https://orcid.org/0009-0008-3322-535X
Figures
Abstract
Hot-start polymerase chain reaction (hot-start PCR) effectively inhibits non-specific product amplification during PCR, with hot-start Taq DNA polymerase (HS Taq) serving as the critical component. Current HS Taq preparation methods, including chemical modification and aptamer-based approaches, exhibit limitations such as prolonged activation time, reduced enzyme activity, and inadequate blocking specificity. In contrast, antibody-mediated hot-start Taq offers distinct advantages: rapid activation, stable enzymatic performance, and high blocking specificity through targeted binding to functional domains. While antibody-based hot-start Taq DNA polymerases are widely utilized for their high efficiency and specificity, their development often relies on commercial sources or complex genetic engineering. This dependency constrains the independent and customizable development of core PCR components. To address this, our study established a streamlined platform for generating highly effective monoclonal antibodies that inhibit Taq enzyme activity. The key innovation lies in our structure-guided, domain-specific immunization strategy: instead of using the full-length enzyme, we targeted the polymerase domain (Taq-P) to generate a specific monoclonal antibody that acts as a reversible inhibitor. This approach overcomes the major challenge of generating functional antibodies against cryptic epitopes, which are poorly immunogenic in the full-length protein. This antibody sterically blocks the enzyme’s active site at room temperature, preventing non-specific priming during PCR setup, and dissociates upon thermal activation to restore full activity. The resulting hot-start Taq enzyme significantly reduced non-specific amplification in quantitative PCR assays, and its practical utility was successfully demonstrated in detecting fungal pathogens and respiratory viruses using clinical samples. This study provides a feasible and effective strategy for the autonomous development of critical reagents for molecular diagnostics.
Citation: Zhu X, Zhang T, Zhao H, Hou B, Fu M, Zhao Q, et al. (2026) Preparation and application of Taq DNA polymerase monoclonal antibody guided by structural domain analysis. PLoS One 21(3): e0345402. https://doi.org/10.1371/journal.pone.0345402
Editor: Kokouvi Kassegne, Shanghai Jiao Tong University School of Medicine, CHINA
Received: August 7, 2025; Accepted: March 5, 2026; Published: March 18, 2026
Copyright: © 2026 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The study was supported by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering. There was no additional external funding received for this study.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Taq DNA polymerase (Taq) gene is cloned from Thermus aquaticus [1], a thermophilic bacterium found in a hot spring in Yellowstone National Park, USA [2]. Taq DNA polymerase exhibits 5′–3′ exonuclease activity and DNA polymerase activity [3,4], but lacks 3’–5’ exonuclease proofreading activity [5]. Taq DNA polymerase is a molecule composed of 832 residues. It can be divided into two functional domains (UniProt ID: P19821, also see S1 Fig), 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 (S2 Fig. PDB ID: 1TAQ) and structures containing only the polymerase domain (S3 Fig). These structural data indicate that the exonuclease domain occupies residues 1–292 (S4 Fig), whereas the polymerase domain corresponds to residues 293–832. Following the invention of the polymerase chain reaction (PCR) in 1983, the introduction of Taq DNA polymerase in 1988 [6] enabled stable and automated amplification, solidifying PCR as a cornerstone technique in biomedical research due to its precision and specificity [7,8]. However, non-specific products may still be generated during the PCR reaction process.The reason for this phenomenon is that although the optimal temperature of Taq DNA polymerase is 75 ~ 80°C, it retains polymerase activity even at 20 ~ 37°C. This means that non-specific amplification can occur before the amplification of the target product begins. This will reduce the yield of the target product and affect the accuracy of the experimental results. Hot-start PCR is an effective method to solve this problem, in which the hot-start Taq DNA polymerase (HS Taq) serves as the most critical component [9,10].
Currently, there are two methods for preparing HS Taq: internal modification and external modification. Internal modification includes chemical modification [11] of the enzyme and cold-sensitive mutations [12,13], while external modification includes the use of physical barriers, oligonucleotide aptamers, antibodies, or gold nanoparticles [14,15], etc. Each of these methods has its own advantages and disadvantages. For example, the chemical modification method provides stable enzyme activity and no exogenous DNA contamination, making it cost-effective. However, this method requires a relatively long time for enzyme activation, which may affect the enzyme activity, leading to lower PCR product yield. The physical barrier method [16–18] is cumbersome and prone to contamination. In contrast, the ligand-based modification method does not require enzyme activation, the enzyme activity is stable, and it reduces the possibility of sample degradation [19,20].
Among these, the antibody-mediated hot-start approach is one of the most widely used due to its short enzyme activation time, stable enzymatic activity, strong affinity, and effective inhibition [21]. The fundamental principle of antibody-mediated hot-start PCR relies on the reversible inhibition of Taq DNA polymerase through specific antigen-antibody interaction [22,23]. At room temperature, the monoclonal antibody binds with high affinity to the enzyme’s active site or an allosteric site critical for its function. This binding sterically hinders the polymerase from accessing the DNA template and dNTPs [24], effectively placing the enzyme in an “off” state and preventing non-specific primer extension and primer-dimer formation during reaction setup [25]. This inhibitory complex remains stable throughout the subsequent temperature ramp. However, antibodies are proteins that undergo irreversible denaturation at high temperatures. When the PCR reaction reaches the initial denaturation step (typically 94–95°C), the antibody, being a protein itself, denatures and loses its native structure. This structural collapse abolishes its high-affinity binding to the Taq polymerase, causing the complex to dissociate [26]. Consequently, the polymerase is released in its fully active form, ready to perform DNA synthesis during the subsequent annealing and extension steps of the thermal cycling. This process ensures that enzymatic activity is only unleashed at high temperatures, thereby conferring the “hot-start” characteristic. The core component of this method of blocking Taq polymerase activity is the monoclonal antibody with the function of blocking Taq activity through antigen-antibody binding. R. Murali et al. also reported [27] 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 center of Taq DNA polymerase.
However, the efficacy of this method is critically dependent on obtaining a monoclonal antibody that binds precisely to a functionally critical epitope. This critical dependency presents a major bottleneck, as conventional immunization with full-length Taq polymerase often fails to yield such inhibitory antibodies. Therefore, based on previous literature [28,29], this study aimed to develop a high-performance hot-start Taq by generating monoclonal antibodies with high specificity and strong blocking efficiency through a rational, domain-targeted immunization strategy. We hypothesized that immunizing with the isolated polymerase domain would better expose epitopes associated with the active site, thereby maximizing the probability of generating function-blocking antibodies.
In summary, this study seeks to produce hot-start Taq using monoclonal antibodies with high specificity and strong blocking efficiency. The resulting HS Taq is proven to be a robust reagent suitable for demanding diagnostic applications, such as pathogen detection and multiplex PCR.
Methods
Homology modeling and domain definition
Homology modeling was performed to define the structural boundaries between the N-terminal and C-terminal domains of Taq DNA polymerase for antigen design.
Homology modeling is a computational method used to predict 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 employs the MODELLER software to generate 3D protein models by identifying structurally characterized homologs and using their spatial information as templates. The procedure involves: (1) inputting a target protein sequence, (2) searching for the best structural templates with HHpred, (3) aligning the target sequence with these templates, and (4) using MODELLER to build a 3D model based on the alignment. This approach is based on the principle that proteins with high sequence similarity tend to adopt similar overall folds.
Plasmid construction, protein expression and purification
The codon-optimized DNA fragments encoding residues 1–290 and 293–832 of Taq DNA polymerase were individually cloned into the NcoI and XhoI sites of the pET-28a vector to generate the expression plasmids pET28a-Taq(1–290)-His (Taq-N) and pET28a-His-Taq(293–832) (Taq-P) (synthesized by Genewiz).
Each recombinant plasmid was transformed into E. coli BL21(DE3) (Novagen) and plated on LB agar containing kanamycin (50 µg/ml) for overnight incubation at 37°C. Positive colonies were selected and inoculated into 30 mL of kanamycin-supplemented LB medium, followed by shaking at 37°C, 200 rpm for 8 h. A 1% (v/v) inoculum was then transferred into 600 mL LB medium and cultured until OD600 reached 0.8, after which protein expression was induced with 0.2 mM IPTG at 18°C overnight. Cells were harvested by centrifugation at 8,000 × g for 30 min at 4°C, and the pellet was resuspended 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) at a 1:9 (w/v) ratio. The suspension was sonicated at 30% power for 10 min with a 50% duty cycle, followed by centrifugation 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, 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), followed by 10 CV 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, and fractions of high purity were pooled and dialyzed overnight at 4°C against 1 L dialysis buffer (25 mM Tris, pH 9.0, 150 mM NaCl).
Mice
Specific pathogen-free (SPF) female BALB/c mice (6 ~ 8 weeks old, 16 ~ 18 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Animal License No.: SCXK (Shanghai) 2017–0005). The mice used in this study were bred and maintained by the Shanghai Institute of Biological Products Technology under standardized conditions (National Standard GB 14925–2023. “Laboratory animal—Environment and housing facilities”). All animal experiments were approved by the Ethics Committee on Laboratory Animals, Shanghai Institute for Biomedical and Phamaceutical Technologios (Approval No. 2023-20). The mice were housed in a barrier facility with 2–4 animals per individually ventilated cage. All euthanasia procedures were performed in accordance with the AVMA Guidelines for the Euthanasia of Animals (2020). Mice were placed in a pre-charged transparent chamber with 30–50% CO₂ (compressed gas, 99.9% purity) at a flow rate displacing 20–30% of the chamber volume per minute. After loss of consciousness (confirmed by absence of righting reflex), the CO₂ concentration was maintained at ≥70% for ≥5 minutes to ensure respiratory arrest. Death was verified by cervical dislocation. The chamber was cleaned between uses to minimize stress from residual odors.
Mouse immunization
For the primary immunization, Taq-N protein and Taq-P protein at 500 μg/mL were each mixed with an equal volume of Freund’s complete adjuvant (Sigma) and administered via multiple subcutaneous injections along the back at 100 μg per mouse, with 3 ~ 4 mice immunized per protein. Under the same conditions, the antigens were mixed with an equal volume of Freund’s incomplete adjuvant (Sigma) for the secondary immunization, followed by a total of four booster immunizations at two-week intervals. Ten days after the final immunization, tail blood was collected from the mice, and serum antibody titers were measured by indirect ELISA, with titers required to reach at least 1:10,000. Four days before cell fusion, the mice were intraperitoneally injected with a booster immunization at 100 μg per mouse. Subsequently, splenocytes from the mice were fused with SP2/0 myeloma cells (Cell Bank, Chinese Academy of Sciences (Catalog No.: TCM18)) at a 1:10 ratio using 50% PEG1500. The cells were resuspended in 15% FBS-HAT-1640 medium and seeded into 96-well plates pre-coated with feeder cells at 100 μL per well, followed by incubation at 37°C with 5% CO2. Two weeks after fusion, 100 μL of cell culture supernatant from each well was transferred to pre-coated ELISA plates. The serum from immunized mice served as the positive control, while SP2/0 cell culture supernatant was used as the negative control. Positive hybridoma cells were screened using indirect ELISA.
Ascites preparation and antibody purification
Each BALB/c mouse was intraperitoneally injected with 250 μL of liquid paraffin for sensitization. The hybridoma cells were passaged and cultured at 37°C until reaching the logarithmic growth phase. After digestion and washing, the cells were injected into the mouse peritoneal cavity at a density of 1 × 10⁶ cells per mouse. Ascites were collected 7 ~ 10 days later when the mouse abdomen showed significant swelling.
The mouse ascites were subjected to IgG isolation using 50% and 45% ammonium sulfate precipitation, followed by affinity chromatography purification using a Protein G column (Smart-Lifesciences). The purified antibodies were dialyzed in PBS, and the OD₂₈₀ of the supernatant was measured using the NanoDrop Lite Plus spectrophotometer (Thermo Fisher). The IgG concentration was calculated using a mass extinction coefficient of 1.46 (for a 1 mg/mL solution at 280 nm with a 1 cm path length), and the purity of IgG was confirmed by reducing 4 ~ 12% gradient SDS-PAGE.
Enzyme-linked immunosorbent assay (ELISA) for the detection and validation of Taq polymerase antibody
The antibody titer was determined using an indirect ELISA method. Full-length Taq DNA polymerase protein (Yaxin Bio), Taq-N protein, and Taq-P protein were separately diluted to 5 μg/mL in sodium bicarbonate buffer (pH 9.6) and coated onto ELISA plates (100 μL/well), followed by overnight incubation at 4°C. The plates were washed three times with phosphate-buffered saline with Tween 20 (PBST) (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 h and washed three times. The purified Taq DNA polymerase-specific monoclonal antibody was serially diluted in PBST with 8 concentration gradient (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 (100 μL/well) was added as the primary antibody, with PBST alone serving as the negative control, and incubated at 37°C for 2 h, followed by three washes. Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000) (Jiangsu Cowin Biotech Co., Ltd (Catalog No.: CW0102S)) was added as the secondary antibody (100 μL/well) and incubated at 37°C for 30 min, followed by another washing step. Finally, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added per well and allowed to develop for 15 min before the reaction was stopped with 50 μL of 1N H₂SO₄. The OD₄₅₀ values were measured using a microplate reader (Bio-Rad).
Validation of monoclonal antibody blocking performance
To evaluate the blocking efficiency of monoclonal antibodies against Taq polymerase, we designed a hairpin oligonucleotide sequence, TZ (synthesized by Genewiz, The sequence of TZ: 5’-TCTAGAGGGGAATTGTTATCCGCTCACAATT CCCCTATAGTGAGTCGTATTACTATGCTAATACGACTCACTAT-3’), and diluted the lyophilized TZ powder with ddH₂O to a final concentration of 6.25 μM.
We mixed the obtained monoclonal antibodies at different ratios (0.25, 0.5, and 1 μg) with Taq DNA polymerase (10 U, 0.2 μg) and incubated the mixture at 37°C for 0.5 h for blocking. We then diluted the resulting reaction mixtures 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 a final concentration of 0.5 U/μL to prepare the experimental samples. We used commercial hot-start Taq polymerase (Takara Bio) and unblocked Taq polymerase as the positive and negative controls, respectively.
The total reaction volume (20 μL) consisted of: 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, 13.25 μL ddH₂O. Amplification was performed using a real-time PCR system (Thermo Fisher) to monitor the reaction fluorescence. The reaction protocol was as follows: 20 cycles of 70°C for 30 s; 95°C for 30 s; 20 cycles of 74°C for 30 s.
Hot-start PCR specificity assay
Using the antibody-Taq polymerase mixtures obtained from the “Validation of monoclonal antibody blocking performance” experiment as test samples, we diluted the DNA template (10 ng/μL) with ddH₂O to concentrations of 1 ng/μL and 0.1 ng/μL. The DNA template was bisulfite-converted genomic DNA prepared from SiHa cells, and the PCR amplifies a 150 bp fragment. Commercial hot-start Taq polymerase (Takara Bio) was used as the control.
The total reaction volume (20 μL) consisted of: 5 μL 4 × GPX buffer, 1.2 μL 5 μM GPX primer mix, 0.3 μL test sample, 10 μL DNA template (0.1 or 1 ng/μL), 3.5 μL ddH₂O.
Using a real-time PCR system (Thermo Fisher) with the following conditions: 94°C for 1 min; 45 cycles of 95°C for 15 s and 67°C for 30 s; followed by 95°C for 15 s and 60°C for 20 s; and finally a melting curve analysis with a gradual temperature increase from 60°C to 90°C at a rate of 0.03°C per step.
Practical application of monoclonal antibodies in the project
Fungal detection.
Following the method described in “Validation of monoclonal antibody blocking performance,” the antibody (1 μg) was mixed and incubated with Taq DNA polymerase (10 U). The resulting reaction mixture served as the test sample for Lichtheimia corymbifera detection, with a commercially available hot-start Taq enzyme used as the control.
The total reaction volume (25 μL) consisted of: 2.5 μL 10 × PCR buffer, 2 μL 2.5 mM dNTP Mix, 5 μL primer Mix, 0.625 μL Taq polymerase stock solution, 0.25 μL UDG, 0.125 μL test sample, 5 μL Lichtheimia corymbifera DNA (Shanghai GeneoDx Biotech Co., Ltd.), Nuclease-free ddH₂O to adjust the final volume to 25 μL.
Using a real-time PCR system (Thermo Fisher) under the following conditions: 37°C for 2 min; 95°C for 2 min; 40 cycles of 94°C for 15 sec and 58°C for 60 sec.
To quantitatively evaluate amplification efficiency and consistency, the Cycle threshold (Ct) values of eight parallel repeated reactions for the test samples and commercial controls were measured. The mean Ct value and Relative Standard Deviation (RSD) were calculated for each group. PCR reactions were performed using the above reaction components and procedure.
Furthermore, the analytical sensitivity of the prepared HS Taq polymerase was evaluated and compared against a commercial hot-start Taq enzyme (Takara Bio) using a Lichtheimia corymbifera DNA detection assay. Serial dilutions of the target DNA template were prepared in nuclease-free water to final concentrations of 300 copies/mL and 75 copies/mL. For each concentration and each polymerase, sixteen (n = 16) independent replicate PCR reactions were performed using the above reaction components and procedure.
Multiplex detection of respiratory viruses.
Following the method described in “Validation of monoclonal antibody blocking performance,” the antibody (1 μg) was mixed and incubated with Taq DNA polymerase (10 U). The reaction mixture was used as the experimental sample for multiplex detection of respiratory viruses, with a commercial hot-start Taq enzyme serving as the control.
The total reaction volume (25 μL) consisted of: 2.5 μL of 10 × PCR buffer, 2 μL of 2.5 mM dNTP Mix, 5 μL of multiplex primer Mix, 0.1 μL of the experimental sample, 5 μL of H1N1 DNA/PIV-1 DNA/PIV-3 DNA (Shanghai GeneoDx Biotech Co., Ltd.), Nuclease-free water to adjust the final volume.
Using a real-time PCR system (Thermo Fisher) under the following conditions: 95°C for 2 min; 10 cycles of 94°C for 15 s and 55°C for 15 s and 72°C for 15 s; 23 cycles of 94°C for 15 s and 50°C for 15 s and 72°C for 15 s; 95°C for 2 min; 40°C for 90 s; then a melting curve analysis with a gradual temperature increase from 40°C to 90°C at 1 °C/step; 37°C for 1 s.
Results
Homology modeling reveals two distinct structural domains of Taq DNA polymerase
Using the MODELLER program implemented in the HHpred web server, the full-length Taq DNA polymerase sequence (green bar, residues 1–832) was aligned with the most homologous structural templates (shown from top to bottom with their corresponding PDB IDs in red boxes; Fig 1). The alignment reveals a clear discontinuity between residues 290 and 293, indicated by two vertical black lines, which likely represents a 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: an N-terminal region (residues 1–290; termed Taq-N) corresponding to the exonuclease domain, and a C-terminal region (residues 293–832; termed Taq-P) containing the polymerase catalytic domain. Model construction was guided by spatial restraints derived from the sequence alignment with the selected templates, under the assumption that proteins with high sequence similarity generally adopt similar overall folds.
The full-length Taq DNA polymerase sequence (green bar, residues 1-832) was analyzed using the MODELLER program. The most homologous structural templates are aligned from top to bottom, with their corresponding PDB IDs shown in red boxes. A clear discontinuity between residues 290 and 293 (indicating by two vertical black lines) marks the structural boundary separating the N-terminal exonuclease domain (residues 1-290) and the C-terminal polymerase domain (residues 293-832).
Protein expression and purification
The purified Taq-N and Taq-P proteins were analyzed by 4–12% gradient SDS-PAGE. Distinct bands corresponding to the target proteins were observed at the expected molecular weights: approximately 60 kDa for Taq-P and 33 kDa for Taq-N. Quantitative analysis using ImageJ software indicated that the purity of the recombinant Taq-P protein exceeded 90% and the purity of the Taq-N protein reached 80% (Fig 2). The original, uncropped gel images for Fig 2 are available in S1 Text.
The purities of Taq-P and Taq-N were analyzed by reducing 4-12% gradient SDS-PAGE, and both proteins were used as antigens for mouse immunization. Lane M: Molecular weight marker; Taq-P protein: ~ 60 kDa; Taq-N protein: ~ 33 kDa.
Preparation and purification of monoclonal antibodies
To generate monoclonal antibodies against Taq DNA polymerase domains, BALB/c mice were immunized with purified Taq-N protein or Taq-P protein. Following cell fusion and screening by ELISA, three positive hybridoma cell lines were finally obtained: IgG Taq-N-A7, IgG Taq-P-B3 and IgG Taq-P-D4. The ascites of the above three cell lines were prepared and purified by Protein G affinity chromatography. Purified IgG was evaluated under reducing conditions using 4–20% gradient SDS-PAGE. The results showed that the purification effect was good, and the antibody protein contained two distinct bands, one at 55 kDa corresponding to the heavy chain and the other at 25 kDa corresponding to the light chain (Fig 3). The original, uncropped gel image for Fig 3 is available in S1 Text. The presence of sharp bands at these expected sizes with minimal background confirms the high purity of the purified monoclonal antibodies.
Antibodies were analyzed by reducing 4-12% gradient SDS-PAGE. Lane M: Molecular weight marker; Lane 1: IgG Taq-N-A7; Lane 2: IgG Taq-P-B3; Lane 3: IgG Taq-P-D4. The observed bands at approximately 55 kDa and 25 kDa correspond to the immunoglobulin heavy chain (HC) and light chain (LC), respectively.
Antibody specificity
The titers of the three purified IgG antibodies were determined by indirect ELISA. The full-length Taq DNA polymerase protein-coated plates were used for detection, and 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 (Fig 4A). When using Taq-N protein-coated plates, the titers of IgG Taq-N-A7, IgG Taq-P-B3, and IgG Taq-P-D4 were greater than 640,000, and the latter two were close to the negative control (Fig 4B). When using Taq-P protein-coated plates, the titers of IgG Taq-N-A7 was close to the negative control, while those of IgG Taq-P-B3 and IgG Taq-P-D4 were approximately 1,280,000(Fig 4C). The original OD values from the ELISA assays shown in Fig 4 are provided in S1 File. Results confirm successful generation of domain-specific monoclonal antibodies.
(A) Titers against full-length Taq DNA polymerase. (B) Titers against the Taq-N protein fragment. (C) Titers against the Taq-P protein fragment. PBST was used as the negative control. The results demonstrate that IgG Taq-N-A7 specifically binds to the Taq-N fragment, while IgG Taq-P-B3 and IgG Taq-P-D4 are specific for the Taq-P fragment.
Detection of IgG blocking performance
For the detection of antibody blocking performance, this study designed a hairpin oligonucleotide sequence TZ as a template and performed real-time monitoring using a fluorescence PCR instrument. The fluorescence increase rate was directly proportional to the DNA template amplification efficiency. As shown in Fig 5, the curve trend of the positive control indicated that the first stage at 70°C (cycle 1 ~ 20) exhibited a flat curve, meaning no change in fluorescence intensity, suggesting that the enzyme activity was completely blocked and the template was not amplified. The second stage (cycle 21) involved antibody denaturation at 95°C and dissociation from the Taq enzyme. The third stage (cycle 21 ~ 40) was the template amplification phase, where Taq enzyme activity was restored at 74°C, and the template was continuously amplified. In contrast, the negative control curve showed a continuous rise starting from cycle 1, with fluorescence intensity increasing alongside template amplification.
(A-C) PCR amplification curves obtained with Taq polymerase pre-incubated 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. The reactions used a hairpin oligonucleotide (TZ) as template. Commercial hot-start Taq polymerase and unblocked Taq polymerase were used as positive and negative controls, respectively. The flat baseline during the initial 20 cycles at 70°C in (A) indicates complete enzyme inhibition by IgG Taq-P-B3 at 0.5 and 1 μg, while its activity was restored upon heating to 95°C. In contrast, IgG Taq-N-A7 and IgG Taq-P-D4 showed no significant blocking effect at any concentration tested.
The results (Fig 5A) demonstrated that HS Taq prepared by mixing IgG Taq-P-B3 at ratios of 0.5 μg and 1 μg with Taq enzyme (10 U, 0.2 μg) effectively blocked Taq enzyme activity, as evidenced by a flat fluorescence curve during the initial 20 cycles that was indistinguishable from the commercial hot-start Taq positive control. This indicates that the blocking efficiency of our antibody at these concentrations is complete and comparable to the established commercial standard. However, at 0.25 μg IgG, Taq polymerase activity was not completely blocked, resulting in a premature rise in the amplification curve, similar to the unblocked Taq negative control. This defines the minimum effective antibody-to-enzyme ratio for full inhibition. In contrast, IgG Taq-N-A7 and IgG Taq-P-D4 failed to block Taq enzyme activity at all three ratios (0.25, 0.5, and 1 μg) (Figs 5B and 5C), as their amplification profiles were virtually superimposable with the negative control, unequivocally confirming their lack of inhibitory function.
Hot-start PCR specificity assay
For the specificity detection of hot-start PCR, this study achieved it through qPCR experiments. The results showed (Fig 6) that the HS Taq enzymes prepared by mixing and incubating IgG Taq P-B3 (1 μg and 0.5 μg) with Taq enzyme (10 U, 0.2 μg) produced melting curves with a single unique peak between 80 ~ 90°C, which was identical in shape and Tm to the peak produced by the commercial hot-start Taq control. The absence of non-specific peaks before 80°C in both the experimental and control groups confirms that our HS Taq achieves a level of specificity equivalent to the commercial product. However, when IgG was 0.25 μg, non-specific product peaks appeared between 74 ~ 80°C, and the peak height between 80 ~ 90°C was lower compared to the other two concentrations. This indicates that the HS Taq prepared with IgG Taq P-B3 at ratios of (1 μg and 0.5 μg) can effectively inhibit non-specific amplification during the heating process of qPCR reactions. This indicates that an insufficient proportion of antibodies can lead to incomplete inhibition of Taq polymerase activity.
(A, B) Melting curves (A1, B1) and amplification curves (A2, B2) from qPCR 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 served as the control.
Additionally, this study compared the prepared HS Taq with a commercial hot-start Taq by amplifying different template concentrations (1 ng/μL and 0.1 ng/μL). The results (Fig 6) showed that distinct amplification was achieved for all template concentrations, demonstrating that this hot-start PCR system exhibits high sensitivity. The single peak in the melting curves and the robust amplification across dilutions indicate that the prepared HS Taq effectively inhibits non-specific amplification and exhibits high specificity comparable to the commercial product.
Application in medical laboratory testing systems
Lichtheimia corymbifera detection system.
For the Lichtheimia corymbifera detection system, the results (Fig 7) showed nearly identical Ct values and amplification curve profiles between the experimental group (IgG Taq P-B3) and the commercial control group. The nearly identical Ct values and curve profiles between the experimental and control groups demonstrate that the antibody-blocked Taq polymerase performs effectively in diagnostic applications without compromising detection efficiency.
Amplification curve analysis for the detection of Lichtheimia corymbifera using (A) the experimental HS Taq (blocked by IgG Taq-P-B3) and (B) a commercial hot-start Taq enzyme (control). The Ct values and curve profiles between the experimental and control groups are nearly identical.
Both the experimental HS Taq and the commercial hot-start Taq DNA polymerase were tested simultaneously, with eight parallel replicates set up for each group. The results (Table 1) showed that the average Ct value obtained from amplification with HS Taq was 32.65, while the average Ct value for the control group was 32.55. The RSD of the Ct values amplified by the HS Taq was 0.82%, compared to 0.99% for the control group. Statistical analysis confirmed no significant difference in performance between the two enzymes (t(14) = 0.697, p = 0.497), with a small effect size (Cohen’s d = 0.34), demonstrating comparable amplification efficiency and reproducibility.
Furthermore, the sensitivity of the HS Taq was evaluated using low template concentrations (75 and 300 copies/mL). As illustrated in Fig 8, at 300 copies/mL, the detection rate of our HS Taq (16/16) was higher than that of the commercial control (12/16), though 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 the commercial enzyme (5/16) (p = 0.026). These results demonstrate that the prepared HS Taq demonstrated a higher detection rate than the commercial enzyme at both concentrations, indicating superior sensitivity for low-abundance targets.
Detection rates for Lichtheimia corymbifera at template concentrations of 300 copies/mL (A, B) and 75 copies/mL (C, D) are shown. (A, C) Results using a commercial hot-start Taq enzyme. (B, D) Results using the experimental HS Taq prepared with IgG Taq-P-B3. Each panel shows the number of positive detections out of 16 replicate reactions (e.g., 12/16).
Multiplex detection system.
In the multiplex detection system of respiratory viruses, the results demonstrated that in both the H1N1 (Fig 9A) and PIV-1/PIV-3 (Fig 9B) systems, the experimental group (IgG Taq P-B3) showed no significant differences in Tm values or peak heights compared to the control group. Only specific target product peaks were observed in all cases. Only specific target product peaks were observed in all cases. The concordance in melting peak profiles between our HS Taq and the commercial control across multiple targets confirms that the prepared HS Taq is suitable for specific multiplex pathogen detection.
(A) Melting curves for H1N1 detection (Peak 1) and (B) for simultaneous detection of PIV-1 (Peak 1) and PIV-3 (Peak 2). The experimental group (A1, B1) used HS Taq prepared with IgG Taq-P-B3, while the control group (A2, B2) used a commercial hot-start Taq enzyme. The absence of non-specific peaks and the concordance in Tm values between experimental and control groups.
Discussion
Non-specific amplification remains a major challenge in conventional PCR, largely stemming from residual Taq polymerase activity at room temperature. While multiple hot-start methods exist, the antibody-based approach offers distinct practical advantages in performance, usability, and cost-effectiveness. Specifically, compared to common alternatives: it eliminates the prolonged activation time and enzyme degradation associated with chemical or mutational methods; it simplifies workflows and reduces contamination risks relative to physical barrier systems; and it provides superior blocking stability and shelf-life compared to aptamers—key factors enhancing reliability and commercial viability for diagnostic applications. The core mechanism involves reversible, temperature-dependent inhibitor: at room temperature, the antibody sterically blocks the active site or prevents critical conformational changes; during initial PCR denaturation, antibody denaturation irreversibly dissociates the complex, fully restoring polymerase activity [23]. However, a critical yet often overlooked limitation is the absolute reliance on antibodies that target functionally critical epitopes.
Our initial failed attempts to generate inhibitory antibodies using the full-length Taq polymerase highlighted this limitation and directly supported our hypothesis that critical epitopes may be conformationally masked. This challenge drove our methodological innovation: a structure-guided, domain-specific immunization strategy designed to overcome the poor immunogenicity of catalytic epitopes in the full-length enzyme. Homology modeling (Fig 1) indicates residues 175−261 contribute a fragment of the 5’-3’ exonuclease domain, distinct from the DNA polymerase active region, suggesting potential epitope overlap between Taq-N and Taq-P domains. We reasoned that using the isolated Taq-P domain as immunogen would better expose active-site associated epitopes, thereby increasing the likelihood of generating function-blocking antibodies [30]. Consequently, we constructed and purified both Taq-N and Taq-P domain proteins for antibody generation and screening.
This domain-specific strategy proved pivotal. We successfully generated three monoclonal antibodies: IgG Taq-N-A7 (Taq-N-specific), and IgG Taq-P-B3 and IgG Taq-P-D4 (both Taq-P-specific). Functional characterization revealed a critical distinction: while both Taq-P antibodies exhibited high ELISA affinity, only IgG Taq-P-B3 effectively inhibited the polymerase activity. This stark functional divergence underscores a fundamental principle in antibody development: high-affinity binding to a catalytic domain does not guarantee inhibiion—epitope localization is decisive [31,32]. We propose IgG Taq-P-B3 targets an epitope at or near the enzyme’s active site, likely obstructing substrate (DNA and dNTP) access or interfering with catalytic conformational dynamics, consistent with prior structural studies [12]. In contrast, IgG Taq-P-D4 or IgG Taq-N-A7 likely bind surface epitopes distal to functional regions. Though non-inhibitory, their specific binding validates our screening process while emphasizing the necessity of coupling binding assays with functional screens to identify true inhibitors [33]. This study confirms that HS Taq (prepared with IgG Taq-P-B3) demonstrates high specificity and sensitivity comparable to commercial products in inhibiting non-specific amplification.
We further evaluated HS Taq polymerase (prepared with IgG Taq-P-B3) in clinical diagnostic settings. In fungal pathogen detection assays, head-to-head comparisons with commercial HS Taq showed no significant differences in amplification curves or Ct values. Additionally, the experimental HS Taq demonstrated strong reproducibility and superior sensitivity for low-abundance targets. Similarly, in single-tube multiplex detection of respiratory pathogens (H1N1, PIV-1, PIV-2), our HS Taq exhibited comparable melting curve Tm values, peak heights, and absence of non-specific products relative to commercial controls. Consistent Ct values, melting curve profiles, and lack of non-specific amplification across these applications confirm the reagent’s suitability for molecular diagnostics.
Conclusion
The principal innovation of this study lies in the structure-guided, domain-specific immunization strategy. Unlike traditional methods that use full-length Taq polymerase as an immunogen—an approach that often fails to generate functional blocking antibodies due to inaccessible “cryptic epitopes”—this strategy significantly enhanced exposure of epitopes associated with the active site. Based on this, we not only produced a highly specific inhibitory monoclonal antibody, which served as the core component of a high-performance hot-start PCR reagent but also validated a fundamental principle: for functional antibodies, epitope location is a more critical determinant of efficacy than binding affinity alone. Our work establishes a novel, generalizable paradigm for core diagnostic reagent development, provides a key raw material for genetic testing and molecular diagnosis, and contributes a valuable methodological framework for advancing enzyme engineering and biotechnology reagent development. Future work will include quantitative determination of the antibody’s binding affinity to further elucidate the mechanistic basis of inhibition.
Supporting information
S1 Fig. The domains of Taq DNA polymerase (Adapted from UniProt ID: P19821).
The protein contains two domains: one is 5′–3′ exonuclease domain (purple) at N-terminal; the other is DNA polymerase domain (orange) at C-terminal.
https://doi.org/10.1371/journal.pone.0345402.s001
(TIF)
S2 Fig. Crystal structure of full-length Taq DNA polymerase (residues 1–832, adapted and modified from PDB ID: 1TAQ).
The 5′–3′ exonuclease domain is colored in green (1–292 aa) while the DNA polymerase domain is colored in orange (293–832 aa).
https://doi.org/10.1371/journal.pone.0345402.s002
(TIF)
S3 Fig. Crystal structure of the DNA polymerase domain of Taq DNA polymerase (residues 293–832, adapted and modified from PDB ID: 1JXE).
The positions of the starting and ending amino acid residue of the polymerase domain are labeled.
https://doi.org/10.1371/journal.pone.0345402.s003
(TIF)
S4 Fig. Crystal structure of the 5’-3’ exonuclease domain of Taq DNA polymerase (residues 1–292, adapted and modified from PDB ID: 1TAQ).
The positions of the starting and ending amino acid residue of the exonuclease domain are labeled.
https://doi.org/10.1371/journal.pone.0345402.s004
(TIF)
S1 Text. Raw Images.
Original, uncropped gel images for all figures. This file contains the original, uncropped gel images corresponding to the main figures in the manuscript. (S1 raw Fig) Purification of Taq-P protein (related to Fig 2). (S2 raw Fig) Purification of Taq-N protein (related to Fig 2). (S3 raw Fig) Purification of monoclonal antibodies (related to Fig 3).
https://doi.org/10.1371/journal.pone.0345402.s005
(PDF)
S1 File. Raw ELISA data underlying Fig 4(A, B, C).
This spreadsheet contains the raw optical density (OD450) values from the indirect ELISA used to determine the titers of monoclonal antibodies IgG Taq-N-A7, IgG Taq-P-B3, and IgG Taq-P-D4 against the full-length Taq polymerase and its isolated domains (Taq-N and Taq-P), as summarized in Fig 4.
https://doi.org/10.1371/journal.pone.0345402.s006
(XLSX)
Acknowledgments
We acknowledge East China University of Science and Technology and Shanghai Yaxin Biotechnology Co., Ltd. (a special thank-you goes to Dr. Chen guan-ming) for providing helpful suggestions.
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