Monoclonal antibodies that neutralize Taq DNA polymerase polymerization activity and their applications

Abstract

This invention proposes a monoclonal antibody that neutralizes the polymerization activity of Taq DNA polymerase and its application. The antibody contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity with it: heavy chain variable region CDR sequence: SEQ ID NO: 4~6, light chain variable region CDR sequence: SEQ ID NO: 10~12. The antibody can specifically act on Taq DNA polymerase or its mutants, thereby significantly reducing the degradation of primers, probes or templates during PCR, and non-specific amplification caused by template or primer mismatch, and significantly improving the reaction efficiency of PCR.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to antibodies that specifically neutralize the polymerization activity of Taq DNA polymerase and their applications, and more specifically, to an antibody or antigen-binding fragment, the use of the antibody, a method for preparing the antibody, a hot-start Taq DNA polymerase, the use of the hot-start Taq DNA polymerase, a method for preparing the hot-start Taq DNA polymerase, a pharmaceutical composition, and a kit. Background Technology

[0002] Taq DNA polymerase-based PCR technology is widely used in biochemical testing and in vitro diagnostics. Taq enzyme is derived from *Thermophyton floccosum*. Thermus Aquaticus The thermostable DNA polymerase isolated from Taq DNA polymerase is a high-temperature enzyme that retains some activity at room temperature. Therefore, before PCR pre-denaturation, the 5'-3' exonuclease activity of Taq DNA polymerase can cause degradation of primers, probes, or templates, while its polymerization activity in the pre-denaturation stage can lead to template or primer mismatches and non-specific amplification. In subsequent PCR cycles, these non-specific products are further amplified, resulting in reduced yield of the target product or even failure to amplify the target band. The hot-start method effectively solves these problems by reversibly inactivating Taq DNA polymerase before PCR pre-denaturation and then activating it during the pre-denaturation stage to restore its activity.

[0003] Currently, commonly used hot-start methods include antibody methods, chemical modification methods, and aptamer methods. Chemical modification methods are more frequently used, offering more complete activity blocking and avoiding the introduction of exogenous contamination; however, their activity is difficult to fully activate, requiring prolonged high-temperature incubation. Aptamer methods, on the other hand, also struggle to completely block activity; activity often recovers at temperatures above 45°C, making it difficult to achieve a truly robust hot start. Compared to these two methods, antibody methods offer certain advantages: Taq DNA polymerase antibodies can completely inhibit Taq DNA polymerase activity at room temperature. With increasing temperature, activity is slowly released above 60°C, and complete release of Taq enzyme activity can be achieved through heat shock at 95°C for 3-5 minutes.

[0004] One-step RT-qPCR is currently the most commonly used molecular diagnostic detection method, and hot-start Taq DNA polymerase is the core enzyme in the one-step RT-qPCR reaction. Therefore, the development of antibodies that neutralize the polymerization activity of Taq DNA polymerase has enormous application potential and market prospects. Summary of the Invention

[0005] This application is based on the inventor's discoveries and understanding of the following facts and problems: Taq DNA polymerase is an enzyme widely used in molecular diagnostics. Hot-start Taq DNA polymerase refers to the enzyme whose activity is inhibited at low temperatures, but is fully released during the pre-denaturation stage as the temperature rises. This method can effectively reduce non-specific amplification before PCR pre-denaturation and improve the sensitivity and yield of PCR / RT-PCR reactions. The inventors used mice to prepare hybridoma cells by immunizing them with the active region of Taq DNA polymerase (i.e., KlenTaq DNA polymerase). Through screening, they obtained antibodies that could neutralize Taq enzyme polymerization activity. Surprisingly, they found that this antibody could reversibly inactivate Taq DNA polymerase before PCR pre-denaturation and reactivate its activity during the PCR pre-denaturation stage, restoring its activity. This antibody can be applied to one-step RT-qPCR reactions and has significant advantages.

[0006] Therefore, in a first aspect, the present invention provides an antibody or antigen-binding fragment, characterized in that the antibody contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity with it: heavy chain variable region CDR sequence: SEQ ID NO: 1-6, light chain variable region CDR sequence: SEQ IN NO: 7-12. According to embodiments of the present invention, the antibody containing the CDR sequence can bind to Taq DNA polymerase or a Taq DNA polymerase mutant, causing the Taq DNA polymerase or the Taq DNA polymerase mutant to reversibly lose activity before PCR pre-denaturation, and activating Taq enzyme activity during the PCR pre-denaturation stage, thereby reducing the degradation of primers, probes, or templates, reducing non-specific amplification caused by template or primer mismatches during PCR, and significantly improving PCR reaction efficiency.

[0007] According to embodiments of the present invention, the above-mentioned antibody or antigen-binding fragment may further include at least one of the following additional technical features: According to embodiments of the present invention, the antibody comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 1, 2, and 3, or amino acid sequences having at least 95% identity with SEQ ID NO: 1, 2, and 3, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 4, 5, and 6, or amino acid sequences having at least 95% identity with SEQ ID NO: 4, 5, and 6, respectively. The hypervariable region is a small subset of amino acid residues within the variable region, where the amino acid residues vary particularly drastically, and the composition and sequence of these amino acid residues are more prone to variation. The hypervariable region is located on the molecular surface and consists of a maximum of 17 amino acid residues, and a minimum of only 2-3. The amino acid sequence of the hypervariable region determines the specificity of the antibody in binding to the antigen.

[0008] According to embodiments of the present invention, the antibody comprises: light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 7, 8, and 9, or amino acid sequences having at least 95% identity with SEQ ID NO: 7, 8, and 9; or light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 10, 11, and 12, or amino acid sequences having at least 95% identity with SEQ ID NO: 10, 11, and 12.

[0009] According to embodiments of the present invention, the antibody contains a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14. According to embodiments of the present invention, the antibody containing the heavy chain variable region can reversibly inactivate Taq DNA polymerase or Taq DNA polymerase mutants before PCR pre-denaturation, and activate Taq enzyme activity during the PCR pre-denaturation stage, thereby reducing the degradation of primers, probes, or templates, reducing non-specific amplification caused by template or primer mismatches during PCR, and significantly improving PCR reaction efficiency.

[0010] According to embodiments of the present invention, the antibody has a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16. According to embodiments of the present invention, the antibody containing the light chain variable region can reversibly inactivate Taq DNA polymerase or Taq DNA polymerase mutants before PCR pre-denaturation, and activate Taq enzyme activity during the PCR pre-denaturation stage, thereby reducing the degradation of primers, probes, or templates, reducing non-specific amplification caused by template or primer mismatches during PCR, and significantly improving PCR reaction efficiency.

[0011] According to embodiments of the present invention, the antibody can bind to Taq DNA polymerase or a mutant thereof. According to embodiments of the present invention, the antibody can specifically inhibit 96%–99% of the polymerization activity of Taq DNA polymerase or a mutant thereof at a temperature of 48–55°C.

[0012] According to an embodiment of the present invention, the amino acid sequence of the Taq DNA polymerase is SEQ ID NO: 17.

[0013] According to an embodiment of the present invention, the amino acid sequence of the Taq DNA polymerase mutant is SEQ ID NO:18.

[0014] In a second aspect, the present invention provides a DNA polymerase composition comprising: the antibody described in the first aspect and Taq DNA polymerase or a mutant thereof. According to embodiments of the present invention, the DNA polymerase composition can reduce non-specific amplification caused by template or primer mismatch during PCR, and significantly improve PCR reaction efficiency.

[0015] According to embodiments of the present invention, the above-described DNA polymerase composition may further include at least one of the following additional technical features: According to an embodiment of the present invention, the mass ratio of the antibody to the Taq DNA polymerase or the Taq DNA polymerase mutant is 1:1 to 4:1. According to an embodiment of the present invention, when the mass ratio of the antibody to the Taq DNA polymerase or the Taq DNA polymerase mutant is 1:1 to 4:1, the antibody can bind to most of the Taq DNA polymerase or the Taq DNA polymerase mutant, thereby inhibiting the activity of most of the Taq DNA polymerase or the Taq DNA polymerase mutant.

[0016] In a third aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises the DNA polymerase composition described in the second aspect. According to embodiments of the present invention, the kit can be used for any PCR amplification, significantly reducing non-specific amplification caused by template or primer mismatch during PCR, and significantly improving PCR reaction efficiency.

[0017] In a fourth aspect, the present invention provides a PCR method. According to an embodiment of the invention, the DNA polymerase composition described in the second aspect is mixed with a DNA template; the mixture is subjected to a PCR reaction to amplify the DNA template. According to an embodiment of the invention, the DNA polymerase retains a certain activity at room temperature. Therefore, before PCR pre-denaturation, the 5'-3' exonuclease activity of Taq DNA polymerase can cause degradation of primers, probes, or templates, and its polymerization activity can cause template or primer mismatches leading to non-specific amplification during the pre-denaturation stage of PCR; this results in reduced yield of the target product during subsequent PCR cycles, or even failure to amplify the target band. The PCR method, comprising a DNA polymerase composition, reversibly loses its activity by binding to the antibody before PCR pre-denaturation, and its activity is activated during the PCR pre-denaturation stage, thereby reducing the degradation of primers, probes, or templates, reducing non-specific amplification caused by template or primer mismatches during PCR, and significantly improving the PCR reaction efficiency.

[0018] In a fifth aspect, the present invention provides a method for preparing an antibody for binding to Taq DNA polymerase or a mutant thereof, comprising the following steps: 1) mixing spleen cells and myeloma cells to obtain hybridoma cells, wherein the spleen cells and myeloma cells are derived from mice immunized with Taq DNA polymerase; 2) subjecting the hybridoma cells to a first screening treatment to obtain positive clones; 3) subjecting the positive clones to limiting dilution treatment to obtain monoclonal lines, wherein each well contains only one monoclonal line; subjecting the monoclonal lines to a second screening treatment to obtain dominant subclonal hybridoma cell lines, wherein an OD value of not less than 1.0 in the ELISA result is an indicator of the dominant subclonal hybridoma cell line; and obtaining a supernatant of the culture medium of the dominant subclonal hybridoma cell line, wherein the supernatant contains the target antibody. According to embodiments of the present invention, the antibody obtained by the method can bind to Taq DNA polymerase or a Taq DNA polymerase mutant, causing the Taq DNA polymerase or the Taq DNA polymerase mutant to reversibly lose its activity before PCR pre-denaturation, and to activate Taq enzyme activity during the PCR pre-denaturation stage, thereby reducing the degradation of primers, probes or templates, reducing non-specific amplification caused by template or primer mismatch during PCR, and significantly improving the reaction efficiency of PCR.

[0019] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features: According to an embodiment of the present invention, the supernatant is further purified.

[0020] According to an embodiment of the present invention, the amino acid sequence of the Taq DNA polymerase is SEQ ID NO: 19.

[0021] According to an embodiment of the present invention, the spleen cells and myeloma cells are derived from mice immunized at least three times with Taq DNA polymerase. According to an embodiment of the present invention, immunized mice with a serum titer of 1:128000 can be obtained after three immunizations.

[0022] According to an embodiment of the present invention, the mice immunized with Taq DNA polymerase at least three times are pre-screened.

[0023] According to an embodiment of the present invention, the purification is magnetic bead purification.

[0024] According to embodiments of the present invention, the pre-screening, first, and second screening processes are performed using ELISA, fluorescent probe method, and M13-7P substrate method. The fluorescent probe method, based on the principle of PCR reaction, is used to detect whether the PCR amplification reaction is proceeding normally. If the amplification reaction proceeds normally in the PCR amplification system containing the antibody to be tested, it indicates that the antibody to be tested cannot neutralize the polymerization activity of Taq DNA polymerase. Furthermore, when the amplification reaction in the PCR amplification system containing the antibody fails to proceed normally, the M13-7P substrate method can further detect whether the failure to proceed normally is due to the antibody to be tested neutralizing the polymerization activity of Taq DNA polymerase, thereby achieving the purpose of screening target antibodies.

[0025] According to an embodiment of the present invention, when the ELISA method is used to perform the pre-screening, first and second screening processes on the sample to be tested, an OD value of not less than 0.8 is an indication that the sample to be tested is the target sample. According to an embodiment of the present invention, during the pre-screening, the sample to be tested is mouse serum immunized at least three times with KlenTaq DNA polymerase. The target sample is an immunized mouse serum sample containing an OD value of not less than 0.8, coated with the KlenTaq DNA polymerase immunogen and detected by ELISA. During the first screening process, the sample to be tested is the supernatant of hybridoma cell culture medium. The target sample is a hybridoma cell culture medium supernatant coated with the KlenTaq DNA polymerase immunogen and detected by ELISA, with an OD value of not less than 0.8. During the second screening process, the sample to be tested is the supernatant of a monoclonal culture medium obtained after a first or second round of limiting dilution. The target sample is a monoclonal culture medium coated with the KlenTaq DNA polymerase immunogen and detected by ELISA, with an OD value of not less than 0.8.

[0026] According to an embodiment of the present invention, the probe set used in the fluorescent probe method has the nucleic acid sequences shown in SEQ ID NO: 20~22.

[0027] According to an embodiment of the present invention, the fluorescent probe method is implemented as follows: 1) the probe group is subjected to a first mixing treatment in equal proportion and annealed to form a probe substrate; 2) the probe substrate is subjected to a second mixing treatment with Taq DNA polymerase-antibody premix, dNTPs and fluorescent probe to obtain a reaction system. Optionally, based on a 20 μL reaction system, the amount of probe substrate is 1~2 μL and the amount of Taq DNA polymerase-antibody premix is ​​1.5~2.5 μL; 3) the reaction system is heated and the FAM channel fluorescence signal is detected. The heating conditions are as follows: 40~50℃, 15~30 min, 55~65℃, 15~30 min; 4) the FAM channel fluorescence signal is detected, wherein no fluorescence signal is detected as an indication that the antibody to be tested is the target antibody.

[0028] According to an embodiment of the present invention, the annealing conditions are: an initial temperature of 80°C to 90°C, followed by a temperature decrease to 25°C to 35°C at a gradient of 0.5°C / min.

[0029] According to an embodiment of the present invention, the Taq DNA polymerase-antibody premix is ​​obtained by mixing Taq DNA polymerase with a sample of antibody to be tested and incubating at 20°C to 28°C for 13 to 16 minutes. According to an embodiment of the present invention, the Taq DNA polymerase in the Taq DNA polymerase-antibody premix is ​​wild-type Taq DNA polymerase or a Taq DNA polymerase mutant, and the antibody to be tested is derived from the serum of immunized mice, hybridoma cell culture medium, monoclonal cell culture medium, or dominant subclonal hybridoma cell line culture medium. The incubation temperature and time according to the embodiments of the present invention allow the Taq DNA polymerase to effectively bind to the target antibody contained in the serum of immunized mice.

[0030] According to an embodiment of the present invention, the mass ratio of the antibody sample to be tested to the Taq DNA polymerase is 1:1 to 8:1.

[0031] According to an embodiment of the present invention, the primer set used in the M13-7P substrate method has the sequences shown in SEQ ID NO:23~29.

[0032] According to an embodiment of the present invention, the M13-7P substrate method is implemented as follows: 1) M13 DNA and the primer set are sequentially reacted at 95°C for 5 min, 75°C for 10 min, 60°C for 10 min, 50°C for 10 min, 40°C for 10 min, and 25°C for 30 min to obtain the M13-7P substrate, wherein the cooling rate from 95°C to 60°C is 0.1°C / s; 2) The M13-7P substrate, Taq DNA polymerase-antibody premix, and dNTPs are mixed to obtain a reaction system. Optionally, based on a 25 μL reaction system, the amount of the M13-7P substrate is 2-3 μL, and the amount of the Taq DNA polymerase-antibody premix is ​​1-3 μL; 3) The reaction system is heated.

[0033] According to an embodiment of the present invention, the heat treatment is carried out at 45-60°C for 8-18 minutes. By heating the reaction system under the conditions described in this embodiment of the present invention, target antibodies that inhibit Taq DNA polymerase at that temperature can be screened.

[0034] According to an embodiment of the present invention, the M13-7P substrate method further includes stopping the reaction in the reaction system.

[0035] In a sixth aspect of the invention, a nucleic acid molecule is provided. According to an embodiment of the invention, the nucleic acid molecule is used to encode the antibody or antigen-binding fragment described in the first aspect. According to a specific embodiment of the invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule can bind to Taq DNA polymerase or a Taq DNA polymerase mutant, such that the Taq DNA polymerase or the Taq DNA polymerase mutant reversibly loses its activity in the pre-denaturation stage of PCR, and activates Taq enzyme activity in the pre-denaturation stage of PCR, thereby reducing the degradation of primers, probes, or templates, reducing non-specific amplification caused by template or primer mismatch during PCR, and significantly improving the reaction efficiency of PCR.

[0036] According to embodiments of the present invention, the above-mentioned nucleic acid molecule may further include at least one of the following additional technical features: According to embodiments of the present invention, the nucleic acid molecule comprises the nucleotide sequences shown in SEQ ID NO: 30-34.

[0037] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0038] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 These are structural diagrams of Taq DNA polymerase and KlenTaq DNA polymerase according to embodiments of the present invention, wherein: Taq DNA polymerase contains both a polymerization active domain and an exonuclease active domain, while KlenTaq DNA polymerase contains only a polymerization active domain. Figure 2 This is a schematic diagram illustrating the principle of detecting the neutralizing activity of antibody-containing serum against Taq DNA polymerase using a fluorescent probe method according to an embodiment of the present invention. Figure 3 This is a graph showing the results of testing the neutralizing activity of Taq DNA polymerase in the serum of five immunized mice after immunization with KlenTaq DNA polymerase as an antigen, using a fluorescent probe method according to an embodiment of the present invention. Among them, the PC group is the positive control group, and the fluorescent probe reaction system of this group does not contain antibodies; the NC group is the negative control group, and the fluorescent probe reaction system of this group does not contain Taq DNA polymerase. Figure 4 This is an experimental flowchart of the fluorescent probe method for detecting the neutralizing activity of 200 subclonal cell lines against Taq DNA polymerase according to an embodiment of the present invention. Figure 5 This is a graph showing the experimental results of detecting the neutralizing activity of 200 subclonal cell lines against Taq DNA polymerase at 50℃ and 60℃ using the fluorescent probe method according to an embodiment of the present invention. Figure 6 This is a graph showing the results of detecting the blocking effect of the dominant antibodies secreted by two dominant subclonal cell lines selected in the first round of subcloning screening on Taq DNA polymerase using the fluorescent probe method according to an embodiment of the present invention. Figure 7 This is a graph showing the results of detecting the recovery effect of the dominant antibodies secreted by two dominant subclonal cell lines selected in the first round of subcloning screening on Taq DNA polymerase using the fluorescent probe method according to an embodiment of the present invention. Figure 8 This is a schematic diagram illustrating the principle of detecting the neutralizing activity of antibody against Taq DNA polymerase using the M13-7P method according to an embodiment of the present invention. Figure 9 This is a graph showing the effect of the amount of 6D7F8H4 antibody added during the second round of subcloning screening on Taq DNA polymerase activity, as determined by the fluorescent probe method according to an embodiment of the present invention. Figure 10This is a graph showing the effect of the amount of 6D7F8H4 antibody added during the second round of subcloning screening on the activity of Taq DNA polymerase mutants, as determined by the fluorescent probe method according to an embodiment of the present invention. Figure 11 This is a graph showing the effect of the amount of 6D7F9G7 antibody added during the second round of subcloning screening on the activity of Taq DNA polymerase and its mutants, as determined by the fluorescent probe method according to an embodiment of the present invention. Figure 12 This is a graph showing the results of SDS-PAGE analysis of the purified 6D7F8H4 and 6D7F9G7 antibodies obtained from the second round of subcloning screening, detected by the fluorescent probe method according to an embodiment of the present invention. Figure 13 This is a diagram showing the results of the fluorescence probe method according to an embodiment of the present invention, detecting the amplification of the light and heavy chain variable region sequences of the 6D7F8H4 and 6D7F9G7 antibodies selected in the second round of subcloning screening; Figure 14 According to an embodiment of the present invention, the average Ct value and amplification curve of hot-start Taq DNA polymerase prepared from the 6D7F8H4 antibody selected in the second round of subcloning screening, and the competitor A antibody-hot-start Taq DNA polymerase, competitor B antibody-hot-start Taq DNA polymerase and non-hot-start Taq DNA polymerase were detected by fluorescent probe method under different template amounts using one-step RT-qPCR amplification. Figure 15 This image shows the results of nucleic acid electrophoresis of the hot-start Taq DNA polymerase prepared from the 6D7F8H4 antibody selected in the second round of subcloning screening according to an embodiment of the present invention, under different template amounts, using a one-step RT-qPCR amplification product and comparing it with that of competitor A antibody-hot-start Taq DNA polymerase, competitor B antibody-hot-start Taq DNA polymerase, and non-hot-start Taq DNA polymerase. Detailed Implementation

[0039] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0040] In this article, the term "antibody" refers to an immunoglobulin molecule capable of binding to a specific antigen. It consists of two lighter light chains and two heavier heavy chains, linked by disulfide bonds to form a tetrapeptide molecule. The amino acid sequence at the N-terminus of the peptide chain varies considerably and is called the variable region (V-terminus), while the carboxyl terminus (C-terminus) is relatively stable and changes very little, called the constant region (C-terminus). The V-termini of the L-chain and H-chain are referred to as VL and VH, respectively.

[0041] Within the variable region, certain areas exhibit a higher degree of variation in amino acid composition and sequence, known as hypervariable regions (HVRs). Located on the molecular surface, these HVRs consist of a maximum of 17 amino acid residues, but can range from only 2 to 3. The HVR is the site where antigens and antibodies bind, determining the specificity of the antibody binding to the antigen; therefore, it is also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDRs.

[0042] In this invention, for comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. The percentage is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position).

[0043] Alternatively, standard settings can be used to calculate the degree of sequence identity between two or more nucleotide sequences using known computer algorithms for sequence alignment, such as NCBI Blastv2.0.

[0044] Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB2357768-A.

[0045] In this invention, for comparing two or more amino acid sequences, the percentage of “sequence identity” between a first amino acid sequence and a second amino acid sequence can be calculated by dividing the number of amino acid residues in the first amino acid sequence by the number of amino acid residues at the corresponding position in the second amino acid sequence, which is the same as the total number of nucleotides in the first amino acid sequence, and then multiplying by 100%, where each deletion, insertion, substitution, or addition of amino acid residues in the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (position), i.e., an “amino acid difference” as defined herein.

[0046] Alternatively, the standard setup can be used again, employing known computer algorithms to calculate the degree of sequence identity between two amino acid sequences, such as the algorithm described above for determining the degree of sequence identity of nucleotide sequences.

[0047] Typically, in order to determine the percentage of “sequence identity” between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated as the “first” amino acid sequence, and the other amino acid sequence is designated as the “second” amino acid sequence.

[0048] Similarly, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called “conserved” amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure that has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art, for example from WO 04 / 037999, GB-A-2357768, WO 98 / 49185, WO 00 / 46383, and WO 01 / 09300; and WO 01 / 09300. The type and / or combination of such substitutions may be selected and / or (preferred) based on the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0049] In the text, "positive clone" refers to hybridoma cells that are able to produce antibodies that bind to Taq DNA polymerase or its mutants after the first screening treatment (fluorescent probe method and M13-7P substrate method).

[0050] In some embodiments, the present invention provides an antibody or antigen-binding fragment, said antibody containing a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity with it: heavy chain variable region CDR sequence: SEQ ID NO: 1-6, light chain variable region CDR sequence: SEQ IN NO: 7-12. "Antigen-binding fragment" refers to an antibody fragment that retains the ability to specifically bind to antigens.

[0051] In some embodiments, the present invention provides a Taq DNA polymerase antibody, the antibody comprising: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 1, 2, and 3 or amino acid sequences having at least 95% identity with SEQ ID NO: 1, 2, and 3; or heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 4, 5, and 6 or amino acid sequences having at least 95% identity with SEQ ID NO: 4, 5, and 6; the antibody comprising: light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 7, 8, and 9 or amino acid sequences having at least 95% identity with SEQ ID NO: 7, 8, and 9; or light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 10, 11, and 12 or amino acid sequences having at least 95% identity with SEQ ID NO: 10, 11, and 12.

[0052] According to some specific embodiments of the present invention, the heavy chain variable regions CDR1, CDR2, CDR3 and the light chain variable regions CDR1, CDR2, CDR3 are defined by any one of the Kabat, Chothia, IMGT, or AbM systems. Commonly used CDR numbering schemes in the art include: Kabat numbering, Chothia numbering, IMGT numbering, Martin numbering, and AHo numbering. CDR definition schemes include: Kabat definition, Chothia definition, IMGT definition, and AbM definition. As described herein, “Kabat numbering” and “Kabat definition” refer to the numbering and definition system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). For “Chothia definition”, see Chothia et al., J Mol Biol 196:901-917 (1987). Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR.

[0053] In some embodiments, the antibody contains a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14. The antibody can reversibly inactivate Taq DNA polymerase or Taq DNA polymerase mutants before PCR pre-denaturation, and activate Taq enzyme activity during the PCR pre-denaturation stage, thereby reducing primer, probe, or template degradation, reducing non-specific amplification caused by template or primer mismatches during PCR, and significantly improving PCR reaction efficiency.

[0054] In some specific embodiments, the antibody has a light chain variable region of the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0055] In some specific implementations, the antibody may specifically target Taq DNA polymerase or a mutant thereof.

[0056] In some specific embodiments, the antibody can specifically inhibit the polymerization activity of Taq DNA polymerase or its mutants.

[0057] In some preferred embodiments, the antibody can specifically inhibit the polymerization activity of 96% to 99% of Taq DNA polymerase or its mutants at a temperature of 48°C to 55°C.

[0058] In some specific embodiments, the amino acid sequence of the Taq DNA polymerase is SEQ ID NO: 17.

[0059] In some specific embodiments, the amino acid sequence of the Taq DNA polymerase mutant is SEQ ID NO:18.

[0060] In some embodiments, the present invention provides a DNA polymerase composition comprising: the antibody and Taq DNA polymerase or a mutant thereof, wherein the DNA polymerase composition can reduce nonspecific amplification caused by template or primer mismatch during PCR and significantly improve PCR reaction efficiency.

[0061] In some specific embodiments, the mass ratio of the antibody to the Taq DNA polymerase or the Taq DNA polymerase mutant is 1:1 to 4:1. When the mass ratio of the antibody to the Taq DNA polymerase or the Taq DNA polymerase mutant is 1:1 to 4:1, the antibody can bind to most of the Taq DNA polymerase or the Taq DNA polymerase mutant, thereby inhibiting the activity of most of the Taq DNA polymerase or the Taq DNA polymerase mutant.

[0062] In some embodiments, the present invention provides a kit comprising the aforementioned DNA polymerase composition. In some specific embodiments, the kit, when used for PCR amplification, can significantly reduce nonspecific amplification caused by template or primer mismatches during PCR, and significantly improve PCR reaction efficiency.

[0063] In some implementations, the kit can be used for any PCR test, such as a novel coronavirus test.

[0064] In some embodiments, the present invention provides a PCR method. In some specific embodiments, the DNA polymerase composition is mixed with a DNA template; the mixture is subjected to a PCR reaction to amplify the DNA template. The method can significantly reduce non-specific amplification caused by template or primer mismatch during PCR and significantly improve the reaction efficiency of PCR.

[0065] In some embodiments, the present invention provides a method for preparing an antibody for binding to Taq DNA polymerase or a mutant thereof, comprising the following steps: 1) mixing spleen cells and myeloma cells to obtain hybridoma cells, wherein the spleen cells and myeloma cells are derived from mice immunized with Taq DNA polymerase; 2) subjecting the hybridoma cells to a first screening treatment to obtain positive clones; 3) subjecting the positive clones to limiting dilution treatment to obtain monoclonal lines, wherein each well contains only one monoclonal line; subjecting the monoclonal lines to a second screening treatment to obtain dominant subclonal hybridoma cell lines, wherein an OD value of not less than 0.8 as measured by ELISA is an indicator of the dominant subclonal hybridoma cell line; and obtaining a supernatant of the culture medium of the dominant subclonal hybridoma cell line, wherein the supernatant contains the target antibody. According to embodiments of the present invention, the antibody obtained by the method can reversibly deactivate Taq DNA polymerase or Taq DNA polymerase mutants before PCR pre-denaturation, and activate Taq enzyme activity during the PCR pre-denaturation stage, thereby reducing the degradation of primers, probes or templates, reducing non-specific amplification caused by template or primer mismatch during PCR, and significantly improving the reaction efficiency of PCR.

[0066] In some specific embodiments, the method for preparing antibodies further includes purifying the supernatant.

[0067] In some specific embodiments, the amino acid sequence of the Taq DNA polymerase is SEQ ID NO: 19.

[0068] In some specific implementations, the spleen cells and myeloma cells are derived from mice immunized with Taq DNA polymerase at least three times, and after three immunizations, immunized mice with a serum titer of 1:128000 can be obtained.

[0069] In some specific implementations, the purification is magnetic bead purification.

[0070] In some specific implementations, the pre-screening, first screening, and second screening processes are performed using ELISA, fluorescent probe methods, and the M13-7P substrate method. The fluorescent probe method, based on the PCR reaction principle, is used to detect whether the PCR amplification reaction is proceeding normally. If the amplification reaction proceeds normally in the PCR amplification system containing the antibody to be tested, it indicates that the antibody to be tested cannot neutralize the polymerization activity of Taq DNA polymerase. Furthermore, when the amplification reaction in the PCR amplification system containing the antibody fails to proceed normally, the M13-7P substrate method can further detect whether the failure to proceed normally is due to the antibody to be tested neutralizing the polymerization activity of Taq DNA polymerase, thereby achieving the purpose of screening for target antibodies.

[0071] In some specific embodiments, the probe set used in the fluorescent probe method has the nucleic acid sequences shown in SEQ ID NO: 20~22.

[0072] In some specific embodiments, the fluorescent probe method is implemented as follows: 1) The probe group is first mixed in equal proportions and then annealed to form a probe substrate; 2) The probe substrate is secondly mixed with Taq DNA polymerase-antibody premix, dNTPs, and the fluorescent probe to obtain a reaction system; based on a 20 μL reaction system, the amount of the probe substrate is 1~2 μL, and the amount of the Taq DNA polymerase-antibody premix is ​​1.5~2.5 μL; 3) The reaction system is heated and the FAM channel fluorescence signal is detected, the heating conditions being sequentially: 40~50℃, 15~30 min, 55~65℃, 15~30 min; 4) The FAM channel fluorescence signal is detected, wherein no fluorescence signal is detected as an indication that the antibody to be tested is the target antibody.

[0073] In some preferred embodiments, the annealing conditions are: an initial temperature of 80°C to 90°C, followed by a temperature decrease to 25°C to 35°C at a gradient of 0.5°C / min.

[0074] In some specific embodiments, the Taq DNA polymerase-antibody premix is ​​obtained by mixing Taq DNA polymerase with the antibody sample and incubating at 20℃~28℃ for 13~16 min. The Taq DNA polymerase in the premix is ​​wild-type Taq DNA polymerase or a Taq DNA polymerase mutant. The antibody is derived from the serum of immunized mice, hybridoma cell culture medium, monoclonal cell culture medium, or dominant subclonal hybridoma cell line culture medium. The incubation temperature and time according to specific embodiments of the present invention allow the Taq DNA polymerase to effectively bind to the target antibody contained in the serum of immunized mice, thereby screening for immunized mice containing the target antibody.

[0075] In some specific implementations, the mass ratio of the antibody sample to be tested to the Taq DNA polymerase is 1:1 to 8:1.

[0076] In some specific implementations, the primer set used in the M13-7P substrate method has the sequences shown in SEQ ID NO:23~29.

[0077] In some specific embodiments, the M13-7P substrate method is implemented as follows: 1) annealing the M13 DNA and the primer set to obtain the M13-7P substrate; 2) mixing the M13-7P substrate, Taq DNAase-antibody premix, and dNTPs to obtain a reaction system, optionally, based on a 20 μL reaction system, the amount of the M13-7P substrate is 2-3 μL, and the amount of the Taq DNAase-antibody premix is ​​1-3 μL; 3) heating the reaction system.

[0078] In some specific implementations, the heat treatment is carried out at 45~60°C for 8~18 minutes.

[0079] In some specific implementations, the M13-7P substrate method further includes stopping the reaction in the reaction system.

[0080] In some embodiments, the present invention provides a nucleic acid molecule for encoding the antibody or antigen-binding fragment described in the first aspect.

[0081] In some specific embodiments, the nucleic acid molecule includes the nucleotide sequences shown in SEQ ID NO: 30-34. It should be noted that, due to the principle of codon degeneracy, the nucleotide sequence used to encode the amino acid sequence of the heavy chain variable region is not a unique and constant sequence; any nucleotide sequence that can encode the same amino acid sequence of the heavy chain variable region is a nucleic acid sequence within the scope of this invention.

[0082] The following is a detailed description of the embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. The antigen used in the mouse immunization phase and ELISA detection is KlenTaq DNA polymerase, and other experiments, such as the activity assay, use Taq DNA polymerase.

[0083] Example 1: Detection of mouse immunity and its serum affinity and neutralizing activity 1. Detection of mouse immunity and its serum affinity activity Recombinant KlenTaq DNA polymerase (purity >90%, protein stored in 1×PBS buffer) was used as the antigen. Five BALB / c mice were selected for three rounds of immunization experiments. After immunization, the serum titer was determined by ELISA to confirm the post-immunization serum titer. The specific procedures are as follows: (1) KlenTaq DNA polymerase was used as the antigen. It was diluted to a suitable concentration of 0.5-10 μg / mL and added to the wells of the microplate at a rate of 100 μL / well. The plate was coated overnight at 4°C. The next day, 200 μL of PBST was added and the plate was washed with shaking for 10 min, 3 times. Then, 200 μL of PBS was added and the plate was washed with shaking for 10 min, 3 times. (2) 200 μL of blocking buffer (1% BSA-PBS) was added and the plate was blocked at 37°C for 1-2 h. Then, 200 μL of PBST was added and the plate was washed with shaking for 10 min, 3 times. Then, 200 μL of PBS was added and the plate was washed with shaking for 10 min, 3 times. (3) 100 μL of diluted supernatant was added and the plate was incubated at 37°C for 2 h. Then, 200 μL of PBST was added and the plate was washed with shaking for 10 min, 3 times. Then, 200 μL of PBS was added and the plate was washed with shaking for 10 min, 3 times. (4) Add 100 μL of freshly diluted Goat Anti-Mouse IgG H&L (HRP), product number ab97023, purchased from abcam, and incubate at 37°C for 1 h. Add 200 μL of PBST and wash for 10 min with shaking, wash 3 times, then add 200 μL of PBS and wash for 10 min with shaking, wash 3 times. (5) Use tetramethylbenzidine microporous peroxidase substrate (TMB) as substrate and react at room temperature in the dark for 10 min. (6) Add 50 μL of stop solution (0.1 mol / L sulfuric acid) and measure the absorbance at 450 nm on an ELISA reader. The specific results are shown in Table 1. In the ELISA detection, KlenTaq DNA polymerase was used for immunogenic protein coating, and at least one animal serum titer reached 1:128000, of which the blank control value OD450 was 0.049.

[0084] Table 1:

[0085] 2. Detection of neutralizing activity in the serum of immunized mice The neutralizing activity of antibody-containing serum against Taq DNA polymerase was detected using a fluorescent probe method. The probe sequences used in this detection method are shown in Table 2.

[0086] Table 2:

[0087] The three single-stranded DNA probe sequences shown in Table 2 were mixed in equal proportions and annealed (the annealing method was to gradually decrease the temperature from 90℃ to 30℃ at a rate of 0.5℃ / min) to form a fluorescent probe substrate. The neutralizing activity assay in immunized mouse serum included the following steps: 2 μL of 0.2 mg / mL wild-type Taq enzyme was mixed with 6 μL of immunized mouse serum and incubated at 25℃ for 15 min. Then, 9.4 μL of PBS buffer was added and mixed thoroughly. The resulting mixture was a 0.23 mg / mL Taq enzyme-serum mixture, which was kept on ice for later use. The reaction system consisted of: 2 μL Taq enzyme-serum premix, 4 μL 5× reaction buffer (50 mM Tris-HCl, 250 mM KCl, 7.5 mM MgCl2, pH 8.3), 0.5 μL 10 mM dNTPs, 11.5 μL nuclease-free water, and 2 μL fluorescent probe substrate. The positive control was the reaction system described above without antibodies, and the negative control was the reaction system described above without Taq enzyme. The reaction conditions were 50℃ for 20 min, then 60℃ for 20 min, with continuous collection of fluorescence signals. If the serum contained antibodies with good neutralizing activity, the release of fluorescence signals would be inhibited. Conversely, the rate of change of fluorescence signals would be consistent with the positive control without antibodies.

[0088] Results of serum neutralizing activity assay in mice after immunization: Figure 3 As shown, the reaction was carried out at 50℃ for 20 min, and then at 60℃ for 20 min. Compared with the positive control group (Taq DNA polymerase without serum incubation), the fluorescence signal changes of Taq DNA polymerase containing serum incubation were inhibited. The results suggest that the antibodies contained in the serum of the five mice can inhibit Taq enzyme activity.

[0089] In addition, the M13 method was used for further screening, employing seven M13 primers and M13 (M13mp18 Single-stranded DNA, purchased from NEB). The corresponding M13 annealing primer sequences are shown in Table 3. Single-stranded M13 and the seven primers were annealed using a gradient cooling method: 95℃ for 5 min; 75℃ for 10 min; 60℃ for 10 min; 50℃ for 10 min; 40℃ for 10 min; and 25℃ for 30 min. The cooling rate between each step was 0.1℃ / s, and the heat was set to 103℃. Pre-annealing yielded the M13-7P substrate, and the inhibitory effect of the antibody on Taq polymerase polymerization activity (the ability to synthesize dsDNA; newly synthesized dsDNA can be detected using a qubit kit) was tested. Mice with high affinity and neutralizing activity in their post-immunization serum were screened using the above steps for subsequent cell fusion experiments. Specific procedures are as follows: The M13-7P method reaction system consisted of: 2 μL Taq enzyme-serum premix, 5 μL 5× reaction buffer (50 mM Tris-HCl, 250 mM KCl, 7.5 mM MgCl2, pH 8.3), 2.5 μL M13-7P substrate, 1 μL 10 mM dNTPs, and 14.5 μL nuclease-free water. The positive control was the above reaction solution without antibody, and the negative control was the reaction mixture without Taq enzyme. The reaction was incubated at 50°C for 10 min, followed by termination with 1 μL 0.5 M EDTA. The inhibitory effect of the antibody on Taq DNA polymerase activity was determined by detecting the synthesized double-stranded DNA using the Qubit™ dsDNA HSAssay Kit.

[0090] Table 3:

[0091] The specific results are shown in Table 4. Mouse E showed the best serum neutralizing activity after immunization, inhibiting 97.8% of Taq polymerase polymerization activity at 50℃. Therefore, mouse E was selected for subsequent cell fusion experiments.

[0092] Table 4:

[0093] Example 2: Cell fusion and screening of hybridoma cell lines Mouse spleen cells with satisfactory serum titers and neutralizing activity were fused with mouse myeloma cells. The main purpose of this step was to screen hybridoma cell lines that could stably produce affinity antibodies. First, successfully immunized mice (E) were selected, euthanized, and fixed on a dissecting plate. The spleen was removed by cutting away connective tissue with a scalpel and homogenized in a glass homogenizer containing fresh culture medium. The homogenate was filtered through a 200-mesh screen, washed, and lysed with erythrocyte lysis buffer for 10 min. The lysate was diluted with fresh culture medium, centrifuged to remove the supernatant, and then redissolved in culture medium. Cell density was measured, and the resulting spleen cells were diluted to a specific concentration. SP2 / OAg14 myeloma cells were then mixed with the treated spleen cells, followed by the addition of polyethylene glycol 2000 (PEG2000) for fusion. Hybridoma cells were obtained and suspended in HAT medium containing feeder cells under pressure before being cultured in 96-well plates. The hybridoma master clone was obtained. All fused cells were seeded into 96-well plates for cell culture. The master clone cell supernatant was subjected to ELISA detection using KlenTaq DNA polymerase coated with the antigen protein. Positive clones were selected, with priority given to hybridoma master clones with high affinity (OD value greater than 1.0 after subtracting the blank control under 1ug / ml antigen (Klentag) coating conditions) for subsequent experiments. The specific operation was the same as in Example 1, and the experimental results are shown in Table 5. Table 5:

[0094] Example 3 Subcloning and Screening The positive master clone described in Example 2 was subjected to a first round of limiting dilution and ELISA affinity screening. The specific procedures are as follows: 1. Subclonal affinity activity assay Thirty to forty positive clones obtained from the first round of limiting dilutions were selected as master clones. From each master clone, five to seven subclones were selected to further confirm the affinity activity of their supernatant (using ELISA, with Klen Taq DNA polymerase as the antigen). The specific procedure was the same as in Example 1. The specific experimental results of the first round of subclonal cell antibody ELISA affinity detection are shown in Table 6. This table displays the ELISA results of a total of 200 antibodies, where the Blank OD value was 0.045; the ELISA detection system was 10 μL of antibody-enzyme mixture (0.05 mg / mL enzyme, 0.1 mg / mL antibody). The ELISA results showed that, after subtracting the blank control, the OD values ​​were generally greater than 1.0, proving that the screened antibodies have a certain affinity activity for the Taq DNA polymerization active region (i.e., Klen Taq DNA polymerase). If the antibody binds to the enzyme catalytic center, it is likely to inhibit the polymerization activity of the enzyme, i.e., it has neutralizing activity. If the antibody binds to a site outside the enzyme catalytic center, it is likely that the antibody only has affinity activity and no neutralizing activity.

[0095] Table 6:

[0096] 2. Subclonal neutralization activity assay Its neutralizing activity against Taq DNA polymerase was detected using the fluorescent probe method described in Example 1. The specific procedures are as follows: The specific operating procedure is shown in Figure 4. Antibody and Taq DNA enzyme were mixed at a mass ratio of 2:1 and incubated at 25°C for 15 min to prepare a hot-start Taq enzyme. The incubation buffer was 1×PBS. The reaction system consisted of: 1.8 μL hot-start Taq enzyme, 4 μL 5× reaction buffer (50 mM Tris-HCl, 250 mM KCl, 7.5 mM MgCl2, pH 8.3), 0.5 μL 10 mM dNTPs, 11.7 μL nuclease-free water, and 2 μL fluorescent probe substrate. The positive control was the above reaction system without antibody, and the negative control was the above reaction system without enzyme. The reaction conditions were 50°C for 20 min, then 60°C for 20 min, on a qPCR instrument. Changes in fluorescence signal were detected during the reaction process. Inhibition of Taq enzyme activity inhibits the release of fluorescence signal. Figure 5 It can be seen that only two of the 200 antibodies were able to significantly inhibit the rate of fluorescence change (compared to the positive control), while the remaining 198 antibodies failed to significantly inhibit Taq enzyme activity (results not shown here).

[0097] 3. Detection of the blocking and recovery effects of subclonal dominant antibodies on Taq enzyme. (1) Fluorescent probe method A hot-start enzyme was prepared, and the changes in Taq enzyme activity before and after hot activation were compared. The reaction mixture containing antibody-Taq DNA polymerase was hot-activated (95°C, 5 min). The changes in fluorescence signal before and after hot activation were compared using a fluorescent probe method to determine the blocking effect of the antibody on Taq enzyme and the recovery of activity after activation. The specific operation was the same as that in Part (2) of Example 3. This step was used to further confirm the neutralizing activity of the two antibodies obtained from the initial screening. Finally, it was determined that the two antibodies could inhibit Taq enzyme activity to different degrees. The results are as follows: Figure 6 and Figure 7 As shown, the reaction temperature was 50℃ for the first 20 minutes and 60℃ for the next 20 minutes. The results indicate that both antibodies exhibited the best neutralizing activity against Taq enzyme, almost completely inhibiting Taq enzyme activity at 50℃.

[0098] (2) M13-7p substrate method The dominant antibodies screened by the fluorescent probe method were further confirmed for neutralizing activity using the M13-7p substrate method. The experimental principle is as follows: Figure 8As shown, the specific operation steps are as follows: (1) Antibody: Taq DNA polymerase is mixed at a ratio of 4:1 (w / w) and incubated at 25℃ for 15 min to prepare hot-start Taq DNA polymerase, wherein the final concentration of Taq DNA polymerase is 0.025 mg / mL and the final concentration of the added antibody is 0.1 mg / mL. (2) The reaction system is 1.8 μL hot-start Taq enzyme, 5 μL 5× reaction buffer (50 mM Tris-HCl, 250 mM KCl, 7.5 mM MgCl2, pH 8.3), 1 μL 10 mM dNTPs, 2.5 μL M13-7P substrate, and 14.7 μL nuclease-free water. NC is the reaction mixture without added Taq DNA polymerase. The positive control is the above reaction solution without antibody, and the negative control is the reaction mixture without Taq enzyme. The reaction conditions are incubation at 50℃ for 10 min, and then 1 μL 0.5 M EDTA is added to terminate the reaction. The inhibitory activity of antibodies against Taq DNA polymerase was determined by detecting the synthesized double-stranded DNA using the Qubit™ dsDNA HS Assay Kit. The results are shown in Table 7, and are consistent with the results obtained by probe assay. At 50°C, the dominant antibodies screened showed a 99% inhibitory activity against Taq DNA polymerase polymerization. Since the antibodies were obtained by immunizing mice with KlenTaq DNA polymerase (i.e., the Taq DNA polymerase polymerization active domain), they can only act on the Taq DNA polymerization active region. This method screened two optimal hybridomas (6D7F8 and 6D7F9) and antibodies that specifically act on the Taq DNA polymerization active domain and inhibit its polymerization activity.

[0099] Table 7:

[0100] Note: The table above shows the dsDNA concentrations measured in the reaction system before and after heat activation. The blocking activity effect is calculated as follows: the difference in dsDNA concentration in the reaction solution before and after heat activation divided by the dsDNA concentration in the reaction system after heat activation. All dsDNA concentrations mentioned should be corrected concentrations after subtracting the corresponding negative control values.

[0101] Example 4: Preparation of stable hybridoma cell lines The superior hybridomas selected in Example 3 were subjected to a second round of limiting dilution, and the neutralizing activity of the antibodies was confirmed.

[0102] The antibodies (6D7F8H4, 6D7F9G7) secreted by the second round of subcloned cells were purified using the magnetic bead method. The specific purification procedure was the same as in Example 3.

[0103] 1. Detection of neutralizing activity of secreted antibodies from the dominant hybridoma subclonal cell line 6D7F8H4 (1) Fluorescent probe method 6D7F8H4 was obtained by further subcloning the dominant hybridoma 6D7F8. Cells were cultured and antibodies were prepared in small batches. The neutralizing activity of the obtained antibodies was tested using a fluorescent probe method. The experimental conditions and methods were the same as in Example 3, except that the mass ratio of antibody / Taq DNA polymerase and antibody / Taq DNA polymerase mutant ranged from 1:1 to 8:1. The reaction conditions were 50°C for 20 min, followed by 60°C for 20 min. The effect of the amount of antibody added on the activity of Taq DNA polymerase and its mutant is shown in the figure below. Figure 9 and Figure 10 As shown, adding 6D7F8H4 antibody in equal mass ratio can achieve the purpose of blocking the activity signal of Taq DNA polymerase and its mutants at 50℃.

[0104] (2) M13-7p substrate method The neutralizing ability of the antibody against the polymerization activity of Taq DNA polymerase and its mutants was further confirmed using the M13-7p substrate method. The experimental conditions and methods were the same as in Example 3. The results are shown in Table 8. Adding the antibody at an equal mass ratio (1:1) blocked more than 95% of the polymerization activity of Taq DNA polymerase and its mutants at 50°C. Stable monoclonal cell lines were obtained and preserved for large-scale cell culture to prepare monoclonal antibodies.

[0105] Table 8:

[0106] 2. Detection of neutralizing activity of secreted antibodies from the dominant hybridoma subclonal cell line 6D7F9G7 (1) Fluorescent probe method 6D7F9G7 was obtained by further subcloning the dominant hybridoma 6D7F9. Cells were cultured and antibodies were prepared in small batches. The neutralizing activity of the obtained antibodies was tested using a fluorescent probe method. The experimental conditions and methods were the same as in Example 3, except that the mass ratio of antibody / Taq DNA polymerase and antibody / Taq DNA polymerase mutant varied from 2:1 to 4:1. The reaction conditions were 50°C for 20 min, followed by 60°C for 20 min. The effect of the amount of antibody added on the activity of Taq DNA polymerase and its mutant is shown in the figure below. Figure 11 As shown, under the conditions indicated, the 6D7F9G7 antibody can block the activity signal of Taq DNA polymerase and its mutants at 50℃.

[0107] (2) M13-7p substrate method The neutralizing ability of this antibody against the polymerization activity of Taq DNA polymerase and its mutants was further confirmed using the M13-7p substrate method. The experimental conditions and methods were the same as in Example 3. The results are shown in Table 9. The antibody's neutralizing ability against Taq DNA polymerase activity was superior to its neutralizing ability against Taq enzyme mutants. At 50°C, the antibody could inhibit 99% of Taq enzyme polymerization activity. At an antibody / enzyme mass ratio of (4:1), most of the Taq mutant activity was inhibited.

[0108] Table 9:

[0109] Example 5: Determination of the variable region sequence of the dominant cell line 1. Culture of dominant hybridomas The dominant hybridoma cells obtained in Example 4 were serum-free acclimatized and then subjected to serum-free suspension culture using CD Hybridoma Medium. The specific procedures were as follows: 1) The cell laboratory was routinely sterilized and irradiated with ultraviolet light for 30 min; 2) The culture medium was placed in a constant temperature water bath at 37°C for at least 20 min; 3) The cryovials were removed from the liquid nitrogen tank and immediately placed in a 37°C water bath, and shaken rapidly until the cryopreservation solution was completely thawed; 4) The cell suspension was transferred to a 15 mL centrifuge tube, and 9 mL of culture medium (RPMI 1640 Medium, 20% FBS) was slowly added, followed by centrifugation at 1000 rpm for 5 min; 5) The cells were resuspended in 4 mL of culture medium and transferred to a T25 culture flask, and cultured statically at 37°C to obtain dominant hybridoma cells; The culture medium of the dominant hybridoma cells was centrifuged to collect the supernatant to obtain antibodies, which were then purified. Protein A Agarose Beads (Sino Biological Inc.) were used for purification. Binding buffer was 1×PBS, and elution buffer consisted of 0.1M glycine, 0.02M arginine, and acetic acid, pH 3.0. The loading rate was 8 mL / min, and elution buffer with a UV280 greater than 30 was collected. The purified antibody was then analyzed by SDS-PAGE, and the results are as follows: Figure 12 As shown, the antibody light chain and heavy chain are located around 25 kDa and 55 kDa, respectively.

[0110] 2. Obtain antibody light and heavy chain sequences Sufficient hybridoma cells were obtained for total RNA extraction in Experiment 1 of this embodiment. Specific operational steps for using the kits were detailed in the respective kit instructions. The specific procedures were as follows: 1) Extract total RNA from hybridoma cells (kit: PureLink RNA Mini Kit, Thermofisher); 2) Synthesize cDNA via RT-PCR (kit: SuperScript™ II Reverse Transcriptase, Thermofisher); 3) Amplify the light and heavy chain variable regions separately using cDNA as a template via PCR using the Mouse Igg Library Primer Set; 4) Obtain light and heavy chain fragments by nucleic acid electrophoresis and gel extraction (EZNA® GelExtraction Kit, Omega); 5) Construct the light and heavy chain variable regions into a plasmid vector (pMD19-T Vector Cloning Kit, Takara), transform into *E. coli* DH5α, and then sequence. The amplification results of the light and heavy chain variable region sequences are shown below. Figure 13 As shown, the amplified light and heavy chain variable region DNA fragments are concentrated around 400bp.

[0111] Example 6 Application of hot-start Taq DNA polymerase 1. Hot-start Taq DNA polymerase accelerated stability test The preparation of hot-start enzymes requires an appropriate antibody dosage to effectively block Taq DNA polymerase activity while maintaining good antibody-enzyme complex stability. Hot-start Taq DNA polymerase was prepared using the antibody 6D7F8H4, which exhibited the best neutralizing activity, and accelerated stability was tested at 37°C for one week. The stability of hot-start Taq DNA polymerases prepared under different antibody dosages was tested. The specific procedures were as follows: First, an antibody-Taq hot-start enzyme mixture was prepared, with a Taq enzyme concentration of 0.1 mg / mL and an antibody concentration of 0.1-2 mg / mL. The storage buffer used was 1×PB containing 50% sterile glycerol. The mixture was incubated at 25°C for 15 min to prepare hot-start Taq DNA polymerase. Then, it was subjected to accelerated stability testing at 37°C. After 7 days, the hot-start effect (i.e., the blocking effect of the antibody on Taq enzyme activity, with blocking activity test conditions of 50°C / 20 min) was determined using M13-7p substrate. As shown in Table 10, the hot-start Taq DNA polymerase prepared after 7 days of accelerated stability testing at 37°C still showed good hot-start effect, that is, it could still inhibit more than 90% of Taq DNA polymerase activity after 20 min of reaction at 50°C, which was comparable to that before accelerated stability treatment (activity blocking was above 95% under 50°C conditions). Accelerated stability results showed that an antibody / enzyme ratio of 2:1 was sufficient to ensure that the prepared hot-start Taq DNA polymerase had no polymerization activity at 50°C and could be stored at -20°C for an extended period (1 year).

[0112] Table 10:

[0113] 2. Antibody 6D7F8H4 was applied to one-step RT-qPCR. A hot-start Taq DNA polymerase was prepared using antibody 6D7F8H4 for one-step RT-qPCR, and compared with commercial Taq DNA polymerase antibodies A and B. One-step RT-qPCR was performed using COVID-19 RNA of different copy numbers as templates. The one-step RT-qPCR program was as follows: reaction at 50℃ for 10 min; reaction at 95℃ for 1 min; reaction at 95℃ for 5 s; annealing at 60℃ for 15 s, for a total of 45 cycles. Figure 14 The figures show the average Ct values ​​and amplification curves for one-step RT-qPCR amplification under different template amounts. Table 11 shows the cycle number results of the superior Taq DNA polymerase antibody described in this invention and similar commercial Taq enzyme antibodies A and B. The Ct value generated by the hot-start Taq DNA polymerase prepared with the self-produced antibody for one-step RT-qPCR is significantly lower than that of competing products A and B, meaning that the self-produced antibody can better improve the sensitivity of RT-qPCR detection. Therefore, it can be concluded that the superior Taq DNA polymerase antibody described in this patent has significantly higher sensitivity for RT-qPCR amplification than similar commercial Taq enzyme antibodies A and B.

[0114] Table 11:

[0115] The above RT-qPCR amplification products were analyzed by nucleic acid electrophoresis, and the results are as follows: Figure 15 As shown, the hot-start Taq DNA polymerase prepared from self-produced antibodies was used for RT-qPCR amplification, and the target band and internal standard were still clearly amplified even when the template amount was 10 copies.

[0116] In summary, the 6D7F8H4 antibody can be used in one-step RT-qPCR for COVID-19 detection. Under the same conditions, it has a lower Ct value and higher amplification sensitivity compared with similar products on the market.

[0117] Table 12:

[0118] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0119] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment, characterized in that, The antibody or antigen-binding fragment includes: The heavy chain variable region CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 4, 5, and 6, or amino acid sequences having at least 95% identity with SEQ ID NO: 4, 5, and 6, respectively; and / or The light chain variable regions CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO: 10, 11, and 12, or in amino acid sequences having at least 95% identity with SEQ ID NO: 10, 11, and 12, respectively.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment contains a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 14; and / or The antibody has a light chain variable region as shown in SEQ ID NO: 16, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:

16.

3. The antibody or antigen-binding fragment according to any one of claims 1 to 2, characterized in that, The antibody or antigen-binding fragment binds to Taq DNA polymerase or a mutant thereof; Optionally, the amino acid sequence of the Taq DNA polymerase is SEQ ID NO: 17; Optionally, the amino acid sequence of the Taq DNA polymerase mutant is SEQ ID NO:

18.

4. A DNA polymerase composition, characterized in that, include: The antibody or antigen-binding fragment and Taq DNA polymerase or mutant thereof as described in any one of claims 1 to 3; Optionally, the mass ratio of the antibody or antigen-binding fragment to the Taq DNA polymerase or Taq DNA polymerase mutant is 1:1 to 4:

1.

5. A reagent kit, characterized in that, The composition comprising the DNA polymerase of claim 4.

6. A PCR method, characterized in that, The DNA polymerase composition of claim 4 is mixed with a DNA template; The mixture was subjected to a PCR reaction to amplify the DNA template.

7. A method for preparing an antibody or antigen-binding fragment, said antibody or antigen-binding fragment being used to bind Taq DNA polymerase or a mutant thereof, characterized in that, Includes the following steps: 1) Splenic cells and myeloma cells were mixed to obtain hybridoma cells, wherein the splenic cells and myeloma cells were derived from mice immunized with Taq DNA polymerase; 2) The hybridoma cells are subjected to a first screening treatment to obtain positive clones; 3) The positive clones are subjected to limiting dilution to obtain single clones, with only one single clone in each predetermined culture well; 4) Perform a second screening treatment on the monoclonal strain to obtain a dominant subclonal hybridoma cell line; 5) Obtain the supernatant of the culture medium of the dominant subclonal hybridoma cell line, wherein the supernatant contains the target antibody; Optionally, the process further includes purifying the supernatant; Optionally, the amino acid sequence of the Taq DNA polymerase is SEQ ID NO: 19; Optionally, the spleen cells and myeloma cells are derived from mice immunized with Taq DNA polymerase at least three times; Optionally, the mice immunized with Taq DNA polymerase at least three times are pre-screened. Optionally, the purification is magnetic bead purification.

8. The method according to claim 7, characterized in that, The pre-screening, first screening, and second screening processes were performed using ELISA, fluorescent probe method, and M13-7P substrate method. Optionally, when using the ELISA method to perform the pre-screening, first screening, and second screening on the test sample, an OD value of not less than 0.8 is an indication that the test sample is the target sample. Optionally, the probe set used in the fluorescent probe method has the nucleic acid sequences shown in SEQ ID NO: 20-22; Preferably, the fluorescent probe method is implemented in the following manner: 1) The probe group is subjected to a first mixing process in equal proportion and then annealed to form a probe substrate; 2) The probe substrate is mixed with Taq DNA polymerase-antibody premix, dNTPs and fluorescent probe to obtain a reaction system. Optionally, based on a 20 μL reaction system, the amount of the probe substrate is 1~2 μL and the amount of the Taq DNA polymerase-antibody premix is ​​1.5~2.5 μL. 3) The reaction system was heated and the fluorescence signal of the FAM channel was detected. The heating conditions were as follows: 40℃~50℃, 15~30min, 55℃~65℃, 15~30min. 4) Detect the fluorescence signal in the FAM channel. No fluorescence signal is detected, which indicates that the antibody to be tested is the target antibody. Optionally, the annealing conditions are: an initial temperature of 80℃~90℃, followed by a temperature decrease to 25℃~35℃ at a gradient of 0.5℃ / min; Optionally, the Taq DNA polymerase-antibody premix is ​​obtained by mixing Taq DNA polymerase with the antibody sample and incubating at 20°C to 28°C for 13 to 16 minutes. Optionally, the mass ratio of the antibody sample to be tested to the Taq DNA polymerase is 1:1 to 8:

1.

9. The method according to claim 8, characterized in that, The primer set used in the M13-7P substrate method has the sequences shown in SEQ ID NO: 23~29; Optionally, the M13-7P substrate method is implemented in the following manner: 1) The M13 DNA and the primer set were subjected to the following reactions in sequence: 95℃ for 5 min, 75℃ for 10 min, 60℃ for 10 min, 50℃ for 10 min, 40℃ for 10 min, and 25℃ for 30 min to obtain the M13-7P substrate. The cooling rate from 95℃ to 60℃ was 0.1℃ / s. 2) The M13-7P substrate, Taq DNA enzyme-antibody premix and dNTPs are mixed to obtain a reaction system. Optionally, based on a 25 μL reaction system, the amount of the M13-7P substrate is 2-3 μL and the amount of the Taq DNA enzyme-antibody premix is ​​1-3 μL. 3) The reaction system is subjected to heat treatment, optionally, the heat treatment is carried out at 45℃~60℃ for 8~18 min; Optionally, the reaction system may be further subjected to a reaction termination process.

10. A nucleic acid molecule, characterized in that, Encoding the antibody or antigen-binding fragment as described in any one of claims 1 to 3; Optionally, the nucleic acid molecule has the nucleotide sequences shown in SEQ ID NO:31, 33.

Images