Generation and Characterization of Monoclonal Antibodies Against Tth DNA Polymerase and its Application to Hot-Start PCR

Abstract

Background: As a heat-resistant polymerase, Thermus thermophilus (Tth) DNA polymerase can be widely used in Polymerase Chain Reaction (PCR). However, its non-specific amplification phenomenon is serious, which greatly limits development. Objective: In this study, we prepared Tth monoclonal antibodies against Tth DNA polymerase and researched their application in hot-start PCR. Methods: Tth was recombinantly expressed and purified, and used as an antigen to immunize BALB/c mice to obtain monoclonal antibodies. The qualified monoclonal antibody and Tth are incubated for a period of time at a certain temperature to obtain the hot-start Tth. We tested the polymerase activity and exonuclease activity blocking performance of hot-start Tth. Finally, the hot start Tth was applied to one-step RT-PCR. Results: Tth with a purity of >95% was obtained, and ten monoclonal antibodies were obtained by immunization. After incubation, there were three monoclonal antibodies identified that could inhibit the polymerase activity of Tth at low temperature. Furthermore, these three antibodies have successfully eliminated non-specific amplification in practical applications. Conclusion: Three monoclonal antibodies were successfully validated. Among them, monoclonal antibody 9 has the best overall effect. They have the function of inhibiting at low temperature and releasing at high temperature, which can be used as Tth polymerase inhibitors in the field of molecular diagnostics.

Simulation Study on the Impact of South–North Water Transfer Central Line Recharge on the Water Environment of Bai River

“…The initial conditions for the MIKE21 hydrodynamic model include water level and flow velocity in the river, with different initial values used for different spatial locations. In addition, this simulation also uses a hot start, which means that the results of the previous simulation are used as the initial conditions for the next simulation, and this iterative algorithm helps to improve the speed and accuracy of the model's operations [26].…”

“…MIKE21 hydrodynamic model include water level and flow velocity in the river, wi ferent initial values used for different spatial locations. In addition, this simulatio uses a hot start, which means that the results of the previous simulation are used initial conditions for the next simulation, and this iterative algorithm helps to impro speed and accuracy of the model's operations [26]. erbed roughness at the center of the river channel is limited to 0.025~0.04 [27]; the p itation and evaporation time series and wind field conditions were adopted from the yang meteorological station in 2020.…”