Fig 1.
Identification of structural templates and domain boundaries of Taq DNA polymerase.
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).
Fig 2.
Purification analysis of Taq-P and Taq-N proteins.
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.
Fig 3.
Purification analysis of 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.
Fig 4.
Determination of monoclonal antibody titers by indirect ELISA.
(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.
Fig 5.
Evaluation of Taq polymerase blocking by monoclonal antibodies using qPCR.
(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.
Fig 6.
Specificity analysis of hot-start PCR by qPCR.
(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.
Fig 7.
Performance validation in a fungal detection assay.
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.
Table 1.
Comparison of Ct values and reproducibility between the experimental and commercial hot-start Taq polymerases in the Lichtheimia corymbifera detection assay.
Fig 8.
Comparative sensitivity analysis of hot-start Taq enzymes at low template concentrations.
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).
Fig 9.
Performance validation in a multiplex respiratory virus detection system.
(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.