Title:Generation and Characterization of Monoclonal Antibodies Against Tth DNA Polymerase and its Application to Hot-Start PCR
Volume: 28
Issue: 10
Author(s): Yuting Tang, Xiaoyu Chen, Jian Zhang, Jin Wang, Wenhao Hu, Songbai Liu, Zhidan Luo*Henghao Xu*
Affiliation:
- Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005,China
- Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005,China
Keywords:
Tth DNA polymerase, monoclonal antibody, hot-start, PCR, qPCR, inhibitor.
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 were 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 the 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, three monoclonal antibodies were identified that could inhibit the
polymerase activity of Tth at low temperature. Furthermore, these three antibodies successfully
eliminated non-specific amplification in practical applications.
Conclusion: Three monoclonal antibodies were successfully validated. Among them, monoclonal
antibody 9 had the best overall effect. They possess 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.
