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.

背景：嗜热栖热菌（Tth）DNA聚合酶作为一种耐热聚合酶，可广泛应用于聚合酶链式反应（PCR）。但其非特异性扩增现象严重，极大限制了其发展。

目的：本研究制备抗Tth DNA聚合酶的Tth单克隆抗体，并研究其在热启动PCR中的应用。

方法：重组表达并纯化Tth，作为抗原免疫BALB/c小鼠，获得单克隆抗体。将合格的单克隆抗体与Tth在一定温度下孵育一段时间，得到热启动Tth。我们测试了聚合酶活性和核酸外切酶活性阻断热启动 Tth 的性能。最后，将热启动Tth应用于一步RT-PCR。

结果：获得纯度>95%的Tth，免疫获得10株单克隆抗体。孵育后，鉴定出三种单克隆抗体可以在低温下抑制Tth的聚合酶活性。此外，这三种抗体在实际应用中成功消除了非特异性扩增。

结论：三种单克隆抗体已成功验证。其中单克隆抗体9总体效果最好。它们具有低温抑制、高温释放的功能，可作为分子诊断领域的Tth聚合酶抑制剂。