Anti-taq DNA polymerase monoclonal antibody and use thereof

Abstract

The present invention relates to a novel anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof, which exhibits high sensitivity and high specificity for specifically binding to Taq DNA polymerase. One or more anti-Taq DNA polymerase monoclonal antibodies or antigen-binding fragments thereof according to the present invention are used to bind to Taq DNA polymerase, thereby efficiently blocking the 5'-3' polymerase activity of the Taq DNA polymerase, and also efficiently blocking the 5'-3' exonuclease activity of the Taq DNA polymerase, thus forming a stable and efficiently blocked hot-start Taq DNA polymerase. The dual-blocking of the Taq DNA polymerase can effectively prevent non-specific amplification caused by mismatches or primer dimers, and can also prevent non-specific signals caused by material degradation, thereby improving the specificity and sensitivity of a PCR reaction, and also improving the reagent stability, thus enabling a detection reagent to better withstand transportation and room-temperature use.

Description

Anti-Taq DNA polymerase monoclonal antibodies and their applications Technical Field

[0001] This invention relates to the field of DNA fragment amplification technology, specifically to an antibody against Taq DNA polymerase, an antibody-enzyme complex for amplification, and a method for amplifying DNA fragments using the complex. Background Technology

[0002] Polymerase chain reaction (PCR) is a method for rapidly amplifying specific DNA fragments in vitro, widely used in gene detection, infectious disease detection, prenatal diagnosis of genetic diseases, and detection of carcinogenic mutations. Taq DNA polymerase possesses both 5'-3' polymerase and 5'-3' exonuclease activities, exhibiting good thermostability and able to withstand the thermal denaturation step of PCR without requiring additional additions, making it a commonly used DNA polymerase. However, because Taq DNA polymerase also exhibits partial activity at low temperatures, this can lead to sequence mismatches and primer dimer formation. Its 5'-3' exonuclease activity can also cause material degradation, generating non-specific signals and resulting in non-specific amplification of DNA fragments. Particularly during the PCR reaction system preparation and preheating stages, Taq DNA polymerase catalyzes the extension of mismatched primers or the formation of primer dimers, which is a major reason affecting the synthesis of the target fragment.

[0003] Blocking Taq DNA polymerase activity with antibodies at low temperatures can effectively reduce non-specific DNA fragment synthesis. At low temperatures, Taq DNA polymerase is inactive and does not cause mismatches. At high temperatures, the antibody denatures, the polymerase regains activity, and specific amplification occurs, thus avoiding mismatches, primer dimer formation, and non-specific signals from material degradation. However, the binding affinity between antibodies and enzymes is easily affected by the environment, leading to unstable blocking. There are many types of Taq enzyme mutants, making it difficult to find a suitable antibody that can efficiently block both polymerization and exonuclease activities.

[0004] Therefore, there is still a need in the field for suitable and stable, efficient blocking antibodies to modify Taq DNA polymerase, thereby reducing non-specific amplification and preventing non-specific signals from material degradation, achieving double blocking and thus improving the specificity and sensitivity of PCR reactions. Summary of the Invention

[0005] The inventors immunized mice with wild-type Taq DNA polymerase, fused mouse spleen cells with myeloma cells, and screened for hybridoma cell lines that specifically bind to Taq DNA polymerase using ELISA. Recombinant antibodies were then constructed to obtain recombinant anti-Taq DNA polymerase antibodies. Thus, this invention was achieved.

[0006] Therefore, in a first aspect, the present invention provides an anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising a heavy chain complementarity-determining region V. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determination region V. L CDR1, V L CDR2 and V L CDR3; where:

[0007] V H The amino acid sequence of CDR1 is shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49 or SEQ ID NO:54;

[0008] V H The amino acid sequence of CDR2 is shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50 or SEQ ID NO:55;

[0009] V H The amino acid sequence of CDR3 is shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:56 or SEQ ID NO:60;

[0010] V LThe amino acid sequence of CDR1 is shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52 or SEQ ID NO:57;

[0011] V L The amino acid sequence of CDR2 is shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47 or SEQ ID NO:58; and

[0012] V L The amino acid sequence of CDR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:53 or SEQ ID NO:59.

[0013] In a second aspect, the present invention provides a nucleic acid molecule encoding an anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof, as described in the first aspect.

[0014] In a third aspect, the present invention provides a carrier comprising the nucleic acid molecule of the second aspect.

[0015] In a fourth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.

[0016] In a fifth aspect, the present invention provides a hot-start Taq DNA polymerase comprising Taq DNA polymerase and at least one anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof from the first aspect.

[0017] In a sixth aspect, the present invention provides a method for amplifying a target DNA sequence by polymerase chain reaction, the method comprising the step of amplifying the target DNA sequence using a hot-start Taq DNA polymerase as described in the fifth aspect.

[0018] In a seventh aspect, the present invention provides a kit for polymerase chain reaction, comprising: a hot-start Taq DNA polymerase as described in the fifth aspect; and instructions for use.

[0019] In summary, this invention provides novel anti-Taq DNA polymerase monoclonal antibodies or their antigen-binding fragments, which exhibit high sensitivity and specificity in binding to Taq DNA polymerase, forming a stable and highly efficient blocked hot-start Taq DNA polymerase. Using one or more of the anti-Taq DNA polymerase monoclonal antibodies or their antigen-binding fragments of this invention to bind to Taq DNA polymerase can efficiently block not only the 5'-3' polymerase activity but also the 5'-3' exonuclease activity, thereby forming a stable and highly efficient blocked hot-start Taq DNA polymerase. This dual blocking of both 5'-3' polymerase and 5'-3' exonuclease activities effectively prevents non-specific amplification caused by mismatches or primer dimers, and also prevents non-specific signals generated by material degradation. This not only improves the specificity and sensitivity of the PCR reaction but also enhances reagent stability, allowing the detection reagents to withstand transportation or use at room temperature with ease. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0021] Figure 1 shows the results of polymerase activity detection of three Taq DNA polymerases blocked by recombinant monoclonal antibody T22.

[0022] Figure 2 shows the results of polymerase activity detection of three Taq DNA polymerases blocked by recombinant monoclonal antibody T23.

[0023] Figure 3 shows the results of polymerase activity detection of three Taq DNA polymerases blocked by recombinant monoclonal antibody T24.

[0024] Figure 4 shows the results of polymerase activity detection of three Taq DNA polymerases blocked by recombinant monoclonal antibody T25.

[0025] Figure 5 shows the results of polymerase activity detection for three Taq DNA polymerases blocked by recombinant monoclonal antibody T26.

[0026] Figure 6 shows the results of polymerase activity detection for three Taq DNA polymerases blocked by recombinant monoclonal antibody T27.

[0027] Figure 7 shows the detection results of the exonuclease activity of two Taq DNA polymerases blocked by recombinant monoclonal antibody T23.

[0028] Figure 8 shows the detection results of the exonuclease activity of two Taq DNA polymerases blocked by the recombinant monoclonal antibody T27.

[0029] Figure 9 shows the detection results of the exonuclease activity of two Taq DNA polymerases blocked by the recombinant monoclonal antibody T28.

[0030] Figure 10 shows the detection results of the exonuclease activity of two Taq DNA polymerases blocked by recombinant monoclonal antibody T29.

[0031] Figure 11 shows the detection results of the exonuclease activity of two Taq DNA polymerases blocked by recombinant monoclonal antibody T30.

[0032] Figure 12 shows the detection results of Taq22 DNA polymerase double-blocked by recombinant monoclonal antibodies T24 and T29.

[0033] Figure 13 shows the detection results of KlenTaq10 DNA polymerase double-blocked by recombinant monoclonal antibodies T22 and T30.

[0034] Figure 14 shows the detection results of Taq WT polymerase double-blocked by recombinant monoclonal antibodies T24 and T31. Detailed Implementation

[0035] The present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the following description is merely illustrative and is not intended to limit the scope of the invention; the scope of protection of the invention is defined by the appended claims. Furthermore, those skilled in the art will understand that modifications can be made to the technical solutions of the present invention without departing from its spirit and intent. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0037] In cases where numerical ranges are provided, such as concentration ranges, percentage ranges, or ratio ranges, it should be understood that, unless the context explicitly specifies otherwise, all intermediate values ​​between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other values ​​or intermediate values ​​within the range are included in the subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and such embodiments are also included in the subject matter, limited by any specific excluded limit values ​​within the range. Where the range includes one or two limit values, the range excluding any one or both of those included limit values ​​is also included in the subject matter.

[0038] In the context of this invention, many embodiments use the expressions "comprising," "including," or "basically / mainly composed of." The expressions "comprising," "including," or "basically / mainly composed of" are generally understood as open-ended expressions, indicating that they include not only the elements, components, parts, or method steps specifically listed after the expression, but also other elements, components, parts, or method steps. However, in this document, the expressions "comprising," "including," or "basically / mainly composed of" can also be understood as closed-ended expressions in certain situations, indicating that they only include the elements, components, parts, or method steps specifically listed after the expression, and do not include any other elements, components, parts, or method steps. In this case, the expression is equivalent to the expression "composed of."

[0039] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0040] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes can be defined accordingly as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions (hinge regions) of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C HThe heavy chain constant region consists of three structural domains (C). H1 C H2 and C H3 It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementarity-determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). For each heavy or light chain, its variable region contains three CDRs: CDR1, CDR2, and CDR3. Therefore, each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L Each of these forms an antigen-binding site.

[0041] The rules for allocating amino acids to various regions or domains have been defined in several publications: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)); Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al., Nature 1989; 342: 878-883; Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0042] The precise boundaries of CDRs have been defined differently depending on the system. The Kabat system not only provides a definitive residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs, which are referred to as Kabat CDRs. Chothia discovered that certain sub-regions within Kabat system CDRs, despite significant diversity at the amino acid sequence level, have nearly identical peptide backbone conformations; these sub-regions are referred to as Chothia CDRs, which have overlapping boundaries with Kabat CDRs. These overlapping boundaries are further described by Padlan and MacCallum. CDR boundary definitions may not strictly adhere to the aforementioned systems, such as the AbM definition. In this document, CDRs can be defined according to any of these systems, although the preferred embodiment uses the antibody numbering system of Chothia et al. to define CDRs.

[0043] As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, i.e., a group of identical antibody molecules except for the possibility of spontaneous natural mutations. The antibody molecules can be immunoglobulins, whether they are natural immunoglobulins or partially or wholly obtained through synthetic methods. The antibody molecules may also include all polypeptides or proteins having an antibody-binding domain, and antibody fragments having an antibody domain are molecules such as Fab, scFv, Fv, dAb, Fd, and bifunctional antibodies. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but it can also be obtained using recombinant DNA technology (see US Patent 4,816,567). As used herein, the terms “monoclonal antibody” and “monoclonal antibody” have the same meaning and are used interchangeably; the terms “polyclonal antibody” and “polyclonal antibody” have the same meaning and are used interchangeably; the terms “peptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented as Ala or A. Glycine can be represented by Gly or G, valine by Val or V, leucine by Leu or L, isoleucine by Ile or I, proline by Pro or P, phenylalanine by Phe or F, tyrosine by Tyr or Y, tryptophan by Trp or W, serine by Ser or S, threonine by Thr or T, cysteine ​​by Cys or C, methionine by Met or M, asparagine by Asn or N, glutamine by Gln or Q, aspartic acid by Asp or D, glutamic acid by Glu or E, lysine by Lys or K, arginine by Arg or R, and histidine by His or H.

[0044] As used herein, the term "recombinant antibody" refers to an antibody obtained by cloning an antibody gene into an expression vector using molecular biology techniques and then transfecting that expression vector into a suitable host cell line for expression. The encoding gene of a recombinant antibody may or may not be identical to the encoding gene of a naturally derived antibody. For example, the complete encoding gene of an antibody obtained by immunizing an animal can be cloned into an expression vector for expression, thereby obtaining an antibody identical to the antibody obtained by immunizing the animal. Alternatively, the gene encoding the variable region (including the heavy chain variable region and the light chain variable region) of an antibody obtained by immunizing an animal can be cloned together with the gene encoding the constant region of an antibody from another species (e.g., human) into an expression vector for expression, thereby obtaining an antibody comprising heavy chain and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having mouse heavy chain and light chain variable regions linked to human constant regions. This type of antibody is commonly referred to in the art as a "chimeric antibody."

[0045] As used herein, the term "antigen-binding fragment" refers to a fragment of an antibody capable of binding an antigen, and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding activity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. A "Fab fragment" consists of a light chain, a CH1 region of a heavy chain, and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The “Fab’ fragment” contains a portion of one light chain and one heavy chain (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); thus, interchain disulfide bonds can be formed between the two heavy chains of two Fab’ fragments to form the F(ab’)2 molecule. The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0046] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.

[0047] In this invention, PCR amplification of the nucleotide sequence encoding the antibody was also performed using primer pairs. In the primer sequences, some sites involve only a single base, such as any one of adenine (A), guanine (G), cytosine (C), and thymine (T), while other sites involve combinations of two, three, or four bases. In these cases, these bases are called degenerate bases, primarily determined based on the degeneracy of the codon. Degenerate bases can be represented by the letters R, Y, M, K, S, W, H, B, V, D, and N, where R represents A / G, Y represents C / T, M represents A / C, K represents G / T, S represents C / G, W represents A / T, H represents A / T / C, B represents G / T / C, V represents G / A / C, D represents G / A / T, and N represents A / T / C / G.

[0048] The terms “sequence identity,” “identity,” or “homology” used herein have their generally accepted meanings in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of that molecule (see, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While many methods exist for measuring the identity between two polynucleotides or polypeptides, the term "identity" is known to those skilled in the art to refer to conserved amino acid substitutions in peptides or proteins that can generally be performed without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0049] In this document, the terms “hot start,” “hot activation,” and “hot shock” are used interchangeably and have the meaning commonly understood in the art, referring to the phenomenon that DNA polymerase exerts its polymerization activity only when the sample temperature is at least above a certain temperature, such as at least 90°C.

[0050] As previously stated, the present invention aims to provide a monoclonal antibody against Taq DNA polymerase. As previously stated, the inventors immunized mice with wild-type Taq DNA polymerase, fused mouse spleen cells with myeloma cells, and screened hybridoma cell lines capable of specifically binding to Taq DNA polymerase using ELISA.

[0051] Therefore, in a first aspect, the present invention provides an anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising a heavy chain complementarity-determining region V. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determination region V. L CDR1, V L CDR2 and V L CDR3; where:

[0052] V H The amino acid sequence of CDR1 is shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49 or SEQ ID NO:54;

[0053] V H The amino acid sequence of CDR2 is shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50 or SEQ ID NO:55;

[0054] V H The amino acid sequence of CDR3 is shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:56 or SEQ ID NO:60;

[0055] V LThe amino acid sequence of CDR1 is shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52 or SEQ ID NO:57;

[0056] V L The amino acid sequence of CDR2 is shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47 or SEQ ID NO:58; and

[0057] V L The amino acid sequence of CDR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:53 or SEQ ID NO:59.

[0058] In one specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:1-3. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:4-6. L CDR1, V L CDR2 and V L CDR3.

[0059] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:7-9. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:10-12. L CDR1, V L CDR2 and V L CDR3.

[0060] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:13-15. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:16-18. L CDR1, V L CDR2 and V L CDR3;

[0061] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:19-21. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:22-24. L CDR1, V L CDR2 and V L CDR3.

[0062] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:25-27. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:28-30. L CDR1, V L CDR2 and V L CDR3.

[0063] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:31-33. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:34-36. L CDR1, V L CDR2 and V LCDR3.

[0064] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:37-39. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:40-42. L CDR1, V L CDR2 and V L CDR3.

[0065] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:43-45. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:46-48. L CDR1, V L CDR2 and V L CDR3.

[0066] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:49-51. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity-determining region V, whose amino acid sequence is defined by the light chain complementarity-determining region shown in SEQ ID NO:52, SEQ ID NO:35, and SEQ ID NO:53, respectively. L CDR1, V L CDR2 and V L CDR3.

[0067] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:54-56. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:57-59. L CDR1, VL CDR2 and V L CDR3.

[0068] In yet another specific embodiment, the heavy chain variable region of the antibody or its antigen-binding fragment includes a heavy chain complementarity-determining region V, whose amino acid sequence is defined by the heavy chain complementarity-determining region V shown in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:60, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:57-59. L CDR1, V L CDR2 and V L CDR3.

[0069] In one specific embodiment, the antibody is a complete antibody comprising a variable region and a constant region. For the antibodies of the present invention, any frame region (FR) and any constant region can be used. The amino acid sequence of the FR or constant region used in the antibodies of the present invention can be the original amino acid sequence of the source FR or constant region, or it can be a different amino acid sequence obtained by substituting, deleting, adding, and / or inserting one or more amino acids into the original FR or constant region. The structure used to support the CDR or CDR group of the present invention generally belongs to the antibody heavy chain or light chain sequence or its major portion, wherein the CDR or CDR group is located in relation to the naturally occurring V region encoded by the rearranged immunoglobulin gene. H and V L At the corresponding position of the CDR or CDR group of the antibody variable domain.

[0070] In one specific implementation, the heavy chain variable region further includes heavy chain frame regions HFR1, HFR2, HFR3, and HFR4, which are related to V H CDR1, V H CDR2 and V H CDR3 progresses from the amino terminus to the carboxyl terminus according to HFR1, V H CDR1, HFR2, V H CDR2, HFR3, V H The order of CDR3 and HFR4.

[0071] In a further specific embodiment, the heavy chain frame regions HFR1, HFR2, HFR3 and HFR4 each have a sequence represented by SEQ ID NO: 61-64 or a sequence that is 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identical to the sequence.

[0072] In yet another specific implementation, the light chain variable region further includes light chain framework regions LFR1, LFR2, LFR3, and LFR4, which are related to V L CDR1, V L CDR2 and V L CDR3 progresses from the amino terminus to the carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The order of CDR3 and LFR4.

[0073] In a further specific embodiment, the light chain framework regions LFR1, LFR2, LFR3 and LFR4 each have a sequence represented by SEQ ID NO:65-68 or a sequence having a sequence identity of 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more.

[0074] In one specific implementation, the antibody further includes a constant region sequence, such as, but not limited to, a constant region sequence selected from any one of IgG, IgA, IgM, IgE and IgD, which can be selected by those skilled in the art as needed, and there is no particular limitation herein.

[0075] In yet another specific implementation, the species source of the constant region sequence can be rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose or human, but is not limited thereto.

[0076] In one specific implementation, the constant region of the antibody of the present invention is derived from mice.

[0077] In some implementations, the species source of the constant region sequence is mice.

[0078] The anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment of the present invention exhibits high sensitivity and high specificity in specifically binding to Taq DNA polymerase. Using one or more of the anti-Taq DNA polymerase monoclonal antibodies or their antigen-binding fragments of the present invention to bind to Taq DNA polymerase not only efficiently blocks the 5'-3' polymerase activity of Taq DNA polymerase, but also blocks the 5'-3' exonuclease activity of Taq DNA polymerase, thereby forming a stable and highly efficient blocked hot-start Taq DNA polymerase. This effectively prevents non-specific amplification caused by mismatches or primer dimers, and also prevents non-specific signals generated by material degradation. This not only improves the specificity and sensitivity of the PCR reaction, but also enhances reagent stability, allowing the detection reagent to withstand transportation or use at room temperature with ease.

[0079] In a second aspect, the present invention provides a nucleic acid molecule encoding an anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof, as described in the first aspect.

[0080] For those skilled in the art, knowing the amino acid sequence of a protein, such as the anti-Taq DNA polymerase monoclonal antibody of the present invention, determining its nucleic acid coding sequence is entirely within their capabilities. Furthermore, to obtain a monoclonal antibody via recombination, the nucleic acid molecule can be cloned into a vector, and the vector can be further introduced into expression cells to express the antibody protein.

[0081] In a third aspect, the present invention provides a carrier comprising the nucleic acid molecule of the second aspect of the present invention.

[0082] In a preferred embodiment, the vector may be a plasmid vector, such as pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ.

[0083] In one specific implementation, the vector is a pTOPO vector.

[0084] In yet another specific implementation, the vector is a eukaryotic expression vector.

[0085] In a preferred embodiment, the pcDNA vector may be a pCDNA3.1 vector.

[0086] In a fourth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.

[0087] The expressed cells are prepared by introducing the aforementioned nucleic acid molecules or the aforementioned vectors into host cells using molecular biology methods well known to those skilled in the art.

[0088] As previously described, the inventors immunized mice with wild-type Taq DNA polymerase, fused mouse spleen cells with myeloma cells, and screened hybridoma cell lines capable of specifically binding to Taq DNA polymerase using ELISA. After screening for monoclonal cell lines secreting the target antibody, the heavy and light chain variable region cDNAs were recovered from the cell lines by reverse transcription-PCR, and suitable immunoglobulin constant regions (e.g., human constant regions) were selected. The heavy and light chain variable region cDNAs and the constant region cDNA were then transferred into host cells such as COS or CHO cells, thereby obtaining expression cells expressing the target antibody of the present invention.

[0089] Using the above-mentioned monoclonal antibody and recombinant DNA technologies, other antibodies or chimeric molecules that retain the specificity of the original antibody can be generated. These technologies may include introducing DNA encoding the variable region or complementarity-determining region (CDR) of an antibody immunoglobulin into a eukaryotic expression vector that includes the constant region or constant region plus frame region of different immunoglobulins, or both into a suitable eukaryotic expression vector, and then introducing the eukaryotic expression vector into expression cells such as CHO host cells, thereby obtaining recombinant antibodies against Taq DNA polymerase.

[0090] In one specific implementation, the expressing cells can be mammalian cells, such as Chinese hamster ovary cells, hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells. In a more specific embodiment, the expressing cells may be, for example, monkey kidney cells transformed with SV40 (COS-7, ATCC CRL1651), human embryonic kidney cells (HEK293 or subcloned HEK293 cells for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36: 59), juvenile hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216; e.g., DG44), mouse thymoma cells (NSO), mouse testicular supporting cells (TM4, Mather, 1980, Biol. Reprod. 23: 243-251), monkey kidney cells (CV-1, ATCC CCL70), and African green monkey kidney cells (VERO-76, ATCC). CRL-1587, human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human hepatocytes (HepG2, HB8065), mouse mammary tumors (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals NYAcad. Sci. 383:44-68), MRC5 cells, FS4 cells, etc., but not limited to these.

[0091] In a fifth aspect, the present invention provides a hot-start Taq DNA polymerase comprising Taq DNA polymerase and at least one anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof from the first aspect.

[0092] As previously described, the antigen-binding fragment of the anti-Taq DNA polymerase monoclonal antibody of the present invention can specifically bind to Taq DNA polymerase, forming an antibody-enzyme complex, thereby obtaining a hot-start Taq DNA polymerase. At low temperatures, the Taq DNA polymerase in the hot-start Taq DNA polymerase remains bound to the anti-Taq DNA polymerase antibody or its antigen-binding fragment, thus blocking the activity of the Taq DNA polymerase. This avoids non-specific amplification caused by primer mismatch and material degradation, improving amplification specificity. Furthermore, the hot-start Taq DNA polymerase of the present invention requires a short hot-start time, effectively protecting the template DNA. After a period of hot-start at a relatively high temperature (e.g., heat shock at 95°C for 2 minutes), the anti-Taq DNA polymerase antibody or its antigen-binding fragment denatures at the high temperature and detaches from the active site of the Taq DNA polymerase, releasing the activity of the Taq DNA polymerase and enabling specific amplification reactions. Furthermore, the anti-Taq DNA polymerase antibody or its antigen-binding fragment of the present invention can effectively block not only the 5'-3' polymerase activity of Taq DNA polymerase at room temperature, but also the 5'-3' exonuclease activity of Taq DNA polymerase. This dual blocking of Taq DNA polymerase effectively prevents non-specific amplification caused by mismatches or primer dimerization, and also prevents non-specific signals generated by material degradation, thus doubly enhancing reagent stability and enabling the detection reagent to withstand both transportation and room temperature use.

[0093] One or two activities of Taq DNA polymerase can be blocked using one or two anti-Taq DNA polymerase antibodies or their antigen-binding fragments. For example, the 5'-3' polymerase activity of the enzyme can be blocked using one anti-Taq DNA polymerase antibody or its antigen-binding fragment, and / or the 5'-3' exonuclease activity of the enzyme can be blocked using another anti-Taq DNA polymerase antibody or its antigen-binding fragment.

[0094] Therefore, in a preferred embodiment, the hot-start Taq DNA polymerase of the present invention comprises Taq DNA polymerase and two anti-Taq DNA polymerase monoclonal antibodies or antigen-binding fragments thereof.

[0095] In a further preferred embodiment, the hot-start Taq DNA polymerase comprises Taq DNA polymerase, an anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment as described in any one of items 1) to 6) of the first aspect of the invention as a first antibody, and an anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment as described in any one of items 7) to 11) of the first aspect of the invention as a second antibody.

[0096] In a more specific embodiment, the weight ratio of the first antibody to the second antibody is 5:1 to 1:5, for example, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5, or a range consisting of any two of these values.

[0097] In a preferred embodiment, the weight ratio of the first antibody to the second antibody is 2:1 to 1:2.

[0098] In a more preferred embodiment, the weight ratio of the first antibody to the second antibody is 1:1.

[0099] In this invention, the combination of the first and second antibodies or their antigen-binding fragments achieves dual blocking of the 5'-3' polymerase activity and the 5'-3' exonuclease activity of Taq DNA polymerase. For example, the hot-start Taq DNA polymerase may comprise Taq22 DNA polymerase, antibody T24, and antibody T29, with antibody T24 and antibody T29 blocking the 5'-3' polymerase activity and the 5'-3' exonuclease activity of Taq22 DNA polymerase, respectively. As another example, the hot-start Taq DNA polymerase may comprise KlenTaq10 DNA polymerase, antibody T22, and antibody T30, with antibody T22 and antibody T30 blocking the 5'-3' polymerase activity and the 5'-3' exonuclease activity of KlenTaq10 DNA polymerase, respectively. For example, the hot-start Taq DNA polymerase may include Taq WT polymerase, antibody T24, and antibody T31, wherein antibody T24 and antibody T31 block the 5'-3' polymerase activity and 5'-3' exonuclease activity of Taq WT polymerase, respectively.

[0100] However, not wanting to be bound by theory, the anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment in the hot-start Taq DNA polymerase of the present invention may also include those anti-Taq DNA polymerase monoclonal antibodies or their antigen-binding fragments disclosed in patent application CN117510639A (the disclosure of which is incorporated herein by reference in its entirety) that are capable of efficiently blocking the 5'-3' polymerase activity of Taq DNA polymerase. These can be used in combination with the antibody or its antigen-binding fragment of the present invention that is capable of blocking the 5'-3' exonuclease activity of Taq DNA polymerase to achieve dual blocking of both the 5'-3' polymerase activity and the 5'-3' exonuclease activity of Taq DNA polymerase.

[0101] In yet another specific embodiment, the weight ratio of the anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment to Taq DNA polymerase is 5:1 to 1:5, for example, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5, or a range consisting of any two of these values.

[0102] In a preferred embodiment, the weight ratio of the anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment to Taq DNA polymerase is 2:1 to 1:2.

[0103] In a more preferred embodiment, the weight ratio of the anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment to Taq DNA polymerase is 1:1.

[0104] In another specific embodiment, the Taq DNA polymerase includes, but is not limited to, wild-type Taq DNA polymerase or its mutants, such as KlenTaq10 DNA polymerase, Taq22 DNA polymerase, Klentaq1 DNA polymerase, Stoffel fragment, recombinant Taq-omni, KT-C3 DNA polymerase, S-Taq, and S-Taq(Δ289). Among these, the "wild-type Taq DNA polymerase" (Taq WT) is a full-length Taq DNA polymerase, comprising 832 amino acids. The "KlenTaq10 DNA polymerase" is a mutant lacking 287 amino acids at the N-terminus, exhibiting 10-100 times greater resistance to inhibitors compared to the full-length Taq DNA polymerase. The "Taq22 enzyme" is obtained by mutating three sites (E626K, I170L, and E708Q) of the wild-type Taq DNA polymerase, resulting in enhanced resistance to inhibitors.

[0105] In a sixth aspect, the present invention provides a method for amplifying a target DNA sequence by polymerase chain reaction, the method comprising the step of amplifying the target DNA sequence using a hot-start Taq DNA polymerase as described in the fifth aspect.

[0106] In one specific implementation, the polymerase chain reaction includes, but is not limited to, quantitative real-time PCR (qPCR), reverse transcription-PCR (RT-PCR), reverse transcription-quantitative PCR (RT-qPCR), digital PCR (dPCR), digital titration PCR (ddPCR), microfluidic PCR, etc.

[0107] In yet another specific embodiment, the hot-start Taq DNA polymerase is activated by hot-start before being used in a polymerase chain reaction. The purpose of hot-start is to dissociate the antibody and enzyme in the hot-start Taq DNA polymerase (i.e., the antibody-enzyme complex), releasing the active single enzyme.

[0108] In a further specific embodiment, the hot start employs a temperature of at least 90°C, for example, 95°C.

[0109] In a seventh aspect, the present invention provides a kit for polymerase chain reaction, comprising: a hot-start Taq DNA polymerase as described in the fifth aspect; and instructions for use.

[0110] Example

[0111] The following examples illustrate the preparation method and characterization of the antibodies of the present invention. Unless otherwise specified, all experimental methods used are conventional methods, and all experimental materials used in the following examples were purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0112] It should be noted that the terminology used in this specification is for the purpose of describing specific embodiments only and is not intended to limit the invention. The foregoing summary section and the following detailed description are for illustrative purposes only and are not intended to limit the invention in any way. The scope of the invention is defined by the appended claims without departing from its spirit and intent.

[0113] Example 1: Preparation of anti-Taq DNA polymerase monoclonal antibody

[0114] Immunization: High-purity Taq DNA polymerase (WT sequence, SEQ ID NO: 69) was used as an immunogen for immunizing mice. Female BALB / c mice aged 6-8 weeks were selected. Mice were immunized four times, with each immunization occurring 2 weeks apart, at a dose of 100 μg / mouse. For the first immunization, Taq DNA polymerase was mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich) and injected subcutaneously at multiple sites on the back. For the subsequent three immunizations, Taq DNA polymerase was mixed with an equal volume of Freund's incomplete adjuvant (Sigma-Aldrich) and injected intraperitoneally. Seven days after the fourth immunization, blood was collected from the tails of the mice, and serum was separated. The antibody titer of the antiserum from the immunized mice was detected using an indirect ELISA method to observe the immune response. Mice with serum antibody titers higher than 1:10000 were selected for cell fusion experiments. Three days before the cell fusion experiment, a booster immunization (100 μg / mouse) was administered via intraperitoneal injection of unadjuvanted Taq DNA polymerase.

[0115] The sequence of SEQ ID NO:69 is as follows: MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLCCCDPNLQNIPVRTPLGQRIRRGFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0116] Establishment of hybridoma cells: On the day of fusion, spleens of immunized mice were removed under aseptic conditions and the organ was prepared into a single-cell suspension. Mouse myeloma cells (SP2 / 0) were fused with spleen cells from immunized BALB / c mice at a ratio of 1:5, thoroughly mixed, and washed twice before fusion with PEG. Preheated PEG1500 was added, gently shaken, and the cells were washed with preheated serum-free RPMI-1640 medium, then resuspended in HAT selective medium. The cell suspension was seeded at 200 μL / well into 96-well plates and cultured at 37°C and 5% CO2. After 4 to 7 days of culture, the cells were cultured in HT medium. When the fused cells grew to 1 / 10-1 / 5 of the bottom area of ​​the wells in the 96-well plate, the supernatant was collected for antibody detection.

[0117] Screening for positive hybridoma cells: Taq DNA polymerase was diluted with coating buffer (0.05 mol / L, pH 9.6, PBS) to a final concentration of 1 μg / mL, and 100 μL / well was added to a 96-well plate. The plate was incubated overnight at 4°C. The coating solution was discarded, and the cells were washed three times with phosphate-buffered saline (PBST) and blotted dry. Cells were then screened with a solution containing 2%... BSA was blocked with PBST, 150 μL / well, and incubated at 37°C for 2 h. The cells were washed three times with PBST and blotted dry. Fusion cell supernatant, 1:1000 diluted positive mouse serum (as a positive control), and 1:1000 diluted negative mouse serum (as a negative control) were added to the corresponding wells at 100 μL / well, and incubated at 37°C for 1 h. The cells were washed three times with PBST and blotted dry. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Sigma) diluted 1:5000, 100 μL / well, was added and incubated at 37°C for 1 h. The cells were washed three times with PBST and blotted dry. Tetramethylbenzidine (TMB) substrate, 100 μL / well, was added and incubated at room temperature in the dark for 10 min. The reaction was terminated by adding 50 μL of 2 mol / L sulfuric acid to each well.

[0118] The OD of all wells in the ELISA plate was detected using a microplate reader at a wavelength of 450 nm. 450nm Value. When the OD of negative serum 450nm ≤0.1, to measure the absorbance OD of the orifice. 450nm The value is the negative pore OD. 450nm A value 2.1 times or higher was used as the criterion for a positive result. Positive hybridoma cells were screened out for further cloning.

[0119] Cloning of positive cell lines: Positive cell lines secreting antibodies were sampled and counted from wells, then diluted to 100 cells / 10 mL of culture medium. The diluted cell suspension was seeded at 100 μL / well into 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. After 6-7 days, clonal cell formation was observed under a microscope. Individual clonal growth wells were marked, and the cell supernatant was collected for ELISA detection (same as the fusion detection described above). Positive monoclonal cells were selected. Limiting dilutions were performed on cells from positive wells, with ELISA values ​​measured 5-6 days after each limiting dilution. The OD values ​​obtained from the ELISA results were then collected. 450nm The wells with high positive values ​​were subjected to limiting dilution until the entire 96-well plate was positive by ELISA. Stable monoclonal lines with high positive values ​​were selected. Finally, a series of stable cell lines secreting anti-Taq DNA polymerase antibody were obtained and named T21'-T31'.

[0120] Example 2: Cloning and sequencing of antibody variable region sequence

[0121] Total RNA was isolated from the hybridoma cell lines described above, and cDNA was prepared by reverse transcription to clone immunoglobulin sequences from the hybridoma cell lines. The variable region sequences of antibodies from the hybridoma cell lines were also determined.

[0122] a. RNA extraction: Total RNA was extracted from the above hybridoma cell lines according to the instructions of the M5 Total RNA Extraction Kit (purchased from Beijing Polymer Biotechnology Co., Ltd.).

[0123] b. RNA reverse transcription to cDNA: The total RNA extracted in the previous step was reverse transcribed using the M5 First Strand cDNA Synthesis Kit (purchased from Beijing Jumei Biotechnology Co., Ltd.) to obtain cDNA, which was then frozen at -20℃ for later use.

[0124] c. PCR amplification and recovery of the variable region sequence: Using the cDNA obtained in the previous step as a template, PCR was performed using universal heavy chain primers Mu Ig V. H 5′-A and Mu IgG V H 3′-2, Immunoglobulin heavy chain (IgH) cDNA was amplified by PCR; similarly, the light chain primer Mu IgκV was used. L 5′-A and Mu IgκV L 3′-1, Immunoglobulin light chain (IgK) cDNA was amplified by PCR, and the PCR product was recovered; thermostable Pfu DNA polymerase was used throughout the PCR reaction.

[0125] d. Cloning and sequencing of variable region sequences: Following the instructions of the pTOPO-Blunt Cloning kit (purchased from Beijing Jumei Biotechnology Co., Ltd.), the heavy and light chain variable region genes were ligated into the pTOPO vector, transformed into E. coli DH5α, and positive clones were selected and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0126] The antibody heavy chain variable region gene sequences and light chain variable region gene sequences of the hybridoma cell lines obtained by sequencing were analyzed. The complementarity-determining region sequences of the heavy chain and the light chain are shown in Table 1 below (based on the Chothia numbering system).

[0127] Table 1: Complementarity Determinant Region Sequences of Heavy and Light Chains

[0128] Example 3: Preparation and purification of recombinant antibodies

[0129] Recombinant antibodies were constructed, and cell lines that stably expressed the antibodies were prepared through eukaryotic expression and then cultured and purified on a large scale.

[0130] Specifically, for antibody V L and V H The gene was obtained by sequentially linking the sequenced CDR with the frame region (FR) sequences shown in SEQ ID NO:61-68 in Table 2 to obtain the heavy / light chain variable region. Subsequently, a recombinant antibody eukaryotic expression plasmid was constructed using molecular cloning methods. This eukaryotic expression plasmid was electroporated into CHO host cells, and after electroporation, the cells were cultured in pressure selection medium (50 μM MSX) for 20 days. The supernatant was then used for ELISA detection (using horseradish peroxidase (HRP)-labeled goat anti-mouse IgG as the secondary antibody for screening, using the same method as above) to screen for stably expressing recombinant antibody cell lines.

[0131] Table 2: Frame region sequence of recombinant antibody

[0132] The selected stable cell lines were cultured on a large scale using roller flask culture technology for recombinant antibody preparation. Cells were cultured in Vega CHO medium at a concentration of (0.2-0.3) × 10⁻⁶ cells / mL. 6Cells / mL were seeded into roller bottles, with 1L roller bottle containing 300mL of culture medium (Vega CHO). The number of seeding bottles was determined according to production needs. The seeded roller bottles were placed in a cell culture incubator. The culture conditions were 900 rpm, 37℃, and 5% CO2. After 7-9 days of culture, samples were taken and observed under a microscope. When the cell viability was less than 50%, the samples were centrifuged and collected. The samples were purified by affinity chromatography using a protein A column to obtain antibodies, namely recombinant monoclonal antibodies T21-T31, whose heavy chain and light chain CDRs correspond to antibodies T21'-T31', respectively (see Table 1).

[0133] The concentrations of each recombinant monoclonal antibody T21-T31 were determined using a micro-spectrophotometer, and the results showed that their concentrations were all greater than 4 mg / mL. Each recombinant monoclonal antibody was aliquoted and stored at 4℃-8℃.

[0134] The monoclonal antibodies were identified by SDS-PAGE electrophoresis, and the antibodies had an antibody heavy chain band of about 51 kDa and an antibody light chain band of about 26 kDa.

[0135] Purity detection: Monoclonal antibodies were analyzed using size exclusion chromatography (SEC-HPLC). Under the condition that all components in the sample were eluted, the purity percentage of the main peak was calculated using the peak area normalization method. The purity of all components was greater than 98%.

[0136] Example 4: Binding ability of antibody to Taq DNA polymerase

[0137] Taq DNA polymerase was diluted with 0.05 mol / L carbonate buffer (pH 9.6) to a concentration of 1 μg / mL. 100 μL / well was added to each well of a 96-well microplate and incubated overnight at 4°C. The plates were washed three times with PBST using an automated plate washer and then blotted dry. Blocking was performed with 150 μL / well of PBST containing 2% BSA, and incubated at 37°C for 2 hours. The plates were washed three times with PBST and then blotted dry. The recombinant monoclonal antibody against Taq DNA polymerase was serially diluted with 0.02 M PBS buffer (pH 7.4) at an initial concentration of 5 μg / mL, and then serially diluted three times to obtain a series of monoclonal antibody samples of different concentrations. 100 μL / well of each diluted monoclonal antibody sample was added to each well of the microplate and incubated at 37°C for 1 hour. The plates were washed three times and then blotted dry. Add 100 μL / well of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Sigma) diluted 1:15000, incubate at 37°C for 1 h, wash three times with PBST, and blot dry. Add 100 μL / well of TMB substrate and incubate at room temperature in the dark for 10 min. Stop the reaction by adding 50 μL / well of 2 mol / L sulfuric acid. Measure OD using a microplate reader. 450nmThe EC50 value of the recombinant monoclonal antibody was obtained by analyzing the ELISA results using software. 50 The results are shown in Table 3 below. As can be seen from Table 3, the recombinant monoclonal antibody T21-T31 can bind to all three Taq enzymes.

[0138] Table 3: EC5 binding rates of each recombinant monoclonal antibody to each Taq DNA polymerase 50 value

[0139] Example 5: Blocking detection of Taq DNA polymerase activity and exonuclease activity by the antibody of the present invention

[0140] Antibodies T21-T31 were conjugated with three different Taq DNA polymerases at a mass ratio of 2:1 and incubated at 37°C for 1 hour to prepare antibody-enzyme complexes, i.e., hot-started Taq DNA polymerases. The activity of the hot-started and unhot-started antibody-enzyme complexes was then tested. The hot-starting of the antibody-enzyme complex was achieved by heat shock at 95°C for 2 minutes, the purpose of which was to dissociate the antibody and enzyme in the complex, releasing the active single enzyme.

[0141] The experimental groups included:

[0142] (1) Single enzyme Taq WT without antibody (positive control); without enzyme (negative control); antibody-enzyme (Taq WT) complex without hot start; antibody-enzyme (Taq WT) complex after hot start; wherein the antibody in the antibody-enzyme complex is one of T21-T31;

[0143] (2) Single enzyme Taq22 without antibody (positive control); without enzyme (negative control); antibody-enzyme (Taq22) complex without hot start; antibody-enzyme (Taq22) complex after hot start; wherein the antibody in the antibody-enzyme complex is one of T21-T31;

[0144] (3) Single enzyme Klentaq 10 without antibody (positive control); without enzyme (negative control); antibody-enzyme (Klentaq 10) complex without hot start; antibody-enzyme (Klentaq 10) complex after hot start; wherein the antibody in the antibody-enzyme complex is one of T21-T31.

[0145] First, the blocking effectiveness of the antibody of this invention against Taq DNA polymerase activity and the enzyme activity recovery effect of Taq DNA polymerase were tested. The specific steps are as follows:

[0146] The reaction is carried out according to the following system:

[0147] After the above reaction system was treated at 94℃ for 20 seconds and 55℃ for 30 seconds, 0.5 μL of sample was added to each experimental group for amplification. The signal was read once every 1 min at 37℃, and 60 cycles were detected. Figures 1-6 show the fluorescence curves of each experimental group and control group (negative control and positive control) of antibodies T22-T27, respectively. The fluorescence curves can be used to determine whether the polymerase activity was effectively blocked. A horizontal fluorescence curve indicates that the fluorescence signal has not changed much, indicating that Taq DNAase has not exerted its polymerase activity, while an upward fluorescence curve indicates that the fluorescence signal is continuously increasing, indicating that Taq DNAase has exerted its polymerase activity.

[0148] As shown in Figures 1-6, the amplification results of the antibody-enzyme complexes (i.e., hot-start Taq DNA polymerase) formed by antibodies T22, T23, T24, T25, and T26 with Taq DNA polymerase before hot-start were basically consistent with the results of the negative control, with the fluorescence curves showing a horizontal state. This indicates that the five antibodies of the present invention can almost completely block the polymerase activity of these three Taq DNA polymerases, with a blocking rate of nearly 100%. Furthermore, the results of these hot-start Taq DNA polymerases after hot-start were basically consistent with the results of the positive control, with the fluorescence curves showing an upward trend. This indicates that the five hot-start enzymes of the present invention can almost completely recover their enzyme activity after heat shock at 95°C. Antibody T21 also showed the same effective blocking of the 5'-3' polymerase activity of Taq DNA polymerase as antibodies T22-T26 (data not shown). Furthermore, the inventors have discovered that not all obtained antibodies can effectively block the 5'-3' polymerase activity of Taq DNA polymerase. For example, the T27 antibody not only has no blocking effect on the 5'-3' polymerase activity of Taq WT polymerase (Figure 6), but also has no blocking effect on the other two Taq DNA polymerases (data not shown).

[0149] Next, the blocking performance of the antibody of the present invention against the 5'-3' exonuclease activity of Taq DNA polymerase was tested. The specific steps are as follows:

[0150] Prepare the reaction system according to the following formula:

[0151] The above reaction system was prepared using fluorescent PCR at 37°C for 60 min. Fluorescence signals were monitored in real-time during the reaction, with a reading taken every 1 min per cycle at 37°C, for a total of 60 cycles. Figures 7-11 show the fluorescence curves of the experimental and control groups for antibodies T23, T27, T28, T29, and T30, respectively. These fluorescence curves can be used to determine whether exonuclease activity was effectively blocked. A horizontal fluorescence curve indicates that the fluorescence signal remained essentially unchanged, indicating that Taq DNA polymerase did not exert its 5'-3' exonuclease activity. An upward-sloping curve indicates that the fluorescence signal is continuously increasing, indicating that Taq DNA polymerase exerted its 5'-3' exonuclease activity. As shown in Figures 7-11, antibodies T27, T28, T29, and T30 achieved nearly 100% blocking rates against the 5'-3' exonuclease activities of wild-type Taq DNA polymerase and Taq22 DNA polymerase, while antibody T23 failed to block the 5'-3' exonuclease activities of both. Furthermore, antibody T31 also showed the same effective blocking of Taq DNA polymerase 5'-3' exonuclease activities as antibodies T22-T26 (data not shown).

[0152] Example 6: Specificity test of enzyme composition amplification

[0153] One antibody from T21-T26 and one antibody from T27-T31 were combined at a mass ratio of 1:1, and then each was combined with three polymerases (Taq WT, Taq22, and KlenTaq10) at a mass ratio of 1:1 to form 90 antibody-enzyme complexes. The activities and specificities of these antibody-enzyme complexes and the corresponding three single enzymes were compared and tested.

[0154] Prepare the reaction system in the PCR tube according to the following system:

[0155] Place the PCR tube in a fluorescence PCR instrument and perform the following procedure:

[0156] Figures 12-14 show the detection results of recombinant monoclonal antibodies T24 and T29 blocking Taq22 DNA polymerase, T22 and T30 blocking KlenTaq10 DNA polymerase, and T24 and T31 blocking Taq WT polymerase, respectively. According to the results in Figures 12-14, the three types of Taq DNA polymerases can simultaneously bind to the two antibodies of this invention that block both the polymerase activity and exonuclease activity of Taq DNA polymerase. The antibody-enzyme complexes prepared in this way, compared with unmodified single enzymes, significantly reduce the occurrence of primer dimers. In addition to the three antibody-enzyme complexes shown in Figures 12-14, the other 87 dual-blocking antibody-enzyme complexes can also effectively block both the polymerase activity and exonuclease activity of Taq DNA polymerase (data not shown), achieving a significant reduction in primer dimers.

Claims

1. A monoclonal antibody against Taq DNA polymerase or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising a heavy chain complementarity-determining region V. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determination region V. L CDR1, V L CDR2 and V L CDR3; where: V H The amino acid sequence of CDR1 is shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49 or SEQ ID NO:54; V H The amino acid sequence of CDR2 is shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50 or SEQ ID NO:55; V H The amino acid sequence of CDR3 is shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:56 or SEQ ID NO:60; V L The amino acid sequence of CDR1 is shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52 or SEQ ID NO:57; V L The amino acid sequence of CDR2 is shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47 or SEQ ID NO:58; and V L The amino acid sequence of CDR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:53 or SEQ ID NO:

59.

2. The anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment according to claim 1, wherein: 1) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V shown in SEQ ID NO:1-3. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:4-6. L CDR1, V L CDR2 and V L CDR3; 2) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:7-9. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:10-12. L CDR1, V L CDR2 and V L CDR3; 3) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V shown in SEQ ID NO:13-15. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:16-18. L CDR1, V L CDR2 and V L CDR3; 4) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:19-21. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:22-24. L CDR1, V L CDR2 and V L CDR3; 5) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V shown in SEQ ID NO:25-27. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:28-30. L CDR1, V L CDR2 and V L CDR3; 6) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:31-33. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:34-36. L CDR1, V L CDR2 and V L CDR3; 7) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:37-39. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:40-42. L CDR1, V L CDR2 and V L CDR3; 8) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:43-45. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:46-48. L CDR1, V L CDR2 and V L CDR3; 9) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:49-51. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:52, SEQ ID NO:35 and SEQ ID NO:53, respectively. L CDR1, V L CDR2 and V L CDR3; 10) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:54-56. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:57-59. L CDR1, V L CDR2 and V L CDR3; or 11) The heavy chain variable region includes the heavy chain complementarity-determining region V, whose amino acid sequence is defined by the heavy chain complementarity-determining region shown in SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:60, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:57-59. L CDR1, V L CDR2 and V L CDR3.

3. The anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The heavy chain variable region further includes frame regions HFR1, HFR2, HFR3, and HFR4, which are related to V H CDR1, V H CDR2 and V H CDR3 progresses from the amino terminus to the carboxyl terminus according to HFR1, V H CDR1, HFR2, V H CDR2, HFR3, V H The sequence of CDR3 and HFR4, wherein HFR1, HFR2, HFR3, and HFR4 respectively have the sequence shown in SEQ ID NO:61-64 or a sequence having more than 80%, 85%, 90%, or 95% identity with the sequence, and the light chain variable region further includes frame regions LFR1, LFR2, LFR3, and LFR4, which are related to V L CDR1, V L CDR2 and V L CDR3 progresses from the amino terminus to the carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The sequence of CDR3 and LFR4, wherein LFR1, LFR2, LFR3 and LFR4 each have a sequence represented by SEQ ID NO:65-68 or a sequence that is 80% or more, 85% or more, 90% or more or more identical to the sequence.

4. A nucleic acid molecule encoding an anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-3.

5. A vector comprising the nucleic acid molecule of claim 4; preferably, the vector is a plasmid vector, such as any one of pEE12, pCAGGS, pTOPO, pcDNA such as pCDNA3.1, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

6. An expression cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5, preferably, the expression cell being a mammalian cell, such as selected from Chinese hamster ovary cells, hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells.

7. A hot-start Taq DNA polymerase comprising Taq DNA polymerase and at least one, preferably two, anti-Taq DNA polymerase monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 1-3.

8. The hot-start Taq DNA polymerase according to claim 7, wherein the hot-start Taq DNA polymerase comprises an anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment according to any one of claims 1) to 6) of claim 2 or 3 as a first antibody, and an anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment according to any one of claims 7) to 11) of claim 2 or 3 as a second antibody.

9. The hot-start Taq DNA polymerase according to claim 8, wherein the weight ratio of the first antibody to the second antibody is 5:1 to 1:5, preferably 2:1 to 1:2, and more preferably 1:

1.

10. The hot-start Taq DNA polymerase according to any one of claims 7-9, wherein the weight ratio of the anti-Taq DNA polymerase monoclonal antibody or its antigen-binding fragment to the Taq DNA polymerase is 5:1 to 1:5, preferably 2:1 to 1:2, and more preferably 1:

1.

11. The hot-start Taq DNA polymerase according to any one of claims 7-10, wherein, The Taq DNA polymerases include wild-type Taq DNA polymerases or their mutants, such as KlenTaq10 DNA polymerase, Taq22 DNA polymerase, Klentaq1 DNA polymerase, Stoffel fragment, recombinant Taq-omni, KT-C3 DNA polymerase, S-Taq, and S-Taq(Δ289).

12. A method for amplifying a target DNA sequence by polymerase chain reaction, the method comprising the step of amplifying the target DNA sequence using the hot-start Taq DNA polymerase of any one of claims 7-11.

13. A kit for polymerase chain reaction, comprising: Hot-start Taq DNA polymerase according to any one of claims 7-11; Including instructions for use.

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