Preparation method and application of Taq DNA polymerase monoclonal antibody based on structural domain analysis

Abstract

The invention discloses a preparation method and application of a Taq DNA polymerase monoclonal antibody based on structural domain analysis, which are specifically different from a traditional method using full-length Taq polymerase as an immunogen (the method generally cannot generate a functional blocking antibody due to a hidden epitope which is difficult to approach). According to the invention, by adopting a structure-oriented and domain-specific strategy, the exposure of epitopes related to active sites is remarkably enhanced, and a novel antigen Taq-P protein is developed. Based on the antigen, a high-specificity inhibitory monoclonal antibody is prepared, and the antibody is expected to become a core component of a high-performance hot start PCR reagent. According to the invention, a novel and generalizable mode is established for the development of a core diagnostic reagent, a key raw material is provided for gene detection and molecular diagnosis, and a valuable methodological framework is contributed to the development of propulsor engineering and biotechnology reagents.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation technology, specifically to a method and application for preparing Taq DNA polymerase monoclonal antibodies based on domain analysis. Background Technology

[0002] The Taq DNA polymerase (Taq) gene was cloned from *Thermus aquaticus*, a thermophilic bacterium discovered in a hot spring in Yellowstone National Park, USA. Taq DNA polymerase possesses both 5′–3′ exonuclease and DNA polymerase activities, but lacks 3′–5′ exonuclease proofreading activity. Taq DNA polymerase is an 832-residue molecule divided into two functional domains (UniProt ID: P19821): a 5′–3′ exonuclease domain and a DNA polymerase domain. Several crystal structures of Taq DNA polymerase have been reported, including the full-length enzyme (PDB ID: 1TAQ) and structures containing only the polymerase domain. These structural data indicate that the exonuclease domain contains residues 1–292, while the polymerase domain corresponds to residues 293–832. Following the invention of polymerase chain reaction (PCR) in 1983, the introduction of Taq DNA polymerase in 1988 enabled stable and automated amplification processes. Due to its precision and specificity, PCR has become a cornerstone technology in biomedical research. However, non-specific products can still be generated during the PCR reaction. This phenomenon occurs because, although the optimal temperature for Taq DNA polymerase is 75-80℃, it retains polymerase activity at 20-37℃. This means that non-specific amplification may occur before the target product begins to amplify, thus reducing the yield of the target product and affecting the accuracy of experimental results. Hot-start PCR is an effective method to solve this problem, with hot-start Taq DNA polymerase (HS Taq) being the most critical component.

[0003] Currently, there are two methods for preparing HS Taq: internal modification and external modification. Internal modification includes chemical modification of the enzyme and cold-sensitive mutagenesis, while external modification includes the use of physical barriers, oligonucleotide aptamers, antibodies, or gold nanoparticles. Each method has its advantages and disadvantages. For example, chemical modification provides stable enzyme activity without exogenous DNA contamination and is cost-effective. However, this method requires a long enzyme activation time, which may affect enzyme activity and lead to reduced PCR product yield. Physical barrier methods are cumbersome and susceptible to contamination. In contrast, ligand-based modification methods do not require enzyme activation, provide stable enzyme activity, and reduce the possibility of sample degradation.

[0004] Antibody-mediated hot-start PCR is widely used due to its short enzyme activation time, stable enzyme activity, high affinity, and good inhibitory effect. The basic principle of antibody-mediated hot-start PCR is the reversible inhibition of Taq DNA polymerase through specific antigen-antibody interactions. At room temperature, monoclonal antibodies bind with high affinity to the enzyme's active site or allosteric sites crucial to its function. This binding spatially prevents the polymerase from binding to the DNA template and dNTPs, effectively putting the enzyme in a "shutdown" state, thus preventing nonspecific primer extension and primer dimer formation during reaction setup. This inhibitory complex remains stable during subsequent temperature increases. However, antibodies are proteins and undergo irreversible denaturation at high temperatures. When the PCR reaction reaches the initial denaturation step (typically 94-95°C), the antibody, as a protein, denatures and loses its native structure. This structural disruption causes the loss of its high-affinity binding to Taq polymerase, leading to complex dissociation. Therefore, the polymerase is released in its fully active form, ready for DNA synthesis in the subsequent annealing and extension steps of the thermal cycle. This process ensures that enzyme activity is released only at high temperatures, thus giving it a "hot-start" characteristic. The core component of this method for blocking Taq polymerase activity is a monoclonal antibody capable of blocking Taq activity through antigen-antibody binding. R. Murali et al. also reported that multiple regions of the Taq DNA polymerase protein can bind to the Fab fragment of TP7, which may play a positive role in blocking the active site of Taq DNA polymerase.

[0005] However, the effectiveness of this method hinges on obtaining monoclonal antibodies that can precisely bind to key functional epitopes. This critical dependence has become a major bottleneck because routine immunization with full-length Taq polymerase often fails to produce such inhibitory antibodies. Therefore, based on previous literature, this invention aims to develop high-performance hot-start Taq by preparing monoclonal antibodies with high specificity and strong blocking efficiency through a rational, domain-targeted immunization strategy. We hypothesize that immunization with isolated polymerase domains better exposes epitopes associated with the active site, thereby maximizing the probability of generating functionally blocking antibodies.

[0006] In summary, this invention aims to prepare hot-start Taq using monoclonal antibodies with high specificity and strong blocking efficiency. The resulting HS Taq may be a reliable reagent suitable for demanding diagnostic applications such as pathogen detection and multiplex PCR. Summary of the Invention

[0007] The technical problem this invention aims to solve is overcoming the reliance on commercial sources or complex genetic engineering techniques in the development of antibody-based hot-start Taq DNA polymerases. This reliance limits the independent and customized development of core PCR components. To address this, we have established a simple platform for preparing highly efficient monoclonal antibodies that inhibit Taq enzyme activity. Simultaneously, we obtained a hybridoma cell line whose secreted monoclonal antibody significantly outperforms existing technologies in PCR detection.

[0008] The first objective of this invention is to provide a hybridoma cell line, with accession number CCTCC NO:C2025391.

[0009] A second objective of this invention is to provide a Taq DNA polymerase monoclonal antibody secreted by the aforementioned hybridoma cell line.

[0010] Furthermore, the method for preparing Taq DNA polymerase monoclonal antibody includes the step of immunizing animals with the protein shown in SEQ ID NO.3 as an antigen to prepare monoclonal antibody.

[0011] Furthermore, the specific steps include: S1. Prepare an antigen from the protein shown in SEQ ID NO.3 and use the antigen to immunize animals; S2. Blood samples are collected from immunized animals, and the serum immune titers of the immunized animals are screened. S3. The spleen cells and myeloma cells of the selected immunized animals are fused and cultured to obtain a hybridoma cell line that secretes monoclonal antibodies. This hybridoma cell line is then used to prepare monoclonal antibodies.

[0012] A third object of the present invention is to provide a recombinant antigen for preparing Taq DNA polymerase antibodies, said recombinant antigen containing the sequence shown in SEQ ID NO.3.

[0013] Of course, the present invention also provides: Nucleic acid molecules encoding the recombinant antigen.

[0014] A recombinant plasmid carrying the nucleic acid molecule.

[0015] Monoclonal antibodies prepared using the recombinant antigen.

[0016] A fourth object of the present invention is to provide a complex comprising the monoclonal antibody and Taq DNA polymerase (both of which bind specifically).

[0017] Furthermore, the Taq DNA polymerase is a hot-start Taq DNA polymerase. Hot-start polymerase chain reaction (hot-start PCR) can effectively inhibit the amplification of non-specific products during PCR, and hot-start Taq DNA polymerase (HS Taq) is a key component. Current methods for preparing HS Taq, including chemical modification and aptamer-based methods, have limitations such as long activation time, reduced enzyme activity, and insufficient blocking specificity. In contrast, antibody-mediated hot-start Taq has significant advantages: rapid activation, stable enzyme performance, and high blocking specificity through targeted binding to functional domains.

[0018] A fifth objective of this invention is to provide a hot-start PCR system comprising the monoclonal antibody or complex.

[0019] Furthermore, the hot-start PCR system contains reagents necessary for amplification, including but not limited to dNTPs, primers, and buffer solutions.

[0020] A sixth objective of this invention is to provide the application of the monoclonal antibody, complex, or hot-start PCR system in the preparation of detection products.

[0021] Furthermore, the detection product is used for hot-start PCR detection of target nucleic acids, including the following steps: mixing the nucleic acid sample to be tested with the hot-start PCR system, starting the amplification program, and detecting the content of target nucleic acids (e.g., through fluorescence signals).

[0022] Furthermore, the detection product is a fungal, bacterial, or viral detection product, and correspondingly, the target nucleic acid is DNA or RNA derived from fungi, bacteria, or viruses.

[0023] The beneficial effects of this invention are: This invention employs a structure-guided domain-specific immunoassay strategy: instead of using a full-length enzyme, a specific monoclonal antibody is prepared targeting the polymerase domain (Taq-P), which acts as a reversible inhibitor. This method overcomes the significant challenge of preparing functional antibodies against cryptic epitopes, which exhibit poor immunogenicity in full-length proteins. Through screening, this invention yielded a hybridoma cell line whose secreted antibody exhibits significant advantages in detection: the antibody blocks the enzyme's active site through steric hindrance at room temperature, preventing non-specific primer binding during PCR setup, and dissociates upon thermal activation to restore full enzyme activity. The resulting hot-start Taq enzyme significantly reduces non-specific amplification in quantitative PCR detection and has successfully demonstrated its practical application value in detecting fungal pathogens and respiratory viruses using clinical samples. This invention provides a feasible and effective strategy for the independent development of key molecular diagnostic reagents.

[0024] The hybridoma cell line TAQ22-P8D2E8E5G8 was deposited at the China Center for Type Culture Collection (CCTCC) on December 30, 2025, with accession number CCTCC NO: C2025391, at Wuhan University, China. Attached Figure Description

[0025] Figure 1 Identification of Taq DNA polymerase structural templates and domain boundaries: The full-length Taq DNA polymerase sequence was analyzed using the MODELLER program. The most homologous structural templates were aligned from top to bottom, and the corresponding PDB numbers are shown in red boxes. The obvious discontinuity between amino acid residues 290 and 293 (indicated by two vertical black lines) marks the structural boundary separating the N-terminal exonuclease domain (amino acid residues 1-290) and the C-terminal polymerase domain (amino acid residues 293-832).

[0026] Figure 2 Purification analysis of Taq-P and Taq-N proteins. The purity of Taq-P and Taq-N, both proteins used as antigens for mouse immunization, was analyzed by reducing gradient SDS-PAGE of 4–12%. Lane M: Molecular weight marker; Taq-P protein: approximately 60 kDa; Taq-N protein: approximately 33 kDa.

[0027] Figure 3 Purification analysis of monoclonal antibodies. Antibodies were analyzed by reducing gradient SDS-PAGE from 4% to 12%. Lane M: molecular weight marker; Lane 1: IgG Taq-N-A7; Lane 2: IgG Taq-P-B3; Lane 3: IgG Taq-P-D4. Bands observed at approximately 55 kDa and 25 kDa correspond to the immunoglobulin heavy chain (HC) and light chain (LC), respectively.

[0028] Figure 4 Monoclonal antibody titers were determined by indirect ELISA. (A) Titer against full-length Taq DNA polymerase. (B) Titer against the Taq-N protein fragment. (C) Titer against the Taq-P protein fragment. PBST was used as a negative control. The results showed that IgG Taq-N-A7 specifically binds to the Taq-N fragment, while IgG Taq-P-B3 and IgG Taq-P-D4 specifically bind to the Taq-P fragment.

[0029] Figure 5To evaluate the blocking effect of monoclonal antibodies on Taq polymerase using quantitative PCR, PCR amplification curves were obtained after pre-incubating (A-C) Taq polymerase with different masses (0.25, 0.5, and 1 μg) of (A) IgG Taq-P-B3, (B) IgG Taq-N-A7, and (C) IgG Taq-P-D4. Hairpin oligonucleotides (TZ) were used as templates. Commercial hot-start Taq polymerase and unblocked Taq polymerase were used as positive and negative controls, respectively. The baseline level of the first 20 cycles at 70°C in (A) showed that 0.5 μg and 1 μg of IgG Taq-P-B3 completely inhibited enzyme activity, which was restored upon heating to 95°C. In contrast, IgG Taq-N-A7 and IgG Taq-P-D4 did not show significant blocking effects at any tested concentration.

[0030] Figure 6 To perform specificity analysis of hot-start PCR by quantitative PCR. (A, B) Melting curves (A1, B1) and amplification curves (A2, B2) obtained from quantitative PCR assays using IgG Taq-P-B3 blocked Taq polymerase (HS Taq) at template concentrations of 0.1 ng / μL (A) and 1 ng / μL (B). A commercial hot-start Taq enzyme was used as a control.

[0031] Figure 7 For performance validation in fungal detection assays, amplification curves of *Pterocarya aurea* were analyzed using (A) experimental HS Taq (blocked with IgG Taq-P-B3) and (B) commercial hot-start Taq enzyme (control). The Ct values ​​and curve profiles of the experimental and control groups were almost identical.

[0032] Figure 8 Comparative sensitivity analysis of hot-start Taq enzyme at low template concentrations. The detection rates of *Agrostis umbellatus* are shown at template concentrations of 300 copies / mL (A, B) and 75 copies / mL (C, D). (A, C) Results using commercially available hot-start Taq enzyme. (B, D) Results using experimental HS Taq prepared with IgG Taq-P-B3. Each panel shows the number of positive detections in 16 replicate reactions (e.g., 12 / 16).

[0033] Figure 9Performance validation of the multiplex respiratory virus detection system. (A) Melting curve (peak 1) for H1N1 detection and (B) simultaneous detection of PIV-1 (peak 1) and PIV-3 (peak 2). Experimental groups (A1, B1) used HS Taq prepared with IgG Taq-P-B3, while control groups (A2, B2) used commercially available hot-start Taq enzyme. No nonspecific peaks and consistent Tm values ​​were observed between the experimental and control groups.

[0034] Figure 10 Comparison of different Taq DNA polymerase blocking methods.

[0035] Figure 11 To compare hot-start Taq DNA polymerase and non-hot-start Taq DNA polymerase in different detection systems. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0037] The technical solution involved in this invention is as follows: Nonspecific amplification remains a major challenge in routine PCR, primarily due to the residual activity of Taq polymerase at room temperature. While various hot-start methods exist, antibody-based methods offer significant practical advantages in terms of performance, ease of use, and cost-effectiveness. Specifically, compared to common alternatives: it eliminates the long activation times and enzyme degradation issues associated with chemical or mutagenic methods; compared to physical barrier systems, it simplifies the workflow and reduces the risk of contamination; and compared to aptamers, it provides better blocking stability and shelf life—key factors that improve the reliability and commercial viability of diagnostic applications. Its core mechanism involves reversible, temperature-dependent inhibitors: at room temperature, the antibody blocks the active site or prevents key conformational changes through steric hindrance; during the initial PCR denaturation process, antibody denaturation irreversibly dissociates the complex, fully restoring polymerase activity. However, a key but often overlooked limitation is the absolute reliance on antibodies targeting functionally critical epitopes.

[0038] Our initial failed attempt to prepare inhibitory antibodies using full-length Taq polymerase highlighted this limitation and directly supported our hypothesis that key epitopes may be conformally masked. This challenge spurred our methodological innovation: a structure-guided, domain-specific immunogenic strategy designed to overcome the poor immunogenicity of catalytic epitopes in full-length enzymes. Homology modeling (Figure 1) revealed that residues 175–261 constitute a fragment of the 5'–3' exonuclease domain, distinct from the DNA polymerase active site, suggesting potential epitope overlap between the Taq-N and Taq-P domains. We inferred that using the isolated Taq-P domain as an immunogen would better expose the epitopes associated with the active site, thereby increasing the likelihood of generating functionally blocking antibodies. Therefore, we constructed and purified Taq-N and Taq-P domain proteins for antibody preparation and screening.

[0039] This domain-specific strategy has proven crucial. We successfully prepared three monoclonal antibodies: IgG Taq-N-A7 (Taq-N specific), IgG Taq-P-B3, and IgG Taq-P-D4 (both Taq-P specific). Functional characterization of the Taq-P antibodies showed that they all exhibited high affinity in enzyme-linked immunosorbent assays (ELISA) and effectively inhibited polymerase activity. For example, the Taq-P antibody IgG Taq-P-B3, which we believe targets epitopes at or near the enzyme's active site, may hinder substrate (DNA and dNTP) entry or interfere with catalytic conformational dynamics. This invention demonstrates that HS Taq prepared using the antibodies of this invention (such as IgG Taq-P-B3) exhibits high specificity and sensitivity comparable to commercial products in inhibiting nonspecific amplification.

[0040] We further evaluated the HS Taq polymerase prepared using the antibody of this invention in a clinical diagnostic setting. In fungal pathogen detection assays, direct comparisons with commercial HS Taq showed no significant differences in amplification curves or Ct values. Furthermore, the experimental HS Taq exhibited strong reproducibility and higher sensitivity for low-abundance targets. Similarly, in single-tube multiplex detection of respiratory pathogens (H1N1, PIV-1, PIV-2), our HS Taq showed comparable Tm values ​​and peak heights to the commercial control, with no nonspecific products. In these applications, consistent Ct values, melting curve profiles, and the absence of nonspecific amplification confirm the suitability of this reagent for molecular diagnostics.

[0041] The sequences related to the present invention are as follows: Full-length Taq DNA polymerase protein (SEQ ID NO.1): MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE Taq - N protein (SEQ ID NO.2): GRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREP Taq - P protein (SEQ ID NO.3): DVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLS

[0042] 1. Homology modeling and domain definition To design antigens, homology modeling is performed to determine the structural boundary between the N-terminal and C-terminal domains of Taq DNA polymerase. Homology modeling is a computational method that predicts the three-dimensional (3D) structure of a target protein based on experimentally determined structures of homologous proteins (templates). The HHpred web server (https: / / toolkit.tuebingen.mpg.de / tools / hhpred) integrates sequence alignment and structure prediction tools. It uses MODELLER software to generate protein 3D models by identifying homologs with structural features and using their spatial information as templates. The specific steps include: (1) inputting the target protein sequence; (2) using HHpred to search for the best structural templates; (3) aligning the target sequence with these templates; and (4) using MODELLER to construct a 3D model based on the alignment results. The principle behind this method is that proteins with high sequence similarity often have similar overall folded structures.

[0043] The results showed that homology modeling revealed two distinct domains of Taq DNA polymerase: Using the MODELLER program in the HHpred web server, the full-length Taq DNA polymerase sequence (green band, amino acid residues 1-832) was compared with the most homologous structural template (shown from top to bottom, with the corresponding PDB number marked in red). Figure 1 The alignment results showed a significant discontinuity between amino acid residues 290 and 293, represented by two vertical black lines, which likely represent the structural boundary separating the two domains. Based on these structural alignments and previously characterized functional domains, Taq DNA polymerase can be divided into two distinct domains: the N-terminal region (amino acid residues 1-290; referred to as Taq-N) corresponds to the exonuclease domain, and the C-terminal region (amino acid residues 293-832; referred to as Taq-P) contains the polymerase catalytic domain. The model was constructed under spatial constraints derived from sequence alignment with the selected template, based on the assumption that proteins with high sequence similarity typically employ similar overall folding structures.

[0044] 2. Plasmid construction, protein expression and purification Codon-optimized DNA fragments encoding residues 1-290 and 293-832 of Taq DNA polymerase were cloned into the NcoI and XhoI sites of the pET-28a vector, respectively, to generate expression plasmids pET28a-Taq(1-290)-His (Taq-N) and pET28a-His-Taq(293-832) (Taq-P) (synthesized by Genewiz).

[0045] Each recombinant plasmid was transformed into *E. coli* BL21(DE3) (Novagen) and plated on LB agar plates containing kanamycin (50 µg / ml) and incubated overnight at 37°C. Positive colonies were selected and inoculated into 30 mL of LB medium supplemented with kanamycin, then cultured at 37°C with shaking at 200 rpm for 8 hours. Next, 1% (v / v) of the inoculum was transferred to 600 mL of LB medium and cultured until OD (outcome limit) was reached. 600 The concentration reached 0.8, and protein expression was induced overnight at 18°C ​​with 0.2 mM IPTG. Cells were collected by centrifugation at 4°C, 8,000 × g for 30 min, and the pellet was resuspended at a ratio of 1:9 (w / v) in lysis buffer (25 mM Tris, pH 9.0, 300 mM NaCl, 2 mM MgCl2, 0.5% Tween-20, 20 mM imidazole, and protease inhibitor). The suspension was sonicated at 30% power for 10 min with a duty cycle of 50%, and then centrifuged at 12,000 rpm for 25 min to obtain the soluble fraction. The supernatant was incubated with 1 mL Ni-NTA resin at 4°C for 1 h, and then loaded onto a gravity flow column. The resin was washed sequentially with 5 column volumes (CV) of equilibration buffer (25 mM Tris, pH 9.0, 300 mM NaCl, 2 mM MgCl2, 20 mM imidazole) and 10 column volumes of wash buffer (25 mM Tris, pH 9.0, 1,300 mM NaCl, 0.5% Tween-20, 2 mM MgCl2, 20 mM imidazole). The target protein was eluted with elution buffer (25 mM Tris, pH 9.0, 300 mM NaCl, 2 mM MgCl2, 250 mM imidazole). Protein purity was analyzed by 4%–12% gradient SDS-PAGE. High-purity fractions were combined and dialyzed overnight at 4°C with 1 L of dialysis buffer (25 mM Tris, pH 9.0, 150 mM NaCl).

[0046] The purified Taq-N and Taq-P proteins were analyzed by 4-12% gradient SDS-PAGE. Clear bands corresponding to the target proteins were observed at the expected molecular weight positions: Taq-P was approximately 60 kDa, and Taq-N was approximately 33 kDa. Quantitative analysis using ImageJ software showed that the purity of the recombinant Taq-P protein exceeded 90%, and the purity of the Taq-N protein reached 80%. Figure 2 ).

[0047] (1) Mouse immunization For the initial immunization, Taq-N and Taq-P proteins at a concentration of 500 μg / mL were mixed with an equal volume of Freund's complete adjuvant (Sigma) and administered via multiple subcutaneous injections along the back of mice, with each mouse receiving 100 μg. Three to four mice were immunized with each protein. Under the same conditions, for the second immunization, the antigen was mixed with an equal volume of Freund's incomplete adjuvant (Sigma), followed by booster immunizations every two weeks for a total of four booster immunizations. Ten days after the final immunization, tail blood was collected from the mice, and serum antibody titers were determined using an indirect ELISA method, requiring a titer of at least 1:10,000. Four days before cell fusion, each mouse received a booster immunization via intraperitoneal injection of 100 μg. Subsequently, mouse spleen cells were fused with SP2 / 0 myeloma cells (Chinese Academy of Sciences Cell Bank, product number: TCM18) at a ratio of 1:10 using 50% PEG1500. Cells were resuspended in HAT-1640 medium containing 15% FBS and seeded at a rate of 100 μL per well into 96-well plates pre-coated with feeder cells. The plates were then cultured at 37°C and 5% CO2. Two weeks after confluence, 100 μL of cell culture supernatant per well was transferred to pre-coated ELISA plates. Positive hybridoma cells were screened using indirect ELISA, with serum from immunized mice as a positive control and SP2 / 0 cell culture supernatant as a negative control.

[0048] (2) Ascites preparation and antibody purification Each BALB / c mouse was sensitized by intraperitoneal injection of 250 μL of liquid paraffin. Hybridoma cells were passaged and cultured at 37°C until the logarithmic growth phase. After digestion and washing, each mouse was injected with 1 × 10⁻⁶ liquid paraffin. 6 Cells were injected into the peritoneal cavity of mice at a density of 1,000 cells. Ascites was collected 7-10 days later when the mice's abdomens were significantly swollen.

[0049] IgG was separated from mouse ascites fluid using 50% and 45% ammonium sulfate precipitation methods, followed by affinity chromatography purification using a Protein G column (Smart-Lifesciences). The purified antibody was dialyzed against PBS, and the OD of the supernatant was measured using a NanoDropLite Plus spectrophotometer (Thermo Fisher). 280 The IgG concentration was calculated using an extinction coefficient of 1.46, and the purity of the IgG was confirmed by reducing gradient SDS-PAGE electrophoresis at 4–12%.

[0050] The results show: To prepare monoclonal antibodies targeting the Taq DNA polymerase domain, BALB / c mice were immunized with purified Taq-N or Taq-P protein. After cell fusion and screening by enzyme-linked immunosorbent assay (ELISA), three positive hybridoma cell lines were obtained: IgG Taq-N-A7, IgG Taq-P-B3, and IgG Taq-P-D4. Ascites fluid from these three cell lines was prepared and purified by protein G affinity chromatography. The purified IgG was evaluated using a 4–20% gradient SDS-PAGE under reducing conditions. The results showed good purification, with the antibody protein exhibiting two distinct bands: one at 55 kDa, corresponding to the heavy chain, and the other at 25 kDa, corresponding to the light chain. Figure 3 The presence of clear bands at the expected size with minimal background interference confirms the high purity of the purified monoclonal antibody.

[0051] 1. Enzyme-linked immunosorbent assay (ELISA) for detecting and validating Taq polymerase antibodies. Antibody titers were determined using an indirect ELISA method. Full-length Taq DNA polymerase, Taq-N, and Taq-P proteins were diluted to 5 μg / mL with bicarbonate buffer (pH 9.6) and coated onto ELISA plates at 100 μL per well, then incubated overnight at 4°C. The plates were washed three times with phosphate-buffered saline (PBST) containing Tween 20 (0.01 M PBS, pH 7.4, containing 0.05% (v / v) Tween 20), each wash lasting 3 minutes. Next, the plates were blocked with 1% BSA at 37°C for 2 hours, followed by three more washes. The purified Taq DNA polymerase-specific monoclonal antibody was serially diluted with PBST to create eight concentration gradients (1:10,000, 1:20,000, 1:40,000, 1:80,000, 1:160,000, 1:320,000, 1:640,000, 1:1,280,000). Each dilution was added as primary antibody at 100 μL per well, with PBST used as a negative control. The mixture was incubated at 37°C for 2 hours, followed by three washes. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000) (Jiangsu Kangwei Century Biotechnology Co., Ltd., product number: CW0102S) was added as secondary antibody (100 μL per well), and the mixture was incubated at 37°C for 30 minutes, followed by another wash. Finally, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to each well. After 15 minutes of color development, the reaction was terminated with 50 μL of 1N H2SO4. OD was measured using a microplate reader (Bio-Rad). 450value.

[0052] The results show: The titers of three purified IgG antibodies were determined by indirect enzyme-linked immunosorbent assay (ELISA). Using ELISA plates coated with full-length Taq DNA polymerase protein, the titers of IgG Taq-N-A7, IgG Taq-P-B3, and IgG Taq-P-D4 were approximately 320,000, 640,000, and 320,000, respectively. Figure 4 A). When using an ELISA plate coated with Taq-N protein, the titers of IgG Taq-N-A7, IgG Taq-P-B3, and IgG Taq-P-D4 were greater than 640,000, with the latter two being close to the negative control. Figure 4 B). When using an ELISA plate coated with Taq-P protein, the titer of IgG Taq-NA7 was close to that of the negative control, while the titers of IgG Taq-P-B3 and IgG Taq-P-D4 were approximately 1,280,000 (B). Figure 4 C). The results confirmed the successful preparation of a domain-specific monoclonal antibody.

[0053] 2. Validation of monoclonal antibody blocking performance To evaluate the blocking efficiency of monoclonal antibodies against Taq polymerase, we designed a hairpin oligonucleotide sequence TZ (TCTAGAGGGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTACTATGCTAATACGACTCACTAT, synthesized by Genewiz) and diluted the lyophilized TZ powder with ddH2O to a final concentration of 6.25 μM.

[0054] We mixed the obtained monoclonal antibodies with Taq DNA polymerase (10 U, 0.2 μg) at different ratios (0.25, 0.5, and 1 μg) and incubated at 37°C for 0.5 h to block the reaction mixture. The resulting reaction mixture was then diluted to a final concentration of 0.5 U / μL with Taq storage buffer (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% NP-40, 50% glycerol) to prepare experimental samples. We used commercially available hot-start Taq polymerase (Takara Bio) and unblocked Taq polymerase as positive and negative controls, respectively.

[0055] The total reaction volume (20 μL) included: 2 μL 10× PCR buffer, 0.25 μL 6.25 μM TZ solution, 2 μL 2.5 mM dNTPs, 2 μL 10× SYBR Green, 0.5 μL experimental sample, and 13.25 μL ddH2O. Amplification was performed using a real-time quantitative PCR system (Thermo Fisher Scientific) to monitor the reaction fluorescence. The reaction program was as follows: 70℃ for 30 seconds, 20 cycles; 95℃ for 30 seconds; 74℃ for 30 seconds, 20 cycles.

[0056] The results show: To test the blocking performance of the antibody, a hairpin oligonucleotide sequence TZ was designed as a template, and real-time monitoring was performed using a quantitative real-time PCR instrument. The fluorescence rate increase was directly proportional to the DNA template amplification efficiency. As shown in Figure 5, the curve trend of the positive control indicates that the curve in the first stage (cycles 1-20) at 70℃ is horizontal, meaning that the fluorescence intensity does not change, indicating that the enzyme activity is completely blocked and the template is not amplified. The second stage (cycle 21) involves antibody denaturation and dissociation from Taq enzyme at 95℃. The third stage (cycles 21-40) is the template amplification stage, during which Taq enzyme recovers its activity at 74℃, and the template continues to amplify. Conversely, the negative control curve shows a continuous increase from the first cycle, with the fluorescence intensity increasing with template amplification.

[0057] The results (Figure 5A) showed that HS Taq, prepared by mixing 0.5 μg and 1 μg of IgG Taq-P-B3 with Taq enzyme (10 U, 0.2 μg), effectively blocked Taq enzyme activity. The fluorescence curves were horizontal during the first 20 cycles, showing no difference from the commercial hot-start Taq positive control. This indicates that our antibody blocking efficiency was complete at these concentrations, comparable to existing commercial standards. However, when the IgG dosage was 0.25 μg, Taq polymerase activity was not completely blocked, resulting in a premature rise in the amplification curve, similar to the unblocked Taq negative control. This determined the minimum effective antibody-to-enzyme ratio required for complete inhibition. In contrast, IgG Taq-N-A7 failed to block Taq enzyme activity at all three dosages (0.25, 0.5, and 1 μg) (Figure 5B), as their amplification curves almost overlapped with the negative control, clearly confirming their lack of inhibitory function. Therefore, the IgG Taq-P-B3 cell line was deposited for future performance verification.

[0058] 3. Hot-start PCR specificity detection Using the antibody-Taq polymerase mixture obtained in the "Monoclonal Antibody Blocking Performance Validation" experiment as the test sample (antibody was IgG Taq-P-B3), we diluted the DNA template (10 ng / μL) to concentrations of 1 ng / μL and 0.1 ng / μL with ddH2O. A commercially available hot-start Taq polymerase (Takara Bio) was used as a control.

[0059] The total reaction volume (20 μL) includes: 5 μL 4× GPX buffer, 1.2 μL 5 μM GPX primer mixture, 0.3 μL test sample, 10 μL DNA template (0.1 or 1 ng / μL), and 3.5 μL ddH2O.

[0060] The Thermo Fisher Scientific real-time PCR system was used under the following conditions: 94°C pre-denaturation for 1 minute; 95°C denaturation for 15 seconds, 67°C annealing extension for 30 seconds, for 45 cycles; followed by 95°C denaturation for 15 seconds, 60°C annealing for 20 seconds; finally, melting curve analysis was performed, with the temperature gradually increased from 60°C to 90°C in increments of 0.03°C.

[0061] The results show: To test the specificity of hot-start PCR, this invention employs quantitative PCR experiments. The results (Figure 6) show that the HS Taq enzyme prepared by incubating IgG Taq P-B3 (1 μg and 0.5 μg) with Taq enzyme (10 U, 0.2 μg) exhibits a single, unique peak in its melting curve between 80-90℃. The shape and melting temperature (Tm) of this peak are identical to those of the commercial hot-start Taq control. Neither the experimental nor control groups showed any non-specific peaks before 80℃, confirming that our prepared HS Taq achieved a level of specificity comparable to commercial products. However, when the IgG concentration was 0.25 μg, a non-specific product peak appeared between 74-80℃, and the peak height between 80-90℃ was lower compared to the other two concentrations. This indicates that the HS Taq prepared with IgG Taq P-B3 at (1 μg and 0.5 μg) ratios effectively inhibits non-specific amplification during the heating process of the quantitative PCR reaction. This indicates that an insufficient antibody ratio leads to incomplete inhibition of Taq polymerase activity.

[0062] Furthermore, this invention compared the prepared HS Taq with commercial hot-start Taq by amplifying different template concentrations (1 ng / μL and 0.1 ng / μL). Results ( Figure 6The results showed that significant amplification was achieved at all template concentrations, indicating that the hot-start PCR system has high sensitivity. The single peak in the melting curve and the robust amplification at different dilutions indicate that the prepared HS Taq can effectively suppress nonspecific amplification and has considerably higher specificity compared to commercial products.

[0063] 1. Fungal detection Following the method described in "Monoclonal Antibody Blocking Performance Validation", the antibody (1 μg IgG Taq-P-B3) was mixed with Taq DNA polymerase (10 U) and incubated. The resulting reaction mixture was used as a test sample for detecting *Pterocaryonium aureum*, with a commercially available hot-start Taq enzyme as a control.

[0064] The total reaction volume (25 μL) included: 2.5 μL 10× PCR buffer, 2 μL 2.5 mM dNTP mixture, 5 μL primer mixture, 0.625 μL Taq polymerase stock solution, 0.25 μL uracil-DNA glycosylase (UDG), 0.125 μL test sample, and 5 μL *Pterocarya stenoptera* DNA (Shanghai Geniodi Biotechnology Co., Ltd.). The final volume was adjusted to 25 μL using nuclease-free ddH2O.

[0065] The Thermo Fisher Scientific real-time PCR system was used under the following conditions: incubation at 37°C for 2 minutes; pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 15 seconds; annealing and extension at 58°C for 60 seconds, for 40 cycles.

[0066] To quantitatively assess amplification efficiency and consistency, the cycle threshold (Ct) values ​​of eight parallel replicate reactions for the test samples and commercial controls were measured. The mean Ct value and relative standard deviation (RSD) for each group were calculated. PCR reactions were performed using the reaction components and procedures described above.

[0067] Furthermore, the analytical sensitivity of the prepared hot-start Taq polymerase was evaluated using a *Pterocarya stenoptera* DNA detection assay and compared with that of a commercial hot-start Taq polymerase (Takara Bio). The target DNA template was serially diluted with nuclease-free water to final concentrations of 300 copies / mL and 75 copies / mL. For each concentration and each polymerase, sixteen independent (n = 16) replicate PCR reactions were performed using the above reaction components and procedure.

[0068] The results are as follows: For the detection system of *Pterocaryonium umbellatus*, the results ( Figure 7The results showed that the Ct values ​​and amplification curve profiles of the experimental group (IgG Taq P-B3) and the commercial control group were almost identical. The nearly identical Ct values ​​and curve profiles of the experimental and control groups indicate that antibody-blocked Taq polymerase performs well in diagnostic applications and does not affect detection efficiency.

[0069] Simultaneously, the experimental HS Taq and commercial hot-start Taq DNA polymerase were tested, with 8 replicates in each group. The results (Table 1) showed that the average Ct value obtained by HS Taq amplification was 32.65, while the average Ct value of the control group was 32.55. The relative standard deviation (RSD) of the Ct value of HS Taq amplification was 0.82%, while that of the control group was 0.99%. Statistical analysis confirmed that there was no significant difference in performance between the two enzymes (t(14) = 0.697, p = 0.497), and the effect size was small (Cohen's d = 0.34), indicating that they have comparable amplification efficiency and reproducibility.

[0070] Table 1. Comparison of Ct values ​​and reproducibility between experimental and commercial hot-start Taq polymerase in the detection assay of *Pteris vittata*.

[0071] The table shows the cycle threshold (Ct) values ​​for eight parallel replicate PCR reactions using experimental HS Taq (blocked with IgG Taq-P-B3) and a commercial hot-start Taq control. The mean Ct value and relative standard deviation (RSD) were calculated for each group.

[0072] Furthermore, the sensitivity of HS Taq was evaluated using low template concentrations (75 and 300 copies / mL). As shown in Figure 8, at 300 copies / mL, our HS Taq showed a higher detection rate (16 / 16) than the commercial control (12 / 16), although this difference did not reach statistical significance (p = 0.095). In contrast, at 75 copies / mL, our HS Taq maintained a significantly higher detection rate (12 / 16), compared to (5 / 16) for the commercial enzyme (p = 0.026). These results indicate that the prepared HS Taq achieved higher detection rates at both concentrations than the commercial enzyme, suggesting greater sensitivity to low-abundance targets.

[0073] 2. Multiplex detection of respiratory viruses Following the method described in "Monoclonal Antibody Blocking Performance Validation", the antibody (1 μg IgG Taq-P-B3) was mixed with Taq DNA polymerase (10 U) and incubated. The reaction mixture was used as the experimental sample for multiplex detection of respiratory viruses, with a commercially available hot-start Taq enzyme used as a control.

[0074] The total reaction volume (25 μL) includes: 2.5 μL 10× PCR buffer, 2 μL 2.5 mM dNTP mixture, 5 μL multiplex primer mixture, 0.1 μL experimental sample, and 5 μL H1N1 DNA / PIV-1 DNA / PIV-3 DNA (Shanghai Geniodi Biotechnology Co., Ltd.), adjusted to the final volume with nuclease-free water.

[0075] The following conditions were used in a real-time quantitative PCR system (Thermo Fisher Scientific): pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 15 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 15 seconds, for 10 cycles; denaturation at 94°C for 15 seconds, annealing at 50°C for 15 seconds, extension at 72°C for 15 seconds, for 23 cycles; denaturation at 95°C for 2 minutes; incubation at 40°C for 90 seconds; then melting curve analysis was performed, with the temperature gradually increased from 40°C to 90°C in 1°C increments; incubation at 37°C for 1 second.

[0076] The results are as follows: In the respiratory virus multiplex detection system, results showed that in both the H1N1 (Figure 9A) and PIV-1 / PIV-3 (Figure 9B) systems, there were no significant differences in melting temperature (Tm) or peak height between the experimental group (IgG Taq P-B3) and the control group. Only specific target product peaks were observed in all cases. The consistency of melting peak spectra of our HS Taq and the commercial control across multiple targets confirms that the prepared HS Taq is suitable for the detection of specific multiplex pathogens.

[0077] (1) Comparison of the effects of antibody IgG Taq P-B3 blocking and aptamer blocking See results Figure 10 Taq-Ab group: The antibody of this invention was used for blocking, resulting in the best blocking effect, with all bands being specific and no non-specific bands. Taq-Apt group (aptamer is a marketed product of Shanghai Yaxin Biotechnology): Aptamer blocking was used, resulting in good blocking effect, mainly with specific bands and faint non-specific bands. Taq group: No blocking was used, with poor specificity, very low concentration of specific bands, and relatively obvious non-specific bands.

[0078] (3) Comparison of hot-start Taq DNA polymerase and non-hot-start Taq DNA polymerase in different detection systems In three detection systems (i.e., the H1N1, PIV-1, and PIV-3 systems mentioned above in the "Multiple Detection of Respiratory Viruses" section), hot-start Taq DNA polymerase (red) and non-hot-start Taq DNA polymerase (blue) were compared and tested. The results showed that ( Figure 11 Hot-start Taq DNA polymerase has significant advantages in terms of Ct value, repeatability, and endpoint fluorescence value.

[0079] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A hybridoma cell line, characterized in that, The accession number is CCTCC NO:C2025391.

2. A Taq DNA polymerase monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. A method for preparing a Taq DNA polymerase monoclonal antibody, characterized in that, The method includes the step of preparing monoclonal antibodies by immunizing animals with the protein shown in SEQ ID NO.3 as an antigen.

4. A recombinant antigen for preparing Taq DNA polymerase antibodies, characterized in that, The recombinant antigen contains the sequence shown in SEQ ID NO.

3.

5. The monoclonal antibody prepared from the recombinant antigen of claim 4.

6. A complex, characterized in that, It includes the Taq DNA polymerase monoclonal antibody and Taq DNA polymerase as described in claim 2, or the monoclonal antibody and Taq DNA polymerase as described in claim 5.

7. The complex according to claim 6, characterized in that, The Taq DNA polymerase is a hot-start Taq DNA polymerase.

8. A hot-start PCR system, characterized in that, It includes the Taq DNA polymerase monoclonal antibody of claim 2, the monoclonal antibody of claim 5, or the complex of claim 6 or 7.

9. The application of the Taq DNA polymerase monoclonal antibody of claim 2, the monoclonal antibody of claim 5, the complex of claim 6 or 7, or the hot-start PCR system of claim 8 in the preparation of detection products.

10. The application as described in claim 9, characterized in that: The detection product is used for detecting target nucleic acids using hot-start PCR, and includes the following steps: mixing the nucleic acid sample to be tested with the hot-start PCR system, starting the amplification program, and detecting the content of target nucleic acids; and / or, the detection product is a fungal, bacterial, or viral detection product.

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