1-1), more preferably an amino acid sequence selected from the group consisting of (D-1-2) to (D-1-8) 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.

In one embodiment, the light chain CDR2 of the antibody or fragment thereof of the present invention preferably consists of the amino acid sequence:

an amino acid sequence represented by the formula (E-1) shown in Table 6A,

TABLE 6A

In the formula (E-1), X ^E1 Preferably G, T, D, Y or R, more preferably G, Y or R, or G, T, D or R, further preferably Y, or G, T, D or R, X ^E2 Preferably A, T or V, further preferably A or T, or A or V, X ^E3 Preferably K, D, N or S, more preferably K, D or N, or K or S, further preferably D or N, or K or S.

The light chain CDR2 of the antibody or fragment thereof of the present invention is preferably composed of an amino acid sequence represented by the formula (E-1-1), more preferably composed of an amino acid sequence represented by the formula (E-1-2) or the formula (E-1-3), and still more preferably composed of an amino acid sequence selected from the group consisting of the amino acid sequences (E-1-4) to (E-1-12) or an amino acid sequence having identity of 90% or more 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.

In one embodiment, the C-terminus of the light chain CDR2 of an antibody or fragment thereof of the invention is preferably adjacent to the amino acid sequence of:

An amino acid sequence represented by the formula (E-2) shown in Table 6B,

TABLE 6B

In the formula (E-2), X ^E4 Preferably S, R, N, Y or T, further preferably S, or R, N, Y or T, X ^E5 Preferably L or R, or L, X ^E6 Preferably A, P or Y, more preferably A or P, or A or Y, X ^E7 Preferably S or T, or S.

The C-terminal of the light chain CDR2 of the antibody or fragment thereof of the present invention is preferably adjacent to an amino acid sequence represented by the formula (E-2-1) or (E-2-2), more preferably adjacent to an amino acid sequence selected from the group consisting of (E-2-3) to (E-2-8) or an amino acid sequence having identity of 90% or more 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.

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, and is preferably Y, F or H, and these amino acids are also preferably conservatively substituted.

In one embodiment, the light chain CDR3 of the antibody or fragment thereof of the present invention preferably consists of the amino acid sequence:

an amino acid sequence represented by the formula (F-1) or (F-2) shown in Table 7,

TABLE 7

In the formula (F-1), X ^F1 Preferably L, Q, F or Y, more preferably L, Q or Y, or Q, F or Y, further preferably L, Q or Y, or Q or F, X ^F2 Preferably Q, X ^F3 Preferably S or Y, X ^F4 Preferably G, N, Q or Y, further preferably N, Q or Y; G. q or Y; G. n or Y; or G or Y, X ^F5 Preferably isS, N or I, further preferably S or I, or S or N, X ^F6 Preferably G, S, Y or W, further preferably G, Y or W; s, Y or W; or G or S, X ^F7 Preferably S, P or W, further preferably P or W; s or P; or P, X ^F8 Preferably L, P, H or Y, further preferably L, H, Y or T, or P.

The light chain CDR3 of the antibody or fragment thereof of the present invention is preferably composed of an amino acid sequence represented by the formula (F-1-1) or (F-2-1), more preferably composed of amino acid sequences represented by (F-1-2) to (F-1-5) and (F-2-2) to (F-2-4) or an amino acid sequence having identity of 90% or more 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.

Preferred combinations of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of the antibodies or fragments thereof of the invention are exemplified in table 8A.

TABLE 8A

Preferred combinations of heavy chain CDRs 1 to 3, light chain CDRs 1 to 3, and sequences adjacent to the C-terminus of light chain CDR2 of the antibodies or fragments thereof of the invention are exemplified in table 8B.

TABLE 8B

In the combinations C14 to C26, it is also preferable that 1 to 3 (preferably 1 or 2, more preferably 1) amino acids in at least 1 amino acid sequence among the heavy chain CDRs 1 to 3, the light chain CDRs 1 to 3 and the sequences adjacent to the C-terminus of the light chain CDR2 are mutated (preferably conservatively substituted).

The regions other than the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 are not particularly limited as long as they are usable in an antibody, and may be any amino acid sequences. Examples of the constant region include, but are not limited to, igG1, igG2, igG3, igA1, igA2, and IgM. The constant region may be a constant region derived from any mammal, for example, a mouse, hamster, rat, guinea pig, rabbit, ferret, goat, monkey, or human, and examples thereof include a sequence region comprising the amino acid sequence: amino acid sequences represented by SEQ ID Nos. 51 and 52 (guinea pig-derived heavy and light chain constant regions), amino acid sequences represented by SEQ ID Nos. 53 and 54 (mouse-derived heavy and light chain constant regions), and amino acid sequences having an identity of 80% or more (preferably 85% or more, 90% or more, or 95% or more) to these amino acid sequences.

The equilibrium dissociation constant (K) of the antibody or fragment thereof of the present invention relative to domain E of DNA polymerase _D ) For example, 50nM or less, preferably 10nM or less, for example, 1pM or more. K (K) _D For example, the measurement can be performed using Biacore (trademark) X100 (Cytiva corporation) as shown in examples described later. Specifically, a carboxymethyl dextran immobilized with a ligand (DNA polymerase having a 5 '. Fwdarw.3' exonuclease active domain) on a CM5 sensor chip (Cytiva Co.) was blocked with a 1M ethanolamine hydrochloride solution by an amine coupling reaction of NHS (N-hydroxysuccinimide) and EDC (N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride, and then a flow cell with the immobilized ligand was prepared, and the reaction signal was analyzed to calculate K _D 。

In one embodiment, the antibody or fragment thereof of the present invention is allowed to coexist with a DNA polymerase at 37℃for 24 hours, and the 5 '. Fwdarw.3' exonuclease activity of the DNA polymerase is inhibited by, for example, 50% or more, preferably 60% or more, 70% or more, 80% or more, or 90% or more. This inhibition ability can be calculated by measuring the radioactivity of the labeled base released when incubated at 37℃for 24 hours using a solution containing the following components:

A substrate nucleic acid lambda DNA labeled with a radioisotope;

DNA polymerase alone (1 unit (U)) or DNA polymerase (1U) with the antibody of the present invention or a fragment thereof (0.005. Mu.g/. Mu.L);

10mM Tris-HCl(pH8.6)；

50mM KCl; a kind of electronic device with high-pressure air-conditioning system

1.5mM MgCl ₂ 。

Inhibition ability (%) = (N2-N3)/(N2-N1) ×100 for 5'→3' exonuclease activity

N1: the amount of free nucleotides before incubating the solution free of the antibody or fragment thereof of the present invention at 37℃for 24 hours (or after incubating at-20℃for 24 hours), or the amount of free nucleotides after incubating the solution free of the antibody or fragment thereof of the present invention and free of DNA polymerase at 37℃for 24 hours

N2: amount of free nucleotide after incubation of the solution free of the antibody or fragment thereof of the invention at 37℃for 24 hours

And N3: amount of free nucleotide after incubation of a solution comprising the antibody or fragment thereof of the invention for 24 hours at 37 ℃C

In another embodiment, when the antibody or a fragment thereof of the present invention is allowed to coexist with a substrate DNA (herein, the substrate DNA may be single-stranded or double-stranded, and the substrate DNA may optionally have a function as a probe) and a DNA polymerase at 25℃for 24 hours, the inhibition ability (substrate DNA degradation inhibition ability) of the 5 '. Fwdarw.3' exonuclease activity of the DNA polymerase can be quantitatively confirmed by the method of (1) or (2) below.

(1) The Ct value in real-time PCR was compared with the reaction solution after 24 hours of exposure at 25℃and the control reaction solution (reaction solution after 24 hours of exposure at-20℃or before 24 hours of exposure at 25℃in the absence of the antibody or fragment thereof) by allowing the antibody or fragment thereof of the present invention to coexist with the fluorescent-labeled substrate DNA (for example, fluorescent-labeled probe). The smaller the difference in Ct values, the higher the inhibition ability of the 5 '. Fwdarw.3' exonuclease activity of the DNA polymerase.

(2) The antibody or fragment thereof of the present invention is allowed to coexist with a fluorescent-labeled substrate DNA (for example, a fluorescent-labeled probe), and the initial fluorescence intensity at the cycle in real-time PCR is compared with that of a control reaction solution (a reaction solution after 24 hours of exposure at-20℃or before 24 hours of exposure at 25 ℃) in the absence of the antibody or fragment thereof of the present invention after 24 hours of exposure at 25 ℃. The smaller the difference in fluorescence intensity, the higher the inhibition ability of the 5 '. Fwdarw.3' exonuclease activity of the DNA polymerase.

As the reaction liquid in (1) and (2), for example, a reaction liquid containing the following components can be used:

the antibody or fragment thereof of the present invention (0.06. Mu.g/. Mu.L);

taq polymerase (0.05U/. Mu.L) described by SEQ ID NO. 49;

Anti-polymerase antibody for hot start PCR (0.01. Mu.g/. Mu.L);

10mM Tris-HCl(pH8.3)；

50mM KCl；

1.5mM MgCl ₂ ；

0.3mM dNTPs；

HeLa cell RNA derived cDNA (5 ng/. Mu.L); a kind of electronic device with high-pressure air-conditioning system

Primer and probe solutions (1/20 of the total liquid amount) or reaction solutions containing the following components:

the antibody or fragment thereof of the present invention (0.06. Mu.g/. Mu.L);

tth polymerase described by SEQ ID NO. 50 or Z05 polymerase (0.05U/. Mu.L) described by SEQ ID NO. 55;

anti-polymerase antibody for hot start PCR (0.01. Mu.g/. Mu.L);

10mM Tris-HCl(pH8.3)；

80mM KCl；

1.5mM MgCl ₂ ；

0.5mg/mL BSA；

0.1％(v/v)TritonX-100；

0.1% (w/v) sodium cholate;

0.3mM dNTPs；

Primer and probe solutions (1/20 of the total liquid amount).

As the primer and probe solutions in the reaction solution, for example, the following solutions are used: taqMan (registered trademark) Gene Expression Assays of Thermo Fisher Scientific Co

[ Gene:

interleukin 6, detection ID: hs00985639 _m1 (hereinafter referred to simply as "IL 6")

Cyclin-dependent kinase 10, assay ID: hs00177586 _m1 (hereinafter simply referred to as "CDK 10")

APC, WNT signaling pathway modulator, assay ID: hs01568269 _m1 (hereinafter simply referred to as "APC")

Mitogen-activated protein kinase 8, assay ID: hs00177083 _m1 (hereinafter simply referred to as "MAPK8")

SIVA1 apoptosis-inducing factor, assay ID: hs00276002 _m1 (hereinafter simply referred to as "SIVA 1")

Ribosomal protein S19, assay ID: hs03044115 _g1 (hereinafter referred to simply as "RPS 19") or

Serpin family B member 5, assay ID: hs00985285 _m1 (hereinafter simply referred to as "SERPINB 5")

The reaction liquid preferably satisfies 1, 2 or 3 of the following (a) to (c).

(a) Ct value before 24 hours exposure at 25 ℃ (or after 24 hours exposure at-20 ℃), ct value after 24 hours exposure at 25 ℃ is not less than 0.8

(b) The fluorescence intensity at the initial cycle before 24 hours exposure at 25 ℃ (or after 24 hours exposure at-20 ℃)/the fluorescence intensity at the initial cycle after 24 hours exposure at 25 ℃ is not less than 0.3

(wherein, the initial fluorescence intensity in the cycle refers to the fluorescence intensity in real-time PCR before the amplification curve rises steeply, and the initial fluorescence intensity in the cycle generally refers to the 1 st to 30 th cycles)

(c) The DNA decomposition rate of the fluorescent marked substrate is less than or equal to 40 percent

(wherein the fluorescence labeling substrate DNA degradation rate can be calculated by the following formula:

fluorescence labeling substrate DNA decomposition rate (%) = (F) ₁₃ -F ₁₁ )÷(F ₁₂ -F ₁₁ )×100

F ₁₁ : fluorescence intensity at initial cycle before 24 hours of exposure at 25℃ (or after 24 hours of exposure at-20 ℃) in the absence of the antibody or fragment thereof of the present invention

F ₁₂ : fluorescence intensity at initial cycle after 24 hours of exposure at 25℃in the absence of the antibody or fragment thereof of the invention

F ₁₃ : fluorescence intensity at initial cycle after 24 hours of exposure at 25℃when comprising the antibody or fragment thereof of the invention

The value (Ct value ratio) in (a) is preferably 0.9 or more. The value (fluorescence intensity ratio) in (b) is preferably 0.35 or more, more preferably 0.5 or more, and further preferably 0.7 or more. The value (fluorescence labeling substrate DNA degradation rate) in the above (c) is preferably 30% or less, more preferably 20% or less.

In still another embodiment, when the antibody or a fragment thereof of the present invention is allowed to coexist with a substrate DNA (herein, the substrate DNA may be single-stranded or double-stranded, and the substrate DNA may optionally have a function as a probe) and a DNA polymerase for 24 hours at 25℃or 37℃the inhibition ability (substrate DNA degradation inhibition ability) of the 5 '. Fwdarw.3' exonuclease activity of the DNA polymerase can be quantitatively confirmed by, for example, the following method (3) or (4).

(3) The antibody or fragment thereof of the present invention is allowed to coexist with a substrate DNA (e.g., double-stranded substrate DNA), and the band intensities of the substrate DNA are compared by gel electrophoresis for a solution exposed at 25℃for 24 hours and a control solution (a solution after exposure at-20℃for 24 hours or before exposure at 25℃for 24 hours in the absence of the antibody or fragment thereof of the present invention). The smaller the difference in band intensities, the higher the inhibition ability of the 5 '. Fwdarw.3' exonuclease activity of the DNA polymerase.

(4) The antibody or fragment thereof of the present invention is allowed to coexist with a fluorescent-labeled substrate DNA (for example, a double-stranded substrate DNA having at least one strand fluorescently labeled), and the initial fluorescence intensity in the cycle in real-time PCR or the fluorescence intensity in a spectrophotometer is compared with a control reaction solution (a reaction solution after exposure to-20℃for 24 hours or before exposure to 37℃for 24 hours in the absence of the antibody or fragment thereof) for 24 hours at 37 ℃. The smaller the difference in fluorescence intensity, the higher the inhibition ability of the 5 '. Fwdarw.3' exonuclease activity of the DNA polymerase.

As the solution or reaction liquid in (3) and (4), for example, a liquid containing the following components can be used:

the antibody or fragment thereof of the present invention (0.06. Mu.g/. Mu.L);

taq polymerase (0.05U/. Mu.L) described by SEQ ID NO. 49;

anti-polymerase antibody for hot start PCR (0.01. Mu.g/. Mu.L);

10mM Tris-HCl(pH8.3)；

50mM KCl；

1.5mM MgCl ₂ ；

0.3. Mu.M substrate DNA, or a liquid comprising the following components:

the antibody or fragment thereof of the present invention (0.06. Mu.g/. Mu.L);

anti-polymerase antibody for hot start PCR (0.01. Mu.g/. Mu.L);

10mM Tris-HCl(pH8.3)；

50mM KCl；

1.5mM MgCl ₂ ；

0.3. Mu.M substrate DNA.

In the method of (3), the substrate DNA may or may not be labeled with a fluorescent dye, radioisotope or the like. Examples of the double-stranded substrate DNA among the substrate DNA include, but are not particularly limited to, double-stranded substrate DNA represented by a combination of SEQ ID NO. 56 and SEQ ID NO. 57 in which at least one strand has a 3 '-end protruding from the 5' -end of the other strand. The length of the bases of the projections is, for example, about 3 to 10 bases. The method of gel electrophoresis is not particularly limited, and examples thereof include agarose gel electrophoresis and polyacrylamide gel electrophoresis. An apparatus capable of quantifying the band intensity of nucleic acid is preferably used, and examples of the apparatus include a microchip electrophoresis apparatus for DNA/RNA analysis (MultiNA, shimadzu corporation) and a full-automatic high-throughput electrophoresis system (TapeStation System, agilent technologies Co., ltd.), but are not particularly limited.

In the method of (4), the substrate DNA is preferably fluorescently labeled. Examples of the double-stranded substrate DNA among the substrate DNA include, but are not particularly limited to, double-stranded substrate DNA represented by a combination of SEQ ID NO. 58 and SEQ ID NO. 59, in which at least one strand has a 3 '-end protruding from the 5' -end of the other strand and at least one strand has a fluorescence-labeled end. The length of the bases of the projections is, for example, about 3 to 10 bases. The change in fluorescence value is measured by, for example, a real-time PCR device or a spectrophotometer, but is not particularly limited.

The solution or the reaction solution is preferably a liquid satisfying 1 or 2 of the following (d) and (e).

(d) The decomposition rate of the substrate DNA is less than or equal to 40 percent

(wherein the substrate DNA degradation rate can be calculated by the following formula:

substrate DNA decomposition rate (%) = (S) ₁₁ -S ₁₃ )÷(S ₁₁ -S ₁₂ )×100

S ₁₁ : strip intensity before 24 hours of exposure at 25 ℃ (or after 24 hours of exposure at-20 ℃) in the absence of the antibodies or fragments thereof of the invention

S ₁₂ : band intensity after 24 hours of exposure at 25℃in the absence of the antibody or fragment thereof of the invention

S ₁₃ : band intensity after 24 hours of exposure at 25 ℃ when comprising an antibody or fragment thereof of the invention

(e) The DNA decomposition rate of the fluorescent marked substrate is less than or equal to 40 percent

fluorescence labeling substrate DNA decomposition rate (%) = (F) ₂₃ -F ₂₁ )÷(F ₂₂ -F ₂₁ )×100)

F ₂₁ : fluorescence intensity at initial cycle before 24 hours of exposure at 37℃or after 24 hours of exposure at-20℃in the absence of the antibody or fragment thereof of the present invention

F ₂₂ : fluorescence intensity at initial cycle after 24 hours of exposure at 37℃in the absence of the antibody or fragment thereof of the invention

F ₂₃ : when the antibody or the fragment thereof of the present invention is contained, the temperature is 37 DEG CFluorescence intensity at initial cycle after 24 hours of lower exposure

At the initial stage of the cycle: usually 1 to 30 cycles

Fluorescence intensity: fluorescence intensity in real-time PCR)

The substrate DNA degradation rate in (d) is preferably 30% or less, more preferably 20% or less. The degradation rate of the fluorescent-labeled substrate DNA in (e) is preferably 30% or less, more preferably 20% or less.

The antibody or fragment thereof of the present invention can inhibit the degradation of nucleic acid by the 5 '. Fwdarw.3' exonuclease activity of DNA polymerase even when it is allowed to coexist with nucleic acid such as DNA polymerase and nucleic acid template, primer, probe or the like at 25℃for 24 hours, 48 hours or 72 hours or at 37℃for 24 hours. Therefore, the antibody or fragment thereof of the present invention can be preferably used for improving the stability of a reagent for nucleic acid amplification or the like.

The antibody or fragment thereof of the present invention can be obtained, for example, by immunizing an animal with a part or all of the DNA polymerase comprising domain E as an immunogen. The immunogen is preferably a part of the DNA polymerase comprising domain E, more preferably a part of the DNA polymerase comprising 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 part of the Tth polymerase comprising domain E. Examples of animals include mammals such as mice, hamsters, rats, guinea pigs, rabbits, ferrets, goats, monkeys, and humans, but are not limited thereto.

The antibody of the present invention can be obtained by screening the antibodies produced by the above-mentioned animals immunized with the immunogen. For example, when a part of the DNA polymerase containing domain E is used as an immunogen, the screening may be performed using the binding ability to the entire DNA polymerase as an index, and when all of the DNA polymerase is used as an immunogen, the screening may be performed using the binding ability to a part of the DNA polymerase containing domain E as an index, or the screening may be performed using the difference between the binding ability to the entire DNA polymerase and the binding ability to the DNA polymerase excluding a part of the DNA polymerase containing domain E as an index. In one embodiment, the screening is preferably performed using a part of the DNA polymerase including the domain E as an immunogen and the binding ability to the entire DNA polymerase as an index, and more preferably the screening is performed using a part of the Tth polymerase including the domain E as an immunogen and the binding ability to the entire DNA polymerase as an index.

Specific screening methods may be, for example: a hybridoma method in which mammalian spleen cells and myeloma cells are subjected to cell fusion; phage display methods for selecting antibodies having affinity for a target molecule from a phage library of antibodies, etc., may be the following methods: antigen-specific plasma cells are selected from the immunized animal, and antibody genes (a part of the whole length or variable region, etc.) are isolated to obtain a recombinant antibody having high affinity with the antigen.

Examples of the method for screening antigen-specific plasma cells include a method described in U.S. patent application publication No. 2014/031528 (incorporated herein by reference in its entirety) and a method described in U.S. patent application publication No. 2018/292407 (incorporated herein by reference in its entirety). In the former method, a fluorescent-labeled antigen and an endoplasmic reticulum affinity fluorescent dye are allowed to act on a cell suspension prepared from an immunized animal, and an antibody expressed on the cell surface is fluorescently labeled, whereby antigen-specific plasma cells can be identified. In the latter method, an antigen-specific plasma cell can be identified by immobilizing a cell population containing antibody-producing cells with a crosslinking agent and subjecting the cell population to a cell membrane lysis treatment with a surfactant to bind an antibody expressed in the cell to a fluorescent-labeled antigen. In this method, at least 1 plasma cell bound to the target antigen can be isolated by single cell analysis using a sorter. In this method, a fluorescent probe having high selectivity of staining the endoplasmic reticulum of cells and capable of distinguishing plasma cells and plasmablasts from other cells can be used. As the fluorescent probe, for example, those described in U.S. patent application publication No. 2013/029325 (incorporated herein by reference in its entirety) can be used.

Examples of the method for obtaining antibody genes from antigen-specific plasma cells include a hybridoma method and a cloned antibody gene, but are not limited thereto. The latter method includes, for example, a method of extracting mRNA from antigen-specific plasma cells, performing reverse transcription, and synthesizing cDNA to obtain antibody genes, and may be a method described in U.S. patent application publication No. 2011/020879 (incorporated herein by reference in its entirety). The method is a method of extracting mRNA from antigen-specific plasma cells using magnetic beads and obtaining antibody genes by RT-PCR, and the method uses a reaction jig capable of performing a plurality of stepwise reactions such as cDNA synthesis from mRNA, DNA amplification, and the like, which may optionally include washing steps, in parallel.

The method for obtaining a recombinant antibody from an antibody gene may be, for example, a method of preparing an antibody expression vector containing an antibody gene and expressing an antibody from the antibody expression vector. Examples of the method include a method described in U.S. patent application publication No. 2013/02399 (incorporated herein by reference in its entirety) and a method described in U.S. patent application publication No. 2011/117609 (incorporated herein by reference in its entirety). In the former method, one or more double-stranded DNA fragments are ligated to a PCR amplification product containing a target gene sequence, whereby a ligated DNA fragment containing a sequence derived from a target gene of interest can be specifically produced without purifying the PCR amplification product. In the latter method, the homologous recombination regions at both ends of the linearized vector are kept in the inner sequence of the amplification primer and the amplification primer sequence present only in the target gene, whereby homologous recombination can be selectively performed on the target DNA fragment, thereby constructing a vector.

The antibody or fragment thereof of the present invention can also be obtained by genetic engineering methods based on the amino acid sequence information of the antibody or fragment thereof obtained by the above-described method. For example, the antibody or a fragment thereof of the present invention can also be obtained by expressing an expression vector or the like in which an antibody gene designed in the following manner is integrated in any host cell known in the art: the amino acid sequences of the light chain CDRs 1 to 3 and optionally the C-terminal adjacent regions of the light chain CDRs 2, respectively, have 80% or more identity with respect to the antibody of which the C-terminal adjacent regions of the light chain CDRs 1 to 3 and optionally the light chain CDRs 2 have been obtained by the above-described method, and the amino acid sequences of the heavy chain CDRs 1 to 3, respectively, have 80% or more identity with respect to the antibody of which the heavy chain CDRs 1 to 3 have been obtained by the above-described method.

3. Polynucleotide

The polynucleotide of the invention preferably comprises: the coding sequence of the antibody or a fragment thereof according to the above 2.

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

The promoter is not particularly limited, and may be appropriately selected according to the type of host cell. As the promoter, for example, various promoters among pol II-based promoters can be used. The pol II-based promoter is not particularly limited, and examples thereof include a CMV promoter, an EF1 promoter, an SV40 promoter, and an MSCV promoter. Examples of the promoter include tryptophan promoters such as trc and tac; a lac promoter; a T7 promoter; a T5 promoter; a T3 promoter; the SP6 promoter; an arabinose-inducible promoter; a cold shock promoter; tetracycline inducible promoters, and the like.

The expression cassette may contain other elements as desired. Examples of the other elements include a Multiple Cloning Site (MCS), a drug-resistance gene, an origin of replication, an enhancer sequence, a repressor sequence, an insulator sequence, a reporter protein coding sequence, and a drug-resistance gene coding sequence. These may be used singly or in combination of two or more.

The polynucleotide of the present invention may be in the form of a vector, for example. The appropriate vector may be selected according to the purpose of use, the kind of host cell, and the like. Examples of vectors that host E.coli include M13 phage or a variant thereof, lambda phage or a variant thereof, pBR322 or a variant thereof (e.g., pB325, pAT153, pUC 8), etc., examples of vectors that host yeast include pYepSec1, pMFa, pYES2, pPICC 3.5K, etc., examples of vectors that host insect cells include pAc, pVL, etc., and examples of vectors that host mammalian cells include pcDNA, pCDM8, pMT2PC, etc.

4. Cells

The cell of the present invention preferably comprises the polynucleotide of 3 above. Examples of the cells include insect cells such as Escherichia coli K12, bacillus bacteria such as Bacillus subtilis MI114, yeasts such as Saccharomyces cerevisiae AH22, sf cell lines derived from Spodoptera frugiperda (Spodoptera frugiperda), highfive cell lines derived from Trichoplusia ni (Trichoplusia ni), and olfactory nerve cells, and animal cells. The animal cells are preferably cultured cells derived from mammals, and specifically include COS7 cells, CHO cells, HEK293 cells, expi293 cells, 293F cells, 293T cells, 293FT cells, hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells, and the like.

In one embodiment, the cells of the invention preferably express an antibody or fragment thereof that specifically binds to domain E of DNA polymerase.

In one embodiment, the cells of the invention preferably have antibodies or fragments thereof secreted or located on the cell surface that specifically bind to domain E of DNA polymerase.

5. Reagent(s)

The agent of the present invention preferably comprises the antibody or fragment thereof described in the above 2, the polynucleotide described in the above 3 or the cell described in the above 4. The agent of the invention preferably further comprises excipients or carriers and/or additives.

Examples of the excipient or carrier include starch, lactose, crystalline cellulose, sorbitol, dibasic calcium phosphate, water, ethanol, (poly) ethylene glycol, (poly) propylene glycol, glycerin, vegetable oil, and the like. These may be used singly or in combination of two or more.

Examples of the additives include buffers, isotonic agents, thickeners, chelating agents, emulsifiers, colorants, and preservatives. These may be used singly or in combination of two or more.

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

In one embodiment, the agent of the invention preferably comprises an antibody or fragment thereof that specifically binds to a DNA polymerase having domain E and 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 effect of the present invention can be exerted, and is desirably about 1:1 to about 500:1. the reagent may further comprise a DNA polymerase without domain E. The reagent is preferably a reagent for nucleic acid amplification.

In one embodiment, the agent 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 fragment thereof that specifically binds to domain E of the DNA polymerase, preferably an antibody that binds to at least 1 (e.g., 1 or 2) epitopes present in any one of the amino acid regions a to D in domain E. The reagent may further comprise a metal salt such as manganese, magnesium, a buffer, etc. to enhance the DNA polymerase activity. The reagent is preferably a reagent for nucleic acid amplification.

When the reagent of the present invention comprises a DNA polymerase having a domain E, the DNA polymerase described in the above 2 can be exemplified as the DNA polymerase.

When the reagent of the present invention comprises a primer, the primer may be at least 2 primers. The at least 2 primers may be: an oligonucleotide which is substantially complementary to a nucleic acid sequence to be amplified, defines both ends of the nucleic acid sequence to be amplified, and functions as a template for further synthesis when an extension product synthesized from each primer is separated from its complement. The primer can be appropriately selected, designed and used according to the target nucleic acid, and is not particularly limited. Furthermore, degenerate primers may be used when the target nucleic acid to be targeted is expected to be a subtype. In general, the primer may be an oligonucleotide having a nucleotide number of 12 to 60. Primers may be synthesized by a DNA synthesis device or isolated from a biological supply.

In the case where the reagent of the present invention comprises a probe, the probe may be a hybridization probe labeled with at least 1 labeling substance. By using such a probe, analysis of a nucleic acid amplification product can be monitored by monitoring a fluorescent signal, and the amount of analysis labor can be reduced without using ordinary electrophoresis. Furthermore, the reaction vessel does not need to be opened, and the risk of contamination can be further reduced. For example, each hybridization probe corresponding to a subtype of a nucleic acid sequence to be detected can be labeled with a different fluorescent dye, so that the subtype of the target nucleic acid can be identified. Examples of hybridization probes include TaqMan hydrolysis probes [ U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,538,848, U.S. Pat. No. 5,487,972, U.S. Pat. No. 5,804,375 (the entire contents of which are incorporated herein by reference) ], molecular beacons [ U.S. Pat. No. 5,118,801 (the entire contents of which are incorporated herein by reference) ], FRET hybridization probes [ International publication No. 97/46707, international publication No. 97/46712, international publication No. 97/46714 (the entire contents of which are incorporated herein by reference) ], and the like.

The reagent of the present invention may comprise a double-stranded DNA binding fluorescent compound instead of a probe. Examples of the double-strand DNA binding fluorescent compound include SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), evaGreen (registered trademark; biotium), LCGreen (Idaho), lightCycler (registered trademark) 480ResoLight (Roche Applied Science), and the like, but are not limited thereto.

When the reagent of the present invention comprises deoxyribonucleoside-5 '-phosphate, the deoxyribonucleoside-5' -phosphate is, for example, dATP, dCTP, dTTP, dGTP or a mixture of these. Terms such as dATP include those in which chemical modification is performed.

When the reagent of the present invention is a reagent for nucleic acid amplification, examples of the nucleic acid amplification method include, but are not limited to, a PCR method, a Loop-mediated isothermal amplification (Loop-Mediated Isothermal AmplificationL, LAMP) method, a transcription-reverse transcription cooperative reaction (Transcriprtion Reverse Transcription Concerted Reaction, TRC) method, a nucleic acid sequence-dependent amplification (Nucleic Acid Sequence-Based Amplification, NASBA) method, and the like. The nucleic acid amplification method is preferably a PCR method. Among the PCR methods, for example, a PCR method in which primer annealing is suppressed at a predetermined temperature or lower by a DNA polymerase-specific monoclonal antibody, a so-called hot start PCR method, is preferable. The hot-start PCR reagent of the present invention can more effectively suppress a nonspecific reaction by combining an antibody that specifically binds to the polymerase activity domain of DNA polymerase with an antibody that specifically binds to the domain E of DNA polymerase. The hot-start PCR reagent of the present invention preferably comprises a primer, deoxyribonucleoside-5' -phosphate, DNA polymerase, an antibody specifically binding to the polymerase activity domain of DNA polymerase, and an antibody specifically binding to domain E of DNA polymerase. The reagent is mixed with a reagent containing a target nucleic acid, and the resulting mixture is heated to 60℃or higher (e.g., heated at 95℃for 20 seconds or higher), and any antibody is inactivated, whereby a primer extension product can be formed.

Examples

The present invention will be specifically described below with reference to test examples. Of course, the present invention is not limited to the following test examples.

Test example 1 preparation of antigen

When the whole DNA polymerase is used as an antigen, taq polymerase having an amino acid sequence of SEQ ID NO. 49 (TAP-201, toyobo Co., ltd., hereinafter referred to as "whole Taq") and Tth polymerase having an amino acid sequence of SEQ ID NO. 50 (TTH-301, toyobo Co., ltd., hereinafter referred to as "whole Th") are used. The sequence identity of whole Taq and whole Tth was about 87%.

When the domain E of DNA polymerase is used as an antigen, a polypeptide having the amino acid sequence of SEQ ID NO. 1 (amino acid from the N-terminus to the 290 th position of Whole Taq) (hereinafter referred to as "Taq exo") and a polypeptide having the amino acid sequence of SEQ ID NO. 2 (amino acid from the N-terminus to the 292 nd position of Whole Tth) (hereinafter referred to as "Tth exo") are expressed by using E.coli JM109 strain, respectively, and purified by heparin-agarose chromatography. Any antigen was dissolved in phosphate buffer.

Test example 2 immunization of guinea pigs

To Slc: the back subcutaneous (lumbar) injection of Hartley guinea pigs (7 week old males) was 0.8mL of antigen preparation containing 400 μg antigen. The antigen preparation used an antigen solution prepared by dissolving the antigen in phosphate buffer in test example 1 and an adjuvant TiterMAXgold (TiterMAX Co.) at a ratio of 1:1 (liquid amount ratio) and liquefying. After 3 weeks, additional immunization was performed by injecting 0.8mL of the antigen preparation containing 400. Mu.g of antigen. Additional immunization was performed by injecting 0.4mL of an antigen solution containing 400. Mu.g of antigen after 3 weeks. Lymphadenectasis in immunized guinea pigs sequentially increases in the order of whole Taq, taq exo, whole Th, tth exo.

Test example 3 preparation of fluorescent marker protein

The whole DNA polymerase and the DNA polymerase lacking the domain E are respectively fluorescent-labeled. The DNA polymerase lacking domain E is: taq polymerase (hereinafter referred to as "ΔTaq") obtained by deleting the amino acid from the N-terminus to the 289-position in SEQ ID NO. 49 and Tth polymerase (hereinafter referred to as "ΔTth") obtained by deleting the amino acid from the N-terminus to the 291-position in SEQ ID NO. 50 were each expressed by E.coli JM109 strain and purified by heparin-sepharose chromatography.

Whole Taq and Whole Tth were fluorescently labeled with Dylight (trademark) 488NHS Ester (Thermo Fisher Scientific). Δtaq and Δtth were fluorescently labeled using DyLight (trademark) 550NHS Ester (Thermo Fisher Scientific).

Test example 4 isolation of Domain E-specific plasma cells and construction of antibody expression vectors

Cell suspensions were prepared from the iliac lymph nodes of guinea pigs immunized in test example 2 using the methods described in U.S. patent application publication No. 2014/031528, U.S. patent application publication No. 2018/292407, and U.S. patent application publication No. 2013/029325, and domain E-specific plasma cells were selected using a flow cytometer. The selection of domain E-specific plasma cells was performed by the following 5 methods, varying the combination of antigen for immunization and fluorescent-labeled protein prepared in test example 3.

[ method 1]

Domain E-specific plasma cells were selected from cells immunized with whole Taq by subtraction using DyLight488 labeled whole Taq and DyLight550 labeled Δtaq. That is, plasma cells that confirmed fluorescence corresponding to DyLight488 and did not confirm fluorescence corresponding to DyLight550 were selected.

[ method 2]

Domain E-specific plasma cells were selected from cells immunized with Taq exo using DyLight488 to label whole Taq.

[ method 3]

Domain E-specific plasma cells were selected from cells immunized with Tth exo using DyLight488 to label whole Taq.

[ method 4]

From cells immunized with whole Tth, domain E-specific plasma cells were selected by subtraction using DyLight488 to label whole Tth and DyLight550 to label Δtth. That is, plasma cells in which fluorescence corresponding to DyLight488 was confirmed and fluorescence corresponding to DyLight550 was not confirmed were selected.

[ method 5]

For cells immunized with Tth exo, the whole Tth selection domain E-specific plasma cells were labeled using DyLight 488.

The number of plasma cells selected and extracted targeting domain E of Taq polymerase was greater in method 3 than in methods 1 and 2, and 288 in method 3. The number of plasma cells to be extracted was 192 in method 4 and 240 in method 5, respectively, with respect to the number of plasma cells to be extracted targeting domain E of Tth polymerase.

The construction of the antibody expression vector was performed by using plasma cells selected and extracted by methods 3 to 5, by methods described in U.S. patent application publication No. 2011/020879, U.S. patent application publication No. 2013/02399, and U.S. patent application publication No. 2011/117609. In this case, the amino acid sequences described by SEQ ID Nos. 51 and 52 were used for the guinea pig heavy chain and the light chain constant regions, respectively. 22 antibody expression vectors were obtained by method 3, 9 antibody expression vectors by method 4, and 66 antibody expression vectors by method 5.

In methods 3 to 5, since the guinea pig has a larger lymphadenectasis and a larger number of isolated plasma cells than in methods 1 and 2, it can be said that the immune response by Tth polymerase as an antigen is more remarkable than that by Taq polymerase. From this, it was found that by using Tth polymerase as an antigen, domain E-specific plasma cells can be efficiently extracted from both Taq polymerase and Tth polymerase. In addition, method 5 resulted in a greater number of isolated plasma cells and antibody expression vectors than method 4. From this, it was found that antibodies that specifically bind to the domain E (anti-domain E antibodies) can be obtained efficiently when the immunization is performed using only the domain E, as compared with when the immunization is performed using the whole DNA polymerase.

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

Test example 5 evaluation of the binding Capacity of antibody Domain E

Antibody expression vectors were introduced into 293FT cells by the method described in U.S. patent application publication No. 2018/292407, and culture supernatants in which antibodies were secreted were recovered. A commercially available hot start antibody (TCP-101, manufactured by Toyobo Co., ltd.) was immobilized on an ELISA plate (Sumitomo electric Co., MS-8896F) using a carbonic acid buffer. For each well, blocking was performed using 1 XTBS (Nacalai Tesque) containing 1% (w/v) bovine serum albumin (Nacalai Tesque, inc. without globulin) after washing. After washing each well, an antigen (whole Taq, whole Tth) diluted with 1 XTBS-T (Nacalai Tesque Co.) was added to each well. After washing each well, culture supernatant was added to each well. After washing each well, a 50000-fold dilution of goat anti-guinea pig IgG H & L (HRP) (Abcam corporation) was added. After washing each well, TMB solution (TMBW-1000-01, SURMODICS) was added to develop a color, 1N sulfuric acid (Nacalai Tesque) was added to stop the reaction, and then the wavelength of 450 to 620nm was measured by an ELISA reader. In this binding capacity assessment, the DNA polymerase active domain has been occupied by the immobilized antibody, and thus the binding capacity of the antibody to domain E can be assessed.

Of the 22 antibodies obtained in 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%).

Of the 9 antibodies obtained in method 4, 1 (hit rate 11%) was found to bind to whole Tth, and this antibody did not show binding to whole Taq.

Of the 66 antibodies obtained in 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%).

Compared with the methods 4 and 5, the method 3 has about 2-8 times higher hit rate to each DNA polymerase and hit rate to two DNA polymerases. From the results, it can be said that the method of extracting domain E-specific plasma cells using fluorescence-labeled whole Taq with Tth Exo as an antigen that strongly induces an immune response was effective for obtaining an antibody that specifically binds to domain E of Taq polymerase. It was also found that, in the case of obtaining an antibody specifically binding to domain E of Tth polymerase, the method of selecting domain E-specific plasma cells using Tth Exo as an antigen and using fluorescence-labeled whole Tth gave unexpected results, and that the domain E-specific antibody could be isolated with a high probability.

Test example 6 decomposition of probes by DNA polymerase with Domain E

It was confirmed that the probe was decomposed by exposing the PCR reaction solution containing the DNA polymerase having domain E at 25℃for 24 hours.

(1) Constituent Components of PCR reaction solution

[ mixture for PCR ]

A PCR mixture 1 having the following composition was prepared.

Mixture 1 for PCR:

taq polymerase (0.05U/. Mu. L, TAP-201, manufactured by Toyo-yo Co., ltd.);

anti-polymerase antibody for hot-start PCR (0.01. Mu.g/. Mu. L, TCP-101),

Manufactured by TOYOBO Co., ltd.);

10mM Tris-HCl(pH8.3)；

50mM KCl；

1.5mM MgCl ₂ the method comprises the steps of carrying out a first treatment on the surface of the A kind of electronic device with high-pressure air-conditioning system

0.3mM dNTPs。

[ primer/Probe ]

As a primer/probe mixture at a concentration of 20-fold, taqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific Co.). The genes that can be amplified/detected by the primers/probes in this mix are IL6, CDK10, APC, MAPK8, SIVA1, RPS19 or SERPINB5.

[ nucleic acid template ]

cDNA made from HeLa cell (human cervical cancer origin) RNA was used. For the extraction of RNA and the synthesis of cDNA, human HeLa Cell Total RNA (636543, takara Shuzo Co., ltd.) and SuperPrep (trademark) II Cell Lysis & RT Kit for qPCR (SCQ-401, toyo-yo Co., ltd.) were used, and the procedure was followed.

(2) Reaction

To the PCR mixture 1, each primer/probe was mixed in a ratio of 1/20, and the nucleic acid template was mixed in a ratio of 1/20, thereby preparing 20. Mu.L of a PCR reaction solution. The PCR reaction solution was stored at-20℃or 25℃for 24 hours. Thereafter, a reaction was carried out using a real-time PCR apparatus (Applied Biosystems 7500Fast real-time PCR system) in accordance with the following temperature cycle. The fluorescence value was read in an extension step at 60℃for 60 seconds.

(temperature cycle)

Step 1:95 ℃ for 1 minute

Step 2:95℃15 sec-60℃60 sec 50 cycles (PCR)

(3) Results

In the reaction system, FAM channels were used to detect each gene. The Ct values at the time of detection of each gene (IL 6, CDK10, APC, MAPK8, SIVA1, RPS19, SERPINB 5) in HeLa cDNA using real-time PCR and the fluorescence values at the 10 th cycle in the multicomponent data (Multicomponent Data) are shown in table 9.

When the PCR reaction solution was exposed to 25℃for 24 hours, it was observed that Ct values of IL6, CDK10 and SIVA1 genes were delayed by 2 or more as compared with those when exposed to-20℃for 24 hours, and that Ct values were not calculated for the RPS19 gene (shown by "-" in the table of results). The reason why the RPS19 gene fails to calculate the Ct value is that the probe is completely decomposed and the fluorescence value corresponding to the amplified product is not increased.

In addition, when the PCR reaction solution was exposed to 25℃for 24 hours, the fluorescence values of the 10 th cycle of all 7 genes were increased as compared with those of the PCR reaction solution exposed to-20℃for 24 hours. The reason why the fluorescence value is raised is presumed to be that, when the probe is exposed to 25℃for 24 hours, the fluorescent label is decomposed and released before the initiation of the cycle, and as a result, extinction by quenching is released to generate fluorescence. Thus, it was found that all of the fluorescent-labeled probes for detecting 7 genes were decomposed when exposed to 25℃for 24 hours.

TABLE 9

Test example 7 inhibition of probe decomposition by anti-domain E antibodies

It was confirmed whether or not the anti-domain E antibody can inhibit the degradation of the probe when the PCR reaction solution containing the DNA polymerase having the domain E and the anti-domain E antibody is exposed to 25℃for 24 hours.

(1) Preparation of anti-domain E antibodies

Antibody expression vectors obtained according to the methods described in test examples 1 to 4 were introduced into 293FT cells by the method described in U.S. patent application publication No. 2018/292407, and culture supernatants in which antibodies were secreted were recovered. The culture supernatant was passed through HiTrap protein A HP column (Cytiva) using AKTA pure 25 (Cytiva). After washing the column with wash buffer (20 mM phosphate buffer, pH 7.4), elution was performed with elution buffer (0.1M citric acid-NaOH, pH 3.5). Antibodies were concentrated using Amicon Ultra-15 (Merck company) and quantified with Nanodrop One (Thermo Fisher Scientific company). Clone numbers Anti-TAQ1 to 5 are Anti-domain E antibodies obtained by method 3 of test example 4, and clone numbers Anti-TTH1 to 5 are Anti-domain E antibodies obtained by method 5 of test example 4.

(2) Constituent Components of PCR reaction solution

[ mixture for PCR ]

The same mixture as the mixture 1 for PCR used in test example 6 was used. In addition, the following 2 PCR-use mixtures 2 and 3 were prepared and used.

Mixture 2 for PCR:

tth polymerase (0.05U/. Mu. L, TTH-301, manufactured by Toyo-yo Co., ltd.);

anti-polymerase antibody for hot start PCR (0.01. Mu.g/. Mu. L, TCP-101, manufactured by Toyo Kagaku Co., ltd.);

10mM Tris-HCl(pH8.3)；

80mM KCl；

1.5mM MgCl ₂ ；

0.5mg/mL BSA；

0.1％(v/v)TritonX-100；

0.1% (w/v) sodium cholate; a kind of electronic device with high-pressure air-conditioning system

0.3mM dNTPs。

Mixture 3 for PCR:

tth polymerase (variant) (0.05U/. Mu.L) described in International publication No. 2018/096961;

10mM Tris-HCl(pH8.3)；

80mM KCl；

1.5mM MgCl ₂ ；

0.5mg/mL BSA；

0.1％(v/v)TritonX-100；

0.3mM dNTPs。

[ primer/Probe ]

As a mixture of the primer and the probe at a concentration of 20-fold, taqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific Co.). The gene that can be amplified/detected using the primer/probe in this mixture is RPS19.

[ nucleic acid template ]

(3) Reaction

(reaction liquid 1)

The primer/probe was mixed in a ratio of 1/20 to the PCR mixture 1, and the nucleic acid template was mixed in a ratio of 1/20, thereby preparing 19. Mu.L of a mixed solution. As a control, 1. Mu.L of 20mM Tris-HCl (pH 7.5) was added to the above mixture and exposed to-20℃or 25℃for 24 hours. In addition, as a control, 1. Mu.L of Platinum Taq monoclonal antibody (10965-028, thermo Fisher Scientific Co.) was added to the above mixture and exposed to 25℃for 24 hours. For anti domain E antibodies, to the above mixture was added 0.8mg/mL solution 1L (carry-over amount: 0.8 μg) and at 25 ℃ exposure for 24 hours.

(reaction liquid 2)

To the PCR mixture 2, the primer/probe was mixed in a ratio of 1/20, and the nucleic acid template was mixed in a ratio of 1/20, thereby preparing 19. Mu.L of a mixed solution. As a control, 1. Mu.L of 20mM Tris-HCl (pH 7.5) was added to the above mixture and exposed to-20℃or 25℃for 24 hours. For anti-domain E antibodies, 1. Mu.L (carry-over amount: 1.2. Mu.g) of each 1.2mg/mL solution was added to the above mixture and exposed at 25℃for 24 hours.

(reaction liquid 3)

To the PCR mixture 3, the primer/probe was mixed in a ratio of 1/20, and the nucleic acid template was mixed in a ratio of 1/20, thereby preparing 19. Mu.L of a mixed solution. As a control, 1. Mu.L of 20mM Tris-HCl (pH 7.5) was added to the above mixture and exposed to-20℃or 25℃for 24 hours. For anti-domain E antibodies, 1. Mu.L (carry-over amount: 1.2. Mu.g) of each 1.2mg/mL solution was added to the above mixture and exposed at 25℃for 24 hours.

(reaction)

The reaction solutions 1 to 3 were subjected to the following temperature cycles using a real-time PCR apparatus (Applied Biosystems 7500Fast real-time PCR system). The fluorescence value was read in an extension step at 60℃for 60 seconds.

(temperature cycle)

Step 1:95 ℃ for 1 minute

Step 2:95℃15 sec-60℃60 sec 50 cycles (PCR)

(4) Results

The Ct value at the time of detecting the RPS19 gene in the reaction solutions 1, 2 and 3 and the fluorescence value at the 10 th cycle in the multicomponent data are shown in tables 10, 11 and 12, respectively. 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 sequences adjacent to the C-terminus of the L chain CDR2 of Anti-TAQs 1 to 5 and Anti-TTHs 1 to 5 are shown in Table 13.

The reaction solution 1 to which Tris-HCl was added was exposed at 25℃for 24 hours, and the Ct value of the RPS19 gene was delayed by about 5 to 6 as compared with that when it was exposed at-20℃for 24 hours, and therefore, it could be said that the detection sensitivity was lowered by 2 ⁵ ～2 ⁶ About twice as much. In contrast, in the case of reaction solution 1 to which an anti-domain E antibody was added, ct values were all lower than 30. In addition, all clones of the anti-domain E antibody satisfied the probe decomposition inhibition indexes (a) to (c):

(a) Ct value is greater than or equal to 0.8 as compared with Ct value measured after 24 hours of exposure at-20 ℃ (corresponding to before 24 hours of exposure at 25 ℃)/Ct value measured after 24 hours of exposure at 25 ℃)

(b) The fluorescence intensity ratio [ -20 ℃ after 24 hours of exposure (corresponding to 25 ℃ before 24 hours of exposure) ] of the initial fluorescence intensity at the cycle/the initial fluorescence intensity at the cycle after 24 hours of exposure at 25 ℃ is not less than 0.3

(c) Probe decomposition Rate [ (F) ₃₃ -F ₃₁ )÷(F ₃₂ -F ₃₁ )×100]≤40％

F ₃₁ : fluorescence intensity at initial cycle determined after 24 hours of exposure at-20℃in the absence of anti-domain E antibody (corresponding to 24 hours of exposure at 25 ℃)

F ₃₂ : fluorescence intensity at initial cycle determined after 24 hours of exposure at 25℃in the absence of anti-domain E antibody

F ₃₃ : fluorescence intensity at initial cycle determined after 24 hours of exposure at 25℃in the presence of anti-domain E antibody

Thus, it was found that all clones of the anti-domain E antibody had a probe decomposition inhibitory effect.

The RPS19 gene was not detected by exposing the Tris-HCl-added reaction solution 2 to 25℃for 24 hours. In contrast, the RPS19 gene can be detected in the reaction solution 2 to which the anti-domain E antibody was added. In addition, it was found that all clones of the anti-domain E antibody satisfied the probe decomposition inhibition indexes (a) to (c), and had a probe decomposition inhibition effect. It was found that Anti-TTH2 and 3 showed an effect even in the case of reaction solution 1 containing Taq.

The RPS19 gene was not detected by exposing the Tris-HCl-added reaction solution 3 to 25℃for 24 hours. In contrast, when the reaction solution 3 to which the anti-domain E antibody was added, the RPS19 gene was detected. All clones of the anti-domain E antibody were found to satisfy the probe decomposition inhibition indexes (a) to (c) and had a probe decomposition inhibition effect. In the case of reaction solution 3, when the anti-domain E antibody was added, the probe decomposition inhibition index (b) was far more than 1, and the probe decomposition inhibition index (c) was far more than 100%. The reason is considered that the reaction solution reaches normal temperature when the control reagent is prepared, when the control reagent is set in a real-time PCR apparatus, or the like, and the fluorescent-labeled probe is decomposed. Therefore, the present antibody can suppress the degradation of the probe in the reaction solution not only when the reaction solution is stored for a long period of time but also when a usual nucleic acid amplification reagent is prepared.

TABLE 10

Control	-20 ℃/24 hours	25 ℃/24 hours	Platinum Taq antibodies
				Ct value	24.9	30.5	38.4
Fluorescence value	118525	895963	1055881
				(a) Ct value before and after preservation	-	0.79	0.71
(b) Fluorescence intensity before and after storage	-	0.12	0.11
				(c) Probe decomposition rate (%)	0	100	111

Clone number	Anti-TAQ1	Anti-TAQ2	Anti-TAQ3	Anti-TAQ4	Anti-TAQ5	Anti-TTH2	Anti-TTH3
								Ct value	26.5	24.5	24.4	24.1	24.5	25.1	27.8
Fluorescence value	310124	374937	274781	330541	380719	399935	315014
								(a) Ct value before and after preservation	0.94	1.01	1.02	1.03	1.01	0.99	0.89
(b) Fluorescence intensity before and after storage	0.39	0.32	0.44	0.37	0.32	0.30	0.38
								(c) Probe decomposition rate (%)	22	30	18	25	30	33	23

TABLE 11

Control	-20 ℃/24 hours	25 ℃/24 hours
			Ct value	29.4	n.d.
Fluorescence value	382968	1085575
			(a) Ct value before and after preservation	-	0.73
(b) Fluorescence intensity before and after storage	-	0.28
			(c) Probe decomposition rate (%)	0	100

Clone number	Anti-TTH1	Anti-TTH2	Anti-TTH4	Anti-TTH5
					Ct value	29.3	30.2	29.5	29.5
Fluorescence value	383319	399576	448323	333866
					(a) Ct value before and after preservation	0.99	0.96	0.98	0.98
(b) Fluorescence intensity before and after storage	0.76	0.73	0.65	0.87
					(c) Probe decomposition rate (%)	14	18	14	6

TABLE 12

Control	-20 ℃/24 hours	25 ℃/24 hours
			Ct value	23.3	n.d.
Fluorescence value	266199	1104373
			(a) Ct value before and after preservation	-	0.58
(b) Fluorescence intensity before and after storage	-	0.24
			(c) Probe decomposition rate (%)	0	100

Clone number	Anti-TTH1	Anti-TTH2	Anti-TTH4	Anti-TTH5
					Ct value	23.8	25.8	23.8	29.0
Fluorescence value	201522	196316	202495	213829
					(a) Ct value before and after preservation	0.98	0.90	0.98	0.80
(b) Fluorescence intensity before and after storage	1.29	1.48	1.44	1.36
					(c) Probe decomposition rate (%)	-7	-8	-7	-6

TABLE 13

Test example 8 influence of the exposure time of the PCR reaction solution at 25 ℃C

The probe decomposition inhibition effect of the anti-domain E antibody was confirmed by changing the exposure time of the PCR reaction solution at 25 ℃.

(1) Constituent Components of PCR reaction solution

[ mixture for PCR ]

The same mixture as the mixture 1 for PCR used in test example 6 was used.

[ primer/Probe ]

As a primer/probe mixture at a concentration of 20-fold, taqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific Co.). The genes that can be amplified/detected with the primers/probes in this mix are IL6, CDK10 and RPS19.

[ nucleic acid template ]

cDNA made from HeLa cell (human cervical cancer origin) RNA was used. For RNA extraction and cDNA synthesis, human HeLa Cell Total RNA (636543, takara Shuzo Co., ltd.) and SuperPrep (trademark) II Cell Lysis & RT Kit for qPCR (SCQ-401, toyo-yo Co., ltd.) were used, and the procedure was followed.

(2) Reaction

(reaction liquid)

To the PCR mixture 1, each primer/probe was mixed in a ratio of 1/20, and the nucleic acid template was mixed in a ratio of 1/20, thereby preparing 19. Mu.L of a mixture. As a control, 1. Mu.L of 20mM Tris-HCl (pH 7.5) was added to the above mixture and exposed to-20℃or 25℃for 24 hours. For anti domain E antibodies, to the above mixture was added 0.1mg/mL solution 1L (carry-over amount: 0.1 μg) and exposed at 25℃for 24 hours. Then, the reaction was performed using a real-time PCR apparatus (Applied Biosystems 7500Fast real-time PCR system) according to the following temperature cycle. The fluorescence value was read in an extension step at 60℃for 60 seconds.

(temperature cycle)

Step 1:95 ℃ for 1 minute

Step 2:95℃15 sec-60℃60 sec 50 cycles (PCR)

(3) Results

The Ct values when the genes (IL 6, CDK10, RPS 19) were detected are shown in Table 14. When the reaction solution added with Tris-HCl was exposed at 25℃for 24 hours, 3 genes could not be detected. On the other hand, when 0.1. Mu.g of each reaction solution of the anti-domain E antibody was added, no delay in Ct value was observed, and all genes could be detected. In addition, no delay in Ct value was observed even when the reaction solution was exposed to 25 ℃ for 72 hours. Further, the increase in fluorescence value was not confirmed, and it was found that the addition of the anti-domain E antibody suppressed the degradation of the probe. Therefore, it was confirmed that the use of the antibody can store the PCR reaction solution even at 25℃for 72 hours.

TABLE 14

Test example 9 inhibition of probe decomposition by micro anti-Domain E antibodies

It was confirmed whether or not the probe decomposition was inhibited by exposing a PCR reaction solution containing 0.1. Mu.g of an anti-domain E antibody at 25℃for 24 hours.

(1) Constituent components of the reaction solution

[ mixture for PCR ]

The same mixture as the PCR mixture 2 used in test example 7 was used.

[ primer/Probe ]

As a primer/probe mixture at a concentration of 20-fold, taqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific Co.). The genes that can be amplified/detected with the primers/probes in this mix are IL6, CDK10, SIVA1 and RPS19.

[ nucleic acid template ]

(2) Reaction

To the PCR mixture 2, each primer/probe was mixed in a ratio of 1/20, and the nucleic acid template was mixed in a ratio of 1/20, thereby preparing 15. Mu.L of a mixed solution. As a control, 5. Mu.L of 20mM Tris-HCl (pH 7.5) was added to the above mixture and exposed to-20℃or 25℃for 24 hours. For anti domain E antibodies, to the above mixture was added 0.1mg/mL solution 1L (carry-over amount: 0.1 μg) and exposed at 25℃for 24 hours.

For each reaction solution, a real-time PCR apparatus (Applied Biosystems 7500Fast real-time PCR system) was used, and the reaction was carried out according to the following temperature cycle. The fluorescence value was read in an extension step at 60℃for 60 seconds.

(temperature cycle)

Step 1:95 ℃ for 1 minute

Step 2:95℃15 sec-60℃60 sec 50 cycles (PCR)

(3) Results

Table 15 shows Ct values when the respective genes (IL 6, CDK10, SIVA1, RPS 19) were detected.

The delay in Ct value was confirmed for each gene when the Tris-HCl-added reaction solution 4 was exposed at 25℃for 24 hours. On the other hand, when the reaction solution 4 to which 0.1. Mu.g of each anti-domain E antibody was added was exposed at 25℃for 24 hours, no delay in Ct value was observed.

The control reaction solution added with Tris-HCl was exposed to 25℃for 24 hours, and a delay in Ct value was confirmed for each gene. On the other hand, when the reaction solution 5 to which 0.1. Mu.g of the anti-domain E antibody was added was exposed at 25℃for 24 hours, no delay in Ct value was observed.

TABLE 15

Test example 10 expression of chimeric anti-Domain E antibodies

(1) Preparation of antibody expression plasmid

The antibody sequences comprising the CDRs of Anti-TTH4 were designed and oligo DNA was obtained by artificial synthesis. An antibody expression plasmid having mouse-derived heavy and light chain constant regions described in SEQ ID NOS 53 and 54 was prepared using Mammalian PowerExpress System (trademark) (MPH-102 and MPL-202, toyobo Co., ltd.) according to the accompanying protocol.

(2) Antibody expression Using ExpiCHO-S (trade Mark) cells

Antibody expression was performed using expiho (trade mark) Expression System (Thermo Fisher Scientific). As for the culture conditions, the temperature was 37℃at 5% (v/v) CO ₂ Shaking culture at 80 rpm. ExpiCHO-S (trademark) cells were resuscitated according to the instructions attached to the protocol to give a viable cell count of 2.0X10 ⁵ Each cell/mL was cultured with shaking. The passage is continued until the survival rate reaches 95%, and the number of the living cells is prepared to be 6.0x10 ⁶ Culture medium of individual cells/mL. For 25mL of the culture medium, 80. Mu.L of the antibody expression plasmid 1.0. Mu. g, expiFectamine (trademark) CHO Reagent was diluted with 2mL of OptiPro SFM (trademark) and then added thereto, and the mixture was subjected to 5% (v/v) CO at 37 ℃ ₂ Shaking culture was performed at 80 rpm. After 24 hours, 150. Mu.L of ExpiCHO (trademark) Enhancer and 6mL of ExpiCHO (trademark) Feed were added thereto at 37℃with 5% (v/v) CO ₂ Shaking culture was continued at 80rpm until the survival rate reached 50%.

(3) Purification of antibodies using protein A columns

The culture supernatant of the ExpiCHO-S (trade mark) cells was recovered by centrifugation. The culture supernatant was passed through HiTrap protein A HP column (Cytiva) using AKTA pure25 (Cytiva). After washing the column with wash buffer (20 mM phosphate buffer, pH 7.4), elution was performed with elution buffer (0.1M citric acid-NaOH, pH 3.5). The antibodies were concentrated using Amicon Ultra-15 (Merck company) and quantified with Nanodrop One (Thermo Fisher Scientific company).

In the following test examples, chimeric anti-domain E antibodies having mouse-derived constant regions obtained by the above-described methods were used.

Test example 11 inhibition of probe decomposition by chimeric anti-Domain E antibodies

It was confirmed whether or not the chimeric anti-domain E antibody inhibited the degradation of the probe when the PCR reaction solution containing the chimeric anti-domain E antibody was exposed to 25℃for 24 hours.

(1) Constituent components of the reaction solution

[ mixture for PCR ]

The same mixture as the mixture 1 for PCR used in test example 6 and the mixture 2 for PCR used in test example 7 was used.

[ primer/Probe ]

As a primer/probe mixture at a concentration of 20-fold, taqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific Co.). The gene that can be amplified/detected using the primer/probe in this mixture is RPS19.

[ nucleic acid template ]

(2) Reaction

(reaction solution 4 and 5)

The primer/probe was mixed with each of the PCR mixtures 1 and 2 in a ratio of 1/20, whereby 18. Mu.L of a mixed solution (corresponding to the reaction solutions 4 and 5, respectively) was prepared. Will use Nanodrop ^TM The nucleic acid templates were diluted to 100, 10, 1 and 0.1 ng/. Mu.L after One (Thermo Fisher Scientific Co.) quantification, and 1. Mu.L was added to the above mixture (carrying-in amounts: 100, 10, 1 and 0.1 ng). As a control, 1. Mu.L of 20mM Tris-HCl (pH 7.5) was added to the above mixture and exposed to-20℃or 25℃for 24 hours. For chimeric anti-domain E antibodies, 1. Mu.L (carry-over amount: 0.1. Mu.g) of a 0.1mg/mL solution was added to the above mixture and exposed at 25℃for 24 hours. Thereafter, a reaction was carried out using a real-time PCR apparatus (Applied Biosystems 7500Fast real-time PCR system) in accordance with the following temperature cycle. The fluorescence value was read in an extension step at 60℃for 60 seconds.

(temperature cycle)

Step 1:95 ℃ for 1 minute

Step 2:95℃15 sec-60℃60 sec 50 cycles (PCR)

(3) Results

Table 16 shows the Ct value at the time of detecting the RPS19 gene and the fluorescence value at the 10 th cycle in the multicomponent data for reaction solution 4, and Table 17 shows the Ct value at the time of detecting the RPS19 gene and the fluorescence value at the 10 th cycle in the multicomponent data for reaction solution 5.

The RPS19 gene could not be detected when exposed to 25℃for 24 hours in the reaction solutions 4 and 5 to which Tris-HCl was added, and the RPS19 gene could be detected when exposed to 25℃for 24 hours in the reaction solutions 4 and 5 to which chimeric anti-domain E antibody was added at a Ct value equivalent to that when exposed to-20℃for 24 hours in HeLa cDNA 100, 10, 1, 0.1 ng. In addition, when the probe decomposition rate was estimated by the same method as in (c) of test example 7, the value was calculated to be 4.4% for Anti-TAQ2 and 3.3% for Anti-TTH 4.

TABLE 16

TABLE 17

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Test example 12 separation of probes in the Co-presence of Taq polymerase (variant) or Z05 polymerase with anti-Domain E antibody De-inhibition

It was confirmed whether or not the anti-domain E antibody inhibited the degradation of the probe when the PCR reaction solution containing the anti-domain E antibody and containing Taq polymerase (variant) or Z05 polymerase was exposed to 25℃for 24 hours.

(1) Constituent components of the reaction solution

[ mixture for PCR ]

The following 3 PCR mixtures 4 to 6 were prepared and used.

Mixture 4 for PCR:

QuantiNova Probe RT-PCR Kit (QIAGEN Co., 208352) containing Taq polymerase (variant)

Mixture 5 for PCR:

TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, 4444556) containing Taq polymerase (variant)

Mixture 6 for PCR:

z05 polymerase (0.05U/. Mu.L, roche Diagnostics company, hawkZ05, SEQ ID NO: 55);

10mM Tris-HCl(pH8.3)；

80mM KCl；

1.5mM MgCl ₂ ；

0.5mg/mL BSA；

0.1％(v/v)TritonX-100；

0.3mM dNTPs。

[ primer/Probe ]

[ nucleic acid template ]

(2) Reaction

(reaction liquids 6 to 8)

The primer/probe was mixed in a ratio of 1/20 and the nucleic acid template was mixed in a ratio of 1/20 to each of the PCR mixtures 4 to 6, whereby 19. Mu.L of a mixture (corresponding to each of the reaction solutions 6 to 8) was prepared. As a control, 1. Mu.L of 20mM Tris-HCl (pH 7.5) was added to the above mixture and exposed to-20℃or 25℃for 24 hours. For anti domain E antibodies, to the aforementioned mixture was added 0.1mg/mL solution 1L (carry-over amount: 0.1 μg) and exposed at 25℃for 24 hours. Thereafter, a reaction was carried out using a real-time PCR apparatus (Applied Biosystems 7500Fast real-time PCR system) in accordance with the following temperature cycle. The fluorescence value was read during the extension step at 60 ℃.

(temperature cycle of reaction solution 6 containing mixture 4 for PCR)

Step 1:95 ℃ for 2 minutes

Step 2:95℃for 5 seconds to 60℃for 24 seconds for 50 cycles (PCR)

(temperature cycle of reaction solution 7 containing mixture 5 for PCR)

Step 1:95 ℃ for 20 seconds

Step 2:95℃for 3 seconds to 60℃for 30 seconds for 50 cycles (PCR)

(temperature cycle of reaction solution 8 containing mixture 6 for PCR)

Step 1:95 ℃ for 60 seconds

Step 2:95℃15 sec-60℃45 sec 50 cycles (PCR)

(3) Results

Table 18 and Table 19 show Ct values at the time of detecting the RPS19 gene and fluorescence values at the 10 th cycle in the multicomponent data, respectively.

When the reaction solutions 8 to 10 to which Tris-HCl was added were exposed at 25℃for 24 hours, it was observed that the Ct value of the detectable RPS19 gene was delayed, resulting in an undetectable gene. On the other hand, in the case of adding anti domain E antibody reaction solution 8 ~ 10, even at 25 ℃ exposure for 24 hours, can also be used to 20 ℃ exposure 24 hours equal Ct value detection of each gene. From this, it can be seen that the anti-domain E antibody significantly improved the stability of the PCR reaction solution containing Taq variants. In addition, it was found that the anti-domain E antibodies also significantly improved the stability of the PCR reaction solution containing Z05 polymerase.

TABLE 18

TABLE 19

Test example 13 inhibition of 5 '. Fwdarw.3' exonuclease Activity by anti-Domain E antibodies

For the obtained anti-domain E antibody, the inhibitory ability to the 5 '. Fwdarw.3' exonuclease activity of Taq polymerase or Tth polymerase was confirmed.

(1) Reaction

(reaction liquid 9)

Taq enzyme solution containing 1 unit of Taq polymerase (TAP-201, toyo Co., ltd.) and 0.2. Mu.g of a commercially available hot start antibody Anti-Taq high (TCP-101, toyo Co., ltd.) was prepared. A mixture of Taq polymerase enzyme solution and Anti-TAQ 2.1. Mu.g was added to a reaction solution (final concentration: 10mM Tris-HCl (pH 8.6), 50mM KCl, 1.5mM MgCl) containing substrate DNA (9000 cpm, 80% count rate) radiolabelled at the 5' -end with 32P ₂ ) The amount of the liquid was adjusted to 20. Mu.L (sample 3). As a control, sample 1 containing no Taq polymerase enzyme solution and no Anti-TAQ2, and sample 2 containing only Taq polymerase enzyme solution were prepared, and each sample was incubated at 37℃for 24 hours. Then, 10% (w/v) TCA 100. Mu.L was added to each sample to precipitate the substrate DNA, and the radioactivity of the free 32P-labeled base remaining in the supernatant was measured.

(reaction solution 10)

Tth enzyme solution containing 1 unit of Tth polymerase (TTH-301, toyo Co., ltd.) and 0.6. Mu.g of a commercially available hot start antibody Anti-Taq high (TCP-101, toyo Co., ltd.) was prepared. A mixture of Tth polymerase enzyme solution and Anti-TTH 4.1. Mu.g was added to a reaction solution (final concentration: 10mM Tris-HCl (pH 8.6), 50mM KCl, 1.5mM MgCl) containing substrate DNA (9000 cpm, 80% count rate) radiolabelled at the 5' -end with 32P ₂ ) The amount of the liquid was adjusted to 20. Mu.L (sample 3). As a control, sample 1 containing neither Tth polymerase enzyme solution nor Anti-Tth4, and sample 2 containing only Tth polymerase enzyme solution were prepared, and each sample was incubated at 37 ℃ for 24 hours. Thereafter, 10% (w/v) TCA 100. Mu.L was added to each sample to precipitate a substrate DNA, and radioactivity of the free 32P-labeled base remaining in the supernatant was measured.

[ substrate DNA ]

A reaction solution was prepared by mixing 10. Mu.g of lambda.DNA and 30 units of ScaI (manufactured by Toyo-yo Co., ltd.) according to the protocol, and incubated at 37℃for 24 hours. Particles produced by phenol/chloroform/isoamyl alcohol (liquid amount ratio 25:24:1) treatment and ethanol precipitation were dissolved in 100. Mu.L of TE buffer. To 80. Mu.L of this solution were added 5. Mu.L of P-32' -triphosphateadenosine, [ gamma-32P ] - (manufactured by Perkinelmer Co., ltd., NEG 002) and 5. Mu.L of [ 5.mu. L, T4 ] polynucleotide kinase (manufactured by Toyo-yo Co., ltd., PNK-111) and 10X Blunt End Kinase Buffer. Mu.L (manufactured by Toyo-yo Co., ltd., PNK-111) and incubated at 37℃for 1 hour. Particles produced by phenol/chloroform/isoamyl alcohol (liquid amount ratio 25:24:1) treatment and ethanol precipitation were dissolved in 100. Mu.L of TE buffer.

(2) Results

In the reaction solution 9, the substrate DNA was not decomposed to 100% in the case of sample 1 containing no Taq polymerase enzyme solution and no Anti-TAQ2, and the substrate DNA was decomposed to 0% at the maximum in the case of sample 2 containing only Taq polymerase enzyme solution, and the residual rate of the substrate DNA in sample 3 was calculated as the 5 '. Fwdarw.3' exonuclease activity inhibition ability, and the results are shown in Table 20. The activity inhibition capacity of Anti-TAQ2 was calculated to be 91%.

In the reaction solution 10, in the case of sample 1 containing no Tth polymerase enzyme solution and no Anti-TTH4, the substrate DNA was not decomposed to 100%, in the case of sample 2 containing only Tth polymerase enzyme solution, the substrate DNA was decomposed to 0% at the maximum, and the residual rate of the substrate DNA in sample 3 was calculated as the 5 '. Fwdarw.3' exonuclease activity inhibition ability, and the results are shown in Table 21. The activity inhibitory capacity of Anti-TTH4 was calculated to be 98%.

TABLE 20

TABLE 21

Test example 14 ability to inhibit 5 '. Fwdarw.3' exonuclease Activity when the amount of anti-Domain E antibody added was changed

The inhibitory ability of the obtained anti-domain E antibody to the 5 '. Fwdarw.3' exonuclease activity of Taq polymerase was confirmed by changing the amount of the antibody added.

(1) Reaction

(reaction liquid)

Taq polymerase enzyme solution containing 1 unit of Taq polymerase (TAP-201, toyo Co., ltd.) and 0.2. Mu.g of a commercially available hot start antibody Anti-Taq high (TCP-101, toyo Co., ltd.) was prepared. A mixture of Taq polymerase enzyme solution and Anti-TAQ 2.05, 0.1, 0.2 or 0.4. Mu.g was added to a reaction solution (final concentration: 10mM Tris-HCl (pH 8.6), 50mM KCl, 1.5mM MgCl) containing substrate DNA radiolabelled at the 5' -end with 32P (9000 cpm, count rate 80%) ₂ ) The amount of the liquid was adjusted to 20. Mu.L (samples 3 to 6). As a control, sample 1 containing no Taq polymerase enzyme solution and no Anti-TAQ2, and sample 2 containing only Taq polymerase enzyme solution were prepared, and each sample was incubated at 37℃for 24 hours. Thereafter, 10% (w/v) TCA 100. Mu.L was added to each sample to precipitate a substrate DNA, and radioactivity of the free 32P-labeled base remaining in the supernatant was measured.

[ substrate DNA ]

The same substrate DNA as in test example 13 was used.

(2) Results

In the case of sample 1 containing no Taq polymerase enzyme solution and no Anti-TAQ2, the substrate DNA was not decomposed to 100%, and in the case of sample 2 containing only Taq polymerase enzyme solution, the substrate DNA was decomposed to 0% at the maximum, and the residual rate of the substrate DNA in samples 3 to 6 was calculated as 5 '. Fwdarw.3' exonuclease activity inhibition ability, and the results are shown in Table 22.

TABLE 22

Test example 15 inhibition of Tth polymerase cleavage Probe by antibodies specifically binding to Domain E of Taq polymerase Manufacturing process

It was confirmed whether or not the antibody inhibits the cleavage of the probe by Tth polymerase when the PCR reaction solution containing the antibody specifically binding to domain E of Taq polymerase and Tth polymerase was exposed to 25℃for 24 hours.

(1) Constituent Components of PCR reaction solution

[ mixture for PCR ]

[ primer/Probe ]

As a primer/probe mixture at a concentration of 20-fold, taqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific Co.). The genes that can be amplified/detected with the primers/probes in this mix are IL6, CDK10, SIVA1, RPS19, and SERPINB5.

[ nucleic acid template ]

cDNA made from HeLa cell (human cervical cancer origin) RNA was used. Human HeLa Cell Total RNA (product code: 636543, takara Shuzo Co., ltd.) and SuperPrep (trademark) II Cell Lysis & RT Kit for qPCR (SCQ-401, toyo-yo Co., ltd.) were used for the extraction of RNA and the synthesis of cDNA, and the procedure was followed.

(2) Reaction

Each primer/probe was mixed in a ratio of 1/20 to the mixture 1 or 2 for PCR, thereby preparing 18. Mu.L of a mixed solution. The nucleic acid template quantified by nanodrop (TM) One (Thermo Fisher Scientific Co.) was diluted to 100 ng/. Mu.L, and 1. Mu.L (carry-over amount: 100 ng) was added. As a control, 1. Mu.L of 20mM Tris-HCl (pH 7.5) was added and exposed to-20℃or 25℃for 24 hours. For Anti-TAQ2, 4. Mu.L (carry-over amount: 0.4. Mu.g) of a 0.1mg/mL solution was added and exposed at 25℃for 24 hours. Thereafter, a reaction was carried out using a real-time PCR apparatus (Applied Biosystems 7500Fast real-time PCR system) in accordance with the following temperature cycle. The fluorescence value was read in an extension step at 60℃for 45 seconds.

(temperature cycle)

Step 1:95 ℃ for 1 minute

Step 2:95℃15 sec-60℃45 sec 50 cycles (PCR)

(3) Results

The Ct value at the time of detection of each gene and the fluorescence value at the 10 th cycle in the multicomponent data are shown in Table 23. When the reaction solution containing Tris-HCl was exposed to 25℃for 24 hours, it was confirmed that the detection of each gene or Ct value was not significantly delayed, that is, the detection sensitivity was lowered. On the other hand, when the reaction solution containing Anti-TAQ2 was exposed to 25℃for 24 hours, each gene of HeLa cDNA was detected at a Ct value equivalent to that of the reaction solution containing Tris-HCl at-20℃for 24 hours. Further, it was confirmed that Anti-TAQ2 showed the ability to inhibit probe decomposition for both Taq polymerase and Tth polymerase. From this, it was confirmed that by method 3 of screening antibodies against whole Taq using Tth exo as an immunogen, antibodies having neutralizing activity against both Taq and Tth can be obtained.

TABLE 23

Test example 16 inhibition of 5 '. Fwdarw.3' exonuclease Activity by anti-Domain E antibodies when substrate DNA was altered Force of force

The lambda DNA-derived double-stranded substrate DNA described in SEQ ID Nos. 56 and 57 was designed, and the inhibitory ability of the anti-domain E antibody against the 5 '. Fwdarw.3' exonuclease activity of Taq polymerase or Tth polymerase was confirmed.

(1) Sample preparation

The following 2 mixtures 1 and 2 for activity measurement were prepared and used.

Mixture 1 for activity measurement:

taq polymerase (0.05U/. Mu. L, TAP-201, manufactured by Toyo-yo Co., ltd.);

10mM Tris-HCl(pH8.6)；

50mM KCl；

1.5mM MgCl ₂ ；

0.3. Mu.M double-stranded substrate DNA;

mixture 2 for activity measurement:

tth polymerase (0.05U/. Mu. L, TTH-301, manufactured by Toyo-yo Co., ltd.);

10mM Tris-HCl(pH8.6)；

50mM KCl；

1.5mM MgCl ₂ ；

0.3. Mu.M double-stranded substrate DNA;

[ double-stranded substrate DNA ]

Lambda DNA-derived double-stranded substrate DNA having the oligonucleotides described in SEQ ID Nos. 56 and 57 was designed. The oligonucleotides of SEQ ID Nos. 56 and 57 were synthesized individually and mixed in equal amounts for use.

As a control, 1. Mu.L of 20mM Tris-HCl (pH 7.5) was added to 19. Mu.L of each of the activity-determining mixtures, and exposed to-20℃or 25℃for 24 hours. For the anti-domain E antibody, 1. Mu.L of 0.1mg/mL of solution (carry-over amount: 0.1. Mu.g) was added to 19. Mu.L of the mixture 1 for activity measurement and exposed at 25℃for 24 hours, and 1. Mu.L of 0.4mg/mL of solution (carry-over amount: 0.4. Mu.g) was added to 19. Mu.L of the mixture 2 for activity measurement and exposed at 25℃for 24 hours. Thereafter, each sample was analyzed using a microchip electrophoresis apparatus for DNA/RNA analysis (MultiNA, shimadzu corporation) and a DNA-500 kit (S292-27910-91, shimadzu corporation).

(2) Results

The quantitative values of the bands at the time of analyzing each sample are shown in Table 24. In the case of either of the activity measurement mixtures 1 and 2, tris-HCl was added and exposed at 25℃for 24 hours, and the quantitative value of the bands was significantly lowered as compared with that of the bands exposed at-20℃for 24 hours, confirming that the double-stranded substrate DNA was decomposed. On the other hand, when Anti-TAQ2 was added and exposed to 25℃for 24 hours, the quantitative value of the band was the same as that when Tris-HCl was added and exposed to-20℃for 24 hours, and no degradation of the double-stranded substrate DNA was confirmed.

In addition, the 5 '. Fwdarw.3' exonuclease activity inhibitory ability was determined by calculating the DNA degradation rate of the double-stranded substrate (d) described below.

(d) Double-strand substrate DNA degradation rate (%) [ (S) ₂₁ -S ₂₃ )÷(S ₂₁ -S ₂₂ )×100]

S ₂₁ : band intensity after 24 hours of exposure at-20℃without anti-domain E antibody (corresponding to 24 hours before exposure at 25 ℃)

S ₂₂ : band intensity after 24 hours of exposure at 25℃in the absence of anti-domain E antibody

S ₂₃ : band intensity after 24 hours of exposure at 25 ℃ comprising anti-domain E antibody

When Anti-TAQ2 was added and the sample was exposed at 25℃for 24 hours, the DNA degradation rate (%) of (d) double-stranded substrate by Taq polymerase and Tth polymerase was calculated to be 10% or less. From this, it was confirmed that Anti-TAQ2 exhibited a sufficient ability to inhibit the decomposition of double-stranded substrate DNA for both Taq polymerase and Tth polymerase.

TABLE 24

Test example 17 fluorescent labelling of DNA polymerase by antibodies specifically binding to Domain E of Taq polymerase Inhibition of double-stranded substrate DNA (probe)

The inventors confirmed whether or not the antibody specifically bound to domain E of Taq polymerase and DNA polymerase (Taq polymerase or Tth polymerase) inhibited the degradation of the fluorescent-labeled double-stranded substrate DNA by the antibody when the PCR reaction solution was exposed to 37℃for 24 hours.

(1) Constituent components of the reaction solution

[ mixture for PCR ]

The following 2 PCR mixtures 7 and 8 were prepared and used.

Mixture 7 for PCR:

taq polymerase (0.05U/. Mu. L, TAP-201, manufactured by Toyo-yo Co., ltd.);

10mM Tris-HCl(pH8.6)；

50mM KCl；

0.3. Mu.M fluorescence-labeled double-stranded substrate DNA.

Mixture 8 for PCR:

tth polymerase (0.05U/. Mu. L, TTH-301, manufactured by Toyo-yo Co., ltd.);

10mM Tris-HCl(pH8.6)；

50mM KCl；

0.3. Mu.M fluorescence-labeled double-stranded substrate DNA.

[ fluorescence labeling double-stranded substrate DNA ]

A lambda DNA-derived fluorescence-labeled double-stranded substrate DNA having the oligonucleotides described in SEQ ID Nos. 58 and 59 (herein, the 5 '-end of SEQ ID No. 58 is labeled with FAM and the 5' -end is labeled with BHQ 1) was designed. The oligonucleotides of SEQ ID Nos. 58 and 59 were synthesized individually and mixed in equal amounts for use.

(2) Reaction

As a control, a reaction solution obtained by adding 1. Mu.L of 20mM Tris-HCl (pH 7.5) to 19. Mu.L of the PCR mixture was exposed to-20℃or 37℃for 24 hours. 1. Mu.L (carry-over amount: 0.4. Mu.g) of an anti-domain E antibody solution was added to 19. Mu.L of the PCR mixture, and the mixture was exposed to a reaction solution at 37℃for 24 hours. Thereafter, a reaction was carried out using a real-time PCR apparatus (Applied Biosystems 7500Fast real-time PCR system) in accordance with the following temperature cycle. The fluorescence value was read in an extension step at 60℃for 45 seconds.

(temperature cycle)

Step 1:95 ℃ for 1 minute

Step 2:95℃15 sec-60℃45 sec 50 cycles (PCR)

(3) Results

The fluorescence values for the 10 th cycle in the multicomponent data are shown in table 25. The increase in fluorescence value was confirmed by exposing the reaction solution to Tris-HCl at 37℃for 24 hours, as compared with the reaction solution exposed to-20℃for 24 hours. On the other hand, when the reaction solution containing Anti-TAQ2 was exposed to 37℃for 24 hours, the fluorescence value was not observed to increase in the case of Taq polymerase and Tth polymerase, as compared with the case of exposing the reaction solution containing Tris-HCl to-20℃for 24 hours.

Specifically, (e) the DNA degradation rate of the fluorescent-labeled double-stranded substrate can be calculated by the following formula.

(e) Fluorescence-labeled double-stranded substrate DNA degradation Rate [ (F) ₄₃ -F ₄₁ )÷(F ₄₂ -F ₄₁ )×100)]

F ₄₁ : fluorescence intensity at cycle 10 after 24 hours of exposure at-20℃without anti-domain E antibody (corresponding to 24 hours before exposure at 37 ℃)

F ₄₂ : fluorescence intensity at cycle 10 after 24 hours exposure at 37℃in the absence of anti-domain E antibody

F ₄₃ : fluorescence intensity of cycle 10 after 24 hours of exposure at 37 ℃ when comprising anti-domain E antibody

In the case of a sample to which Anti-TAQ2 was added and which was exposed at 37℃for 24 hours, it was calculated that the degradation rate (%) of (e) the fluorescent-labeled double-stranded substrate DNA was 10% or less in the case of Taq polymerase and Tth polymerase. From this, it was confirmed that Anti-TAQ2 exhibited a sufficient ability to inhibit the degradation of the fluorescent-labeled double-stranded substrate DNA for both Taq polymerase and Tth polymerase.

TABLE 25

Test example 18 antibody binding Rate constant ka value, dissociation Rate constant kd value and equilibrium dissociation constant K _D Value of Measurement

Affinity of the antibodies for Tth polymerase was determined using Surface Plasmon Resonance (SPR). The measurement device used was a Biacore X100 device (Cytiva). The running buffer used was 0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.05% (v/v) Surfactant P20 (Cytiva).

(1) Immobilization by amine coupling

Ligands (Tth polymerase) were immobilized on CM5 sensor chips (Cytiva) using EDC and NHS, and blocked with 1M ethanolamine hydrochloride solution. As a result, tth polymerase is immobilized on flow cells 1-4 at a density of 200-500 RU.

(2) Interaction assay

Antibodies were serially diluted in the range of 0.222-81 nM and added to the flow cell. Fitting the obtained sensor map with a Bivalent analysis model of Biacore X100 evaluation software to determine a binding rate constant (ka), a dissociation rate constant (kd) and a equilibrium dissociation constant (K) _D )。

(3) Results

The results of interaction analysis of Anti-TTH2, anti-TTH4, and Anti-TTH5 as Anti-domain E antibodies are shown in Table 24. Anti-domain E antibodies all showed K below 10nM _D 。

TABLE 26

Antibody number	ka1(1/Ms)	kd1(1/s)	ka2(1/RUs)	kd2(1/s)	K _D (nM)
						Anti-TTH2	4.046×10 ⁵	2.532×10 ^-4	0.003132	0.01332	0.63
Anti-TTH4	1.545×10 ⁶	3.082×10 ^-4	2.452×10 ^-4	0.007099	0.20
						Anti-TTH5	4.546×10 ⁴	3.383×10 ^-4	2.992×10 ^-4	0.001252	7.4

Test example 19 epitope mapping of antibodies

Epitope mapping was performed using conformational epitope mapping in PEPperMAP (trademark) Peptide Microarray trusted assay service from PEPperPRINT corporation. Peptides of 7, 10 and 13 amino acids were synthesized on the peptide array by shifting the amino acid sequence of Taq exo shown in SEQ ID NO. 1 (amino acid from the N-terminus to 290 th position of Whole Taq) and the amino acid sequence of Tth exo shown in SEQ ID NO. 2 (amino acid from the N-terminus to 292 nd position of Whole Tth) by 1 amino acid each time in a manner of overlapping 6, 9 and 12 amino acids. Then, detection signals indicating binding of Anti-TAQ2 and Anti-TTH4 were measured for each peptide array, and an epitope that interacted with the antibody was determined.

(results)

It was confirmed that Anti-TAQ2 bound to at least 2 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, in addition, bound to at least 4 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).

It was confirmed that Anti-TTH4 bound to at least 3 regions of the amino acid sequences HEAYGY (SEQ ID NO: 64), EKYGLRPDQWADY (SEQ ID NO: 68) and RAFLERLEFGSLLH (SEQ ID NO: 80) in Taq exo (SEQ ID NO: 1), and bound to at least 5 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).

Anti-TAQ2 is an antibody that recognizes and binds to the following epitopes: an antibody comprising an epitope of the sequence (EDGDAVIVVF (SEQ ID NO: 60) or EDGYKAVFVVF (SEQ ID NO: 62)) in amino acid region A and an epitope of the common sequence (LERLEF (SEQ ID NO: 75)) in amino acid region D, shared or similar in Taq exo and Tth exo.

Anti-TTH4 is an antibody that recognizes and binds to the following epitopes: common or similar epitopes in Taq exo and Tth exo, comprising the sequence in amino acid region B (HEAYGY (SEQ ID NO: 64) or HEAYEAY (SEQ ID NO: 65)), and comprising the common sequence in amino acid region C (EKYGLRPDQWADY (SEQ ID NO: 68), GLRPEQWVDF (SEQ ID NO: 70) or ITPEWLW (SEQ ID NO: 71)), and comprising the common sequence in amino acid region D (RAFLERLEF (SEQ ID NO: 79) or LEFGSLH (SEQ ID NO: 81)).

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

Sequence listing

<110> TOYOBO CO., LTD.)

University French Fushan university (NATIONAL UNIVERSITY CORPORATION UNIVERSITY OF TOYAMA)

<120> antibody specifically binding to 5 '- > 3' exonuclease active domain of DNA polymerase

<130> P21-233WO

<150> JP 2020-206269

<151> 2020-12-11

<150> JP 2021-098632

<151> 2021-06-14

<160> 83

<170> PatentIn version 3.5

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Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly

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Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala

35 40 45

Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val

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Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly

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Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu

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Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu

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Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys

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Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp

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Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly

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Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro

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Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn

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Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu

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Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu

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Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys

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Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val

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Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe

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Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu

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Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala

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Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu

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Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys

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Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg

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Gly His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg

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Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly

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Leu Leu Glu Ala

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Met Lys Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu

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Gly Phe Thr Phe Asp Asp Tyr Gly

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Gly Phe Thr Phe Asn Asn Tyr Tyr

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Gly Phe Thr Phe Ser Ser Tyr Tyr

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Gly Phe Thr Phe Ser Lys Trp Tyr

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Gly Phe Thr Phe Ser His Tyr Tyr

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Gly Phe Thr Phe Ser Asn Tyr Trp

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Gly Phe Thr Phe Ser Ser Tyr Gly

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Ile Ser Leu Ser Gly Gly Ser Ser

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Ile Ser Asn Thr Gly Gly Ser Thr

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Ile Ser Asn Thr Gly Gly Thr Thr

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Ile Ser Asn Ser Gly Ser Ser Thr

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Ile Ser Gly Gly Gly Ser Tyr Ile

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Ile Lys Gly Asp Ser Ser Thr Ile

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Ile Asn Thr Asp Gly Gly Thr Thr

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Val Arg Ala Pro Ile Gly Val Ala Tyr Phe Asp Val

1 5 10

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Val Arg Ala Pro Ile Gly Leu Ala Tyr Phe Asp Thr

1 5 10

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Ala Arg Asp Gly Ala Leu Gly Leu Ala Val Asn Trp Phe Asp Asn

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Val Arg Asp Gly Ala Leu Gly Leu Ala Val Asn Trp Phe Asp Asn

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Ala Thr Ser Asp Asp Tyr Tyr Ala Leu Asn Ile

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Thr Thr Ala Tyr Tyr Ser Arg Tyr Ser Tyr Tyr Met Phe Asp Val

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Thr Thr Ala Leu Arg Asp Val

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Gln Ser Ile Ser Asn Tyr

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Gln Ser Val Lys Asn Tyr

1 5

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Gln Ser Val Lys Ser Tyr

1 5

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<211> 6

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Gln Ser Val Ser Lys Tyr

1 5

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<211> 6

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Gln Gly Ile Ser Ser Tyr

1 5

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Gln Gly Ile Ser Asn Tyr

1 5

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Gln Gly Val Ser Ser Phe

1 5

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<400> 36

Ser Leu Ala Ser

1

<210> 37

<211> 4

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 37

Ser Leu Pro Ser

1

<210> 38

<211> 4

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 38

Arg Arg Ala Thr

1

<210> 39

<211> 4

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 39

Asn Leu Tyr Ser

1

<210> 40

<211> 4

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 40

Tyr Leu Tyr Ser

1

<210> 41

<211> 4

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 41

Thr Arg Ala Thr

1

<210> 42

<211> 8

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 42

Leu Gln Ser Tyr Ile Tyr Pro Leu

1 5

<210> 43

<211> 8

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 43

Gln Gln Tyr Gln Ser Trp Pro Tyr

1 5

<210> 44

<211> 8

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 44

Gln Gln Tyr Gln Ser Trp Pro His

1 5

<210> 45

<211> 8

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 45

Tyr Gln Tyr Asn Ser Gly Trp Thr

1 5

<210> 46

<211> 9

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 46

Gln Gln Tyr Gly Ser Ser Pro Pro Thr

1 5

<210> 47

<211> 9

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 47

Gln Gln Tyr Gly Asn Ser Pro Pro Thr

1 5

<210> 48

<211> 9

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 48

Phe Gln Tyr Tyr Ser Gly Ser Pro Thr

1 5

<210> 49

<211> 832

<212> PRT

<213> Thermus aquaticus (Thermus aquaticus)

<400> 49

Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu

1 5 10 15

Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly

20 25 30

Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala

35 40 45

Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val

50 55 60

Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly

65 70 75 80

Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu

85 90 95

Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu

100 105 110

Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys

115 120 125

Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp

130 135 140

Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly

145 150 155 160

Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro

165 170 175

Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn

180 185 190

Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu

195 200 205

Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu

210 215 220

Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys

225 230 235 240

Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val

245 250 255

Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe

260 265 270

Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu

275 280 285

Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly

290 295 300

Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp

305 310 315 320

Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro

325 330 335

Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu

340 345 350

Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro

355 360 365

Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn

370 375 380

Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu

385 390 395 400

Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu

405 410 415

Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu

420 425 430

Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly

435 440 445

Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala

450 455 460

Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His

465 470 475 480

Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp

485 490 495

Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg

500 505 510

Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile

515 520 525

Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr

530 535 540

Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu

545 550 555 560

His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser

565 570 575

Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln

580 585 590

Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala

595 600 605

Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly

610 615 620

Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr

625 630 635 640

Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro

645 650 655

Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly

660 665 670

Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu

675 680 685

Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg

690 695 700

Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val

705 710 715 720

Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg

725 730 735

Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro

740 745 750

Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu

755 760 765

Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His

770 775 780

Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala

785 790 795 800

Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro

805 810 815

Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu

820 825 830

<210> 50

<211> 834

<212> PRT

<213> Thermus thermophilus (Thermus thermophilus)

<400> 50

Met Glu Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu

1 5 10 15

Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly

20 25 30

Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala

35 40 45

Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe

50 55 60

Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu

65 70 75 80

Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln

85 90 95

Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu

100 105 110

Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys

115 120 125

Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg

130 135 140

Asp Leu Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu

145 150 155 160

Gly His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg

165 170 175

Pro Glu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp

180 185 190

Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu

195 200 205

Leu Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg

210 215 220

Val Lys Pro Glu Asn Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp

225 230 235 240

Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp Leu Pro Leu

245 250 255

Glu Val Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg

260 265 270

Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly

275 280 285

Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro

290 295 300

Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp

305 310 315 320

Ala Glu Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg

325 330 335

Ala Ala Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg Gly

340 345 350

Leu Leu Ala Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp

355 360 365

Leu Val Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro

370 375 380

Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp

385 390 395 400

Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg

405 410 415

Asn Leu Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr

420 425 430

His Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala

435 440 445

Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu

450 455 460

Leu Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala

465 470 475 480

Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu

485 490 495

Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gln Lys Thr Gly

500 505 510

Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His

515 520 525

Pro Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys

530 535 540

Asn Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly

545 550 555 560

Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu

565 570 575

Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu

580 585 590

Gly Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu

595 600 605

Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu

610 615 620

Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Lys Asp Ile

625 630 635 640

His Thr Gln Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val

645 650 655

Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu

660 665 670

Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr

675 680 685

Glu Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys

690 695 700

Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly

705 710 715 720

Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn

725 730 735

Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn

740 745 750

Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val

755 760 765

Lys Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln

770 775 780

Val His Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu

785 790 795 800

Val Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala

805 810 815

Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser Ala

820 825 830

Lys Gly

<210> 51

<211> 326

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 51

Ala Pro Ser Val Phe Pro Leu Ala Ala Ser Cys Val Asp Thr Ser Gly

1 5 10 15

Ser Met Met Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro

20 25 30

Val Thr Val Lys Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

35 40 45

Phe Pro Ala Val Leu Gln Ser Gly Leu Tyr Ser Leu Thr Ser Met Val

50 55 60

Thr Val Pro Ser Ser Gln Lys Lys Ala Thr Cys Asn Val Ala His Pro

65 70 75 80

Ala Ser Ser Thr Lys Val Asp Lys Thr Val Glu Pro Ile Arg Thr Pro

85 90 95

Gln Pro Asn Pro Cys Thr Cys Pro Lys Cys Pro Pro Pro Glu Asn Leu

100 105 110

Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu

115 120 125

Met Ile Ser Leu Thr Pro Arg Val Thr Cys Val Val Val Asp Val Ser

130 135 140

Gln Asp Glu Pro Glu Val Gln Phe Thr Trp Phe Val Asp Asn Lys Pro

145 150 155 160

Val Gly Asn Ala Glu Thr Lys Pro Arg Val Glu Gln Tyr Asn Thr Thr

165 170 175

Phe Arg Val Glu Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Arg

180 185 190

Gly Lys Glu Phe Lys Cys Lys Val Tyr Asn Lys Ala Leu Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Ala Pro Arg Met Pro Asp

210 215 220

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Ser Lys Ser Lys Val

225 230 235 240

Ser Val Thr Cys Leu Ile Ile Asn Phe Phe Pro Ala Asp Ile His Val

245 250 255

Glu Trp Ala Ser Asn Arg Val Pro Val Ser Glu Lys Glu Tyr Lys Asn

260 265 270

Thr Pro Pro Ile Glu Asp Ala Asp Gly Ser Tyr Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Ala Trp Asp Gln Gly Thr Val Tyr Thr Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Val Thr Gln Lys Ala Ile

305 310 315 320

Ser Arg Ser Pro Gly Lys

325

<210> 52

<211> 101

<212> PRT

<213> Guinea pig (Guinea pig)

<400> 52

Thr Ile Ser Leu Phe Pro Pro Ser Ser Glu Glu Val Thr Ala Gly Ser

1 5 10 15

Ala Ser Val Val Cys Phe Ile Asn Ser Phe Tyr Pro Arg Asp Ile Thr

20 25 30

Val Lys Trp Lys Val Asp Gly Ser Glu Arg Ser Gln Gly Ile Leu Asn

35 40 45

Ser Tyr Thr Asp Gln Asp Ser Lys Asp Asn Thr Tyr Ser Leu Ser Ser

50 55 60

Thr Leu Ala Leu Thr Ala Ser Glu Tyr Asn Gln His Glu Arg Tyr Thr

65 70 75 80

Cys Glu Val Ser His Ala Gly Leu Thr Ser Pro Ala Ala Lys Thr Ile

85 90 95

Asn Arg Ser Glu Cys

100

<210> 53

<211> 320

<212> PRT

<213> mice (Mus musculus)

<400> 53

Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn

1 5 10 15

Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro

20 25 30

Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr

35 40 45

Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val

50 55 60

Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val

65 70 75 80

Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg

85 90 95

Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser

100 105 110

Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu

115 120 125

Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro

130 135 140

Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala

145 150 155 160

Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val

165 170 175

Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe

180 185 190

Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr

195 200 205

Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile

210 215 220

Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys

225 230 235 240

Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp

245 250 255

Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp

260 265 270

Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser

275 280 285

Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly

290 295 300

Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

305 310 315 320

<210> 54

<211> 101

<212> PRT

<213> mice (Mus musculus)

<400> 54

Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

1 5 10 15

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

20 25 30

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

35 40 45

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

50 55 60

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr

65 70 75 80

Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe

85 90 95

Asn Arg Gly Glu Cys

100

<210> 55

<211> 834

<212> PRT

<213> Thermus sp ZO5

<400> 55

Met Lys Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu

1 5 10 15

Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly

20 25 30

Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala

35 40 45

Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe

50 55 60

Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu

65 70 75 80

Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln

85 90 95

Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu

100 105 110

Glu Val Pro Gly Phe Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys

115 120 125

Lys Ala Glu Arg Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg

130 135 140

Asp Leu Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu

145 150 155 160

Gly His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Lys

165 170 175

Pro Glu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp

180 185 190

Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu

195 200 205

Leu Lys Glu Trp Gly Ser Leu Glu Asn Ile Leu Lys Asn Leu Asp Arg

210 215 220

Val Lys Pro Glu Ser Val Arg Glu Arg Ile Lys Ala His Leu Glu Asp

225 230 235 240

Leu Lys Leu Ser Leu Glu Leu Ser Arg Val Arg Ser Asp Leu Pro Leu

245 250 255

Glu Val Asp Phe Ala Arg Arg Arg Glu Pro Asp Arg Glu Gly Leu Arg

260 265 270

Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly

275 280 285

Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro

290 295 300

Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp

305 310 315 320

Ala Glu Leu Lys Ala Leu Ala Ala Cys Lys Glu Gly Arg Val His Arg

325 330 335

Ala Lys Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg Gly

340 345 350

Leu Leu Ala Lys Asp Leu Ala Val Leu Ala Leu Arg Glu Gly Leu Asp

355 360 365

Leu Ala Pro Ser Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro

370 375 380

Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp

385 390 395 400

Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ala Glu Arg Leu Gln Gln

405 410 415

Asn Leu Leu Glu Arg Leu Lys Gly Glu Glu Lys Leu Leu Trp Leu Tyr

420 425 430

Gln Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala

435 440 445

Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Lys Ala Leu Ser Leu Glu

450 455 460

Leu Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala

465 470 475 480

Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu

485 490 495

Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gln Lys Thr Gly

500 505 510

Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His

515 520 525

Pro Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys

530 535 540

Asn Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly

545 550 555 560

Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu

565 570 575

Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu

580 585 590

Gly Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu

595 600 605

Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu

610 615 620

Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Lys Asp Ile

625 630 635 640

His Thr Gln Thr Ala Ser Trp Met Phe Gly Val Ser Pro Glu Ala Val

645 650 655

Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu

660 665 670

Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr

675 680 685

Glu Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys

690 695 700

Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly

705 710 715 720

Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn

725 730 735

Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn

740 745 750

Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val

755 760 765

Lys Leu Phe Pro His Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln

770 775 780

Val His Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu

785 790 795 800

Val Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala

805 810 815

Val Pro Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala

820 825 830

Lys Gly

<210> 56

<211> 30

<212> DNA

<213> lambda phage (Bacteriophage lambda)

<400> 56

actcattcgt tttatacctc tgaatcaata 30

<210> 57

<211> 40

<212> DNA

<213> lambda phage (Bacteriophage lambda)

<400> 57

gttgatattg attcagaggt ataaaacgaa tgagtactgc 40

<210> 58

<211> 30

<212> DNA

<213> lambda phage (Bacteriophage lambda)

<400> 58

actaataagc cgatagatag ccacggactt 30

<210> 59

<211> 40

<212> DNA

<213> lambda phage (Bacteriophage lambda)

<400> 59

ctacgaagtc cgtggctatc tatcggctta ttagtacttg 40

<210> 60

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 60

Glu Asp Gly Asp Ala Val Ile Val Val Phe

1 5 10

<210> 61

<211> 11

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 61

Lys Glu Asp Gly Asp Ala Val Ile Val Val Phe

1 5 10

<210> 62

<211> 11

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 62

Glu Asp Gly Tyr Lys Ala Val Phe Val Val Phe

1 5 10

<210> 63

<211> 12

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 63

Lys Glu Asp Gly Tyr Lys Ala Val Phe Val Val Phe

1 5 10

<210> 64

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 64

His Glu Ala Tyr Gly Gly Tyr

1 5

<210> 65

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 65

His Glu Ala Tyr Glu Ala Tyr

1 5

<210> 66

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 66

His Leu Ile Thr Pro Glu Trp Leu Trp

1 5

<210> 67

<211> 12

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 67

Lys Tyr Gly Leu Arg Pro Glu Gln Trp Val Asp Phe

1 5 10

<210> 68

<211> 13

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 68

Glu Lys Tyr Gly Leu Arg Pro Asp Gln Trp Ala Asp Tyr

1 5 10

<210> 69

<211> 12

<212> PRT

<213> Artificial sequence (Artificial seqence)

<220>

<223> epitope

<400> 69

Lys Tyr Gly Leu Arg Pro Asp Gln Trp Ala Asp Tyr

1 5 10

<210> 70

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 70

Gly Leu Arg Pro Glu Gln Trp Val Asp Phe

1 5 10

<210> 71

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 71

Ile Thr Pro Glu Trp Leu Trp

1 5

<210> 72

<211> 22

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 72

Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro

1 5 10 15

Asp Gln Trp Ala Asp Tyr

20

<210> 73

<211> 22

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 73

His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro

1 5 10 15

Glu Gln Trp Val Asp Phe

20

<210> 74

<211> 22

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 74

His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Lys Pro

1 5 10 15

Glu Gln Trp Val Asp Phe

20

<210> 75

<211> 6

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 75

Leu Glu Arg Leu Glu Phe

1 5

<210> 76

<211> 11

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 76

Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His

1 5 10

<210> 77

<211> 13

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 77

Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe

1 5 10

<210> 78

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 78

Leu Arg Ala Phe Leu Glu Arg Leu Glu Phe

1 5 10

<210> 79

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 79

Arg Ala Phe Leu Glu Arg Leu Glu Phe

1 5

<210> 80

<211> 14

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 80

Arg Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His

1 5 10

<210> 81

<211> 8

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 81

Leu Glu Phe Gly Ser Leu Leu His

1 5

<210> 82

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 82

Leu Glu Phe Gly Ser Leu Leu His Glu Phe

1 5 10

<210> 83

<211> 17

<212> PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> epitope

<400> 83

Leu Arg Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu

1 5 10 15

Phe

Claims

1. An antibody or fragment thereof that specifically binds to the 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase.

2. The antibody or fragment thereof of claim 1, wherein the DNA polymerase is selected from the group consisting of Taq polymerase, tth polymerase, and Z05 polymerase.

3. An antibody or fragment thereof, comprising:

GFX ^A3 X ^A4 X ^A5 X ^A6 X ^A7 X ^A8 (A-1)

in the method, in the process of the invention,

X ^A3 is a T or S, and the number of the T or S,

X ^A4 is a compound of formula F, L or I,

X ^A5 is a compound of D, N, S or T,

X ^A6 is a compound which is D, N, S, T, K, R or H,

X ^A7 is a metal-containing alloy of Y, F or W,

X ^A8 g, S, T, W, Y or F;

IX ^B2 X ^B3 X ^B4 X ^B5 X ^B6 X ^B7 X ^B8 (B-1)

in the method, in the process of the invention,

X ^B2 is a compound of G, S, T, K, R, H, D or N,

X ^B3 is a compound of F, Y, L, I, G, S, T, D or N,

X ^B4 is a compound which is G, S, T, D, N, K, R or H,

X ^B5 is a compound of G, S, T or A,

X ^B6 is a compound of G, S, T, D or N,

X ^B7 is a compound which is S, T, F, Y, D, N, K, R or H,

X ^B8 s, T, V, L, I or M;

a heavy chain CDR3 comprising an amino acid sequence represented by any one of 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 method, in the process of the invention,

X ^C1 is a compound of formula A, S, D, K, H or R,

X ^C2 is a compound of formula G, A, D, P, K, H or R,

X ^C3 is a compound of formula S, T, L, Y or I,

X ^C4 is a compound which is A, V, I, R or L,

X ^C5 is a compound of A, V, Y or P,

X ^C6 is a compound which is A, V, S, T or Y,

X ^C7 is a compound of V, I, L, S, T or N,

X ^C8 is A or V;

light chain CDR1, X comprising an amino acid sequence represented by the following formula (D-1) ^D1 X ^D2 X ^D3 X ^D4 X ^D5 X ^D6 (D-1)

In the method, in the process of the invention,

X ^D1 is a compound of E, Q, D or N,

X ^D2 is a compound of G, A, S or T,

X ^D3 is a compound of A, V, L, I or F,

X ^D4 is a compound which is S, T, K, R or H,

X ^D5 is a compound which is S, T, D, N, K, R or H,

X ^D6 f, Y or W;

light chain CDR2, X consisting of an amino acid sequence represented by the following formula (E-1) ^E1 X ^E2 X ^E3 (E-1)

In the method, in the process of the invention,

X ^E1 is a compound which is G, S, T, D, N, F, Y, K, R or H,

X ^E2 is a compound of formula G, A, S, T, V, L or I,

X ^E3 k, R, H, D, N, S or T; and

a light chain CDR3 consisting of an amino acid sequence represented by 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 method, in the process of the invention,

X ^F1 is a metal-containing alloy of L, I, E, Q, F, Y or W,

X ^F3 is a compound which is S, T, F or Y,

X ^F5 is a compound of formula S, T, N, Q, L or I,

X ^F6 is a metal-containing alloy of G, S, T, F, Y or W,

X ^F7 is a metal-containing alloy of S, T, P, Y or W,

X ^F8 is a metal-containing alloy of L, I, P, K, R, H, Y, T or W,

X ^F9 g, S, T, D or E.

4. The antibody or fragment thereof according to claim 3, further comprising a sequence region adjacent to the C-terminus of the light chain CDR2 consisting of an amino acid sequence represented by the following formula (E-2),

X ^E4 X ^E5 X ^E6 X ^E7 (E-2)

in the method, in the process of the invention,

X ^E4 is a compound of G, S, R, H, K, D, N, F, Y or T,

X ^E5 is a compound of formula L, I, K, H or R,

X ^E6 is a compound which is G, A, S, T, P, F or Y,

X ^E7 g, A, D, N, S or T.

5. An antibody or fragment thereof, comprising:

a heavy chain CDR1 comprising an amino acid sequence represented by the following formula (A-1-1):

GFTFX ^A51 X ^A61 X ^A71 X ^A81 (A-1-1)

In the method, in the process of the invention,

X ^A51 is D, N or S, and the number of the components is D, N or S,

X ^A61 is a compound which is D, N, S, K or H,

X ^A81 g, W or Y;

IX ^B21 X ^B31 X ^B41 X ^B51 X ^B61 X ^B71 X ^B81 (B-1-1)

in the method, in the process of the invention,

X ^B21 is a compound of G, S, T, K or N,

X ^B31 is a compound of Y, L, G, T or N,

X ^B41 is a compound which is G, S, T, D or H,

X ^B51 in the form of G or S,

X ^B71 is a compound which is S, T, Y, D or H,

X ^B81 s, T, V, I or M;

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 method, in the process of the invention,

X ^C11 is a group of A or R,

X ^C21 is a group of the formula P or R,

X ^C31 is a group of T or I,

X ^C41 is a group of V or L,

X ^C51 is a compound of the formula P or A,

X ^C61 is a group of T or Y, and the total number of the groups is the same,

X ^C71 is a compound of V, T or N,

X ^C81 is A or V;

light chain CDR1, QX comprising an amino acid sequence represented by the following formula (D-1-1) ^D21 X ^D31 X ^D41 X ^D51 X ^D61 (D-1-1)

In the method, in the process of the invention,

X ^D21 in the form of G or S,

X ^D31 is a group of V or I,

X ^D41 in the form of S or K,

X ^D51 is a compound of S, N or K,

X ^D61 is F or Y;

light chain CDR2, X consisting of an amino acid sequence represented by the following formula (E-1-1) ^E11 X ^E21 X ^E31 (E-1-1)

In the method, in the process of the invention,

X ^E11 is a compound of formula G, T, D, Y or R,

X ^E21 is a compound of A, T or V,

X ^E31 k, D, N or S; and

light chain CDR3, X comprising the amino acid sequence of the following formula (F-1-1) or (F-2-1) ^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 method, in the process of the invention,

X ^F11 is a compound which is L, Q, F or Y,

X ^F31 in the form of S or Y,

X ^F41 is a compound which is G, N, Q or Y,

X ^F51 is a compound of formula S, N or I,

X ^F61 is a metal-containing alloy of G, S, Y or W,

X ^F71 is a metal-containing alloy of S, P or W,

X ^F81 l, P, H, Y or T.

6. The antibody or fragment thereof according to claim 5, further comprising a sequence region adjacent to the C-terminus of the light chain CDR2, which is composed of an amino acid sequence represented by 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 method, in the process of the invention,

X ^E61 is a group A or P, and the group A is a group B,

X ^E42 is a compound of R, N, Y or T,

X ^E52 is a group of L or R,

X ^E62 is a group of A or Y, and the group of B is a group of C,

X ^E72 is S or T.

7. The antibody or fragment thereof according to any one of claims 3 to 6, which specifically binds to the 5'→3' exonuclease active domain of Taq polymerase.

8. An antibody or fragment thereof according to any one of claims 3 to 6 which specifically binds to the 5'→3' exonuclease active domain of Tth polymerase.

9. The antibody or fragment thereof according to any one of claims 3 to 6, which specifically binds to the 5'→3' exonuclease active domain of Z05 polymerase.

10. An antibody or fragment thereof that specifically binds to a 5'→3' exonuclease active domain of a DNA polymerase, said antibody or fragment thereof having at least 1 epitope present in any one of regions a to D: an amino acid region A selected from the region from 56 to 66 th from the N-terminus of SEQ ID NO. 1 and the region from 56 to 67 th from the N-terminus of SEQ ID NO. 2 or 3; an amino acid region B selected from the region from 75 to 81 th from the N-terminus of SEQ ID NO. 1 and the region from 76 to 82 th from the N-terminus of SEQ ID NO. 2 or 3; an amino acid region C selected from the region 161 to 182 from the N-terminus of SEQ ID NO. 1 and the region 162 to 183 from the N-terminus of SEQ ID NO. 2 or 3; selected from the region from 269 to 285 from the N-terminus of SEQ ID NO. 1 or the amino acid region D in the region from 271 to 287 of SEQ ID NO. 2 or 3.

11. The antibody or fragment thereof of claim 10, wherein the at least 1 epitope is present in either of the amino acid regions a or B.

12. The antibody or fragment thereof of claim 10 or 11, wherein the epitope in amino acid region a is any one of sequence numbers 60-63, the epitope in amino acid region B is sequence number 64 or 65, the epitope in amino acid region C is any one of sequence numbers 66-74, and the epitope in amino acid region D is any one of sequence numbers 75-83.

13. The antibody or fragment thereof of any one of claims 10 to 12, wherein the epitope in amino acid region a is seq id No. 61 or 62, the epitope in amino acid region B is seq id No. 64 or 65, the epitope in amino acid region C is seq id No. 66, 67, 68, 70 or 71, and the epitope in amino acid region D is seq id No. 77, 78, 80 or 82.

14. The antibody or fragment thereof according to any one of claims 1 to 13, which is a monoclonal antibody or fragment thereof.

15. The antibody or fragment thereof according to any one of claims 1 to 14, wherein the heavy chain CDR3 is composed of an amino acid sequence represented by any one of sequence numbers 21 to 28 or an amino acid sequence in which 1 to 3 amino acids in these amino acid sequences are mutated,

16. The antibody or fragment thereof of any one of claims 1 to 15, wherein the heavy chain CDR3 consists of an amino acid sequence set forth in any one of sequence numbers 21 to 28,

17. The antibody or fragment thereof according to any one of claims 1 to 16, comprising:

18. The antibody or fragment thereof according to any one of claims 1 to 17, comprising:

19. The antibody or fragment thereof according to claim 17 or 18, further comprising a sequence region adjacent to the C-terminus of the light chain CDR2, which region comprises an amino acid sequence represented by any one of sequence numbers 36 to 41 or an amino acid sequence in which 1 to 3 amino acids in the amino acid sequences have been mutated.

20. The antibody or fragment thereof of any one of claims 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 sequence numbers 36 to 41.

21. The antibody or fragment thereof of any one of claims 15, 17, and 19, wherein the variation is a conservative substitution.

22. A fragment which is a fragment of the antibody of any one of claims 1 to 21, said fragment being a Fab, F (ab') 2 or scFv.

23. A reagent comprising the antibody or fragment thereof of any one of claims 1-21, or comprising the fragment of claim 22.

24. The reagent of claim 23, further comprising at least one selected from the group consisting of a DNA polymerase having a 5' →3' exonuclease active domain, a primer, a probe, and deoxyribonucleoside-5 ' -phosphate.

25. The reagent of claim 24 in which the DNA polymerase is selected from the group consisting of Taq polymerase, tth polymerase and Z05 polymerase.

26. The reagent according to any one of claims 23 to 25, which is a reagent for nucleic acid amplification.

27. A method for producing an antibody or a fragment thereof specifically binding to a 5'→3' exonuclease active domain of a DNA polymerase, comprising the following step a: an antibody having binding ability to the whole DNA polymerase is selected from antibodies produced by animals immunized with an immunogen comprising a portion of the DNA polymerase (herein, the portion comprises a 5 '. Fwdarw.3' exonuclease active domain).

28. The method of claim 27, wherein the immunogen consists of a 5 '. Fwdarw.3' exonuclease active domain of a DNA polymerase.

29. The method according to claim 27 or 28, wherein the DNA polymerase is selected from the group consisting of Taq polymerase, tth polymerase, and Z05 polymerase.

30. The method of any one of claims 27 to 29, wherein the immunogen consists of a 5'→3' exonuclease active domain of Tth polymerase.

31. The manufacturing method according to claim 27, wherein the step a is a step of: from among antibodies produced by animals immunized with an immunogen comprising a portion of Tth polymerase (herein, the portion comprising a 5 '. Fwdarw.3' exonuclease active domain), an antibody having binding ability to Taq polymerase as a whole is selected.

32. The manufacturing method according to claim 27, wherein the step a is a step of: from among antibodies produced by animals immunized with an immunogen composed of the 5 '. Fwdarw.3' exonuclease active domain of Tth polymerase, an antibody having binding ability to Taq polymerase as a whole is selected.

33. A method for producing an antibody fragment, comprising the steps of: based on the amino acid sequence of the antibody obtained by the production method according to any one of claims 27 to 32, a part of the amino acid sequence thereof is expressed by a genetic engineering method.

34. The antibody or fragment thereof according to any one of claims 1 to 21, or the fragment according to claim 22, wherein the 5 '. Fwdarw.3' exonuclease inhibitory activity of the DNA polymerase is 60% or more when present with the DNA polymerase at 37 ℃ for 24 hours.

35. The antibody or fragment thereof according to any one of claims 1 to 21 and 34, or the fragment according to claim 22, wherein the substrate DNA (herein, the substrate DNA is optionally single-stranded or double-stranded, and the substrate DNA is optionally functional as a probe) has a substrate DNA degradation rate of 40% or less when the substrate DNA and the DNA polymerase having a 5'→3' exonuclease activity domain coexist at 25 ℃ for 24 hours.

36. A reagent for stabilizing a composition comprising a DNA polymerase having a 5'→3' exonuclease activity domain and at least one 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 of any one of claims 1 to 21, 34 and 35, or the fragment of claim 22.

37. A method of stabilizing a composition comprising a DNA polymerase having a 5'→3' exonuclease activity domain and at least one nucleic acid selected from the group consisting of a primer, a probe, and a nucleic acid template, comprising the step of adding the antibody or fragment thereof of any one of claims 1 to 21, 34, and 35, or the fragment of claim 22, to the composition.