Taq enzyme antibody
PCR (Polymerase Chain Reaction) technology is the most mature and widely used molecular diagnostic technology in clinical applications. Taq DNA polymerase antibodies are essential materials for hot-start PCR detection, with a huge demand. Xiamen Tongrenxin Biotechnology Co., Ltd (hereinafter referred to as "Tongrenxin") has independently developed a dual-closed hot-start Taq DNA polymerase antibody (patent number: ZL 2021 1 0548532.9). By simultaneously closing the polymerase activity and 5'-3' exonuclease activity of Taq enzyme, it effectively improves the specificity of amplification and the stability of PCR reagents, and has a broad binding capacity for different types of Taq enzymes. During the pandemic, Tongrenxin's Taq DNA polymerase antibody has sold a total of 1,600,000 mg, capable of producing over 1.2 billion COVID-19 test kits, with a market share of up to 20%, outperforming similar products.
(1) Supports the preparation of fully premixed reagents, effectively improving the stability of PCR reagents.
The Taq DNA polymerase antibody from Tongrenxin and two other commercial Taq DNA polymerase antibodies were used to block Taq DNA polymerase separately, forming a fully premixed solution containing primers and probes. The stability test conditions were set as follows: 1) placed at 4°C for 3, 5, and 7 days; 2) placed at 37°C for 3, 5, and 7 days. The same PCR program was run to observe the changes in amplification curves and Ct values.
The stability test results are detailed in Figure 1 (4°C) and Figure 2 (37°C). It can be seen that the double-blocking antibody of Tongrenxin Taq DNA polymerase has superior stability compared to the other two competing products, with no significant changes in amplification curves and Ct values under different temperatures and storage times.
(2) Effectively blocks the activity of Taq DNA polymerase and can quickly release enzyme activity.
The Taq DNA polymerase antibody from Tongrenxin and two other commercial Taq DNA polymerase antibodies were mixed with Taq DNA polymerase in proportion, and the enzyme activity was tested according to the method in Appendix A of "GB/T 35542-2017 Taq DNA Polymerase" to calculate the blocking efficiency of the Tongrenxin Taq DNA polymerase antibody at different temperatures (55°C, 65°C).
As shown in Figure 3, the affinity of the Tongrenxin Taq DNA polymerase antibody for Taq enzyme is very high. At 65°C, the Tongrenxin Taq DNA polymerase antibody can still block the activity of Taq enzyme, thus effectively inhibiting primer dimerization and non-specific amplification.
Using the Tongrenxin Taq DNA polymerase antibody to block Taq enzyme and heat-shocking at 95°C for 30 seconds, the activity of Taq DNA polymerase was detected (refer to the method in Appendix A of "GB/T 35542-2017 Taq DNA Polymerase"). It can be seen that after heat-shocking at 95°C for 30 seconds, the Tongrenxin Taq DNA polymerase antibody can fully release the blocking, releasing 98% of the enzyme activity.
(3) Effectively blocks the exonuclease activity of Taq DNA polymerase.
The Taq DNA polymerase antibody from Tongrenxin and two other commercial Taq DNA polymerase antibodies were mixed with Taq DNA polymerase in proportion to form a fully premixed solution containing primers and probes, placed at 37°C for 0, 3, 5, and 7 days, and the changes in fluorescence signals in the system were observed.
As shown in Figure 4, the double-blocking antibody from Tongrenxin can effectively block the 5'-3' exonuclease activity of Taq DNA polymerase.
(4) Can block various types of Taq enzymes.
Using the Tongrenxin Taq DNA polymerase antibody to block three mainstream Taq DNA polymerases from the market, with a blocking ratio of 1mg:4000U, fluorescence values and blocking efficiency were measured (refer to the method in Appendix A of "GB/T 35542-2017 Taq DNA Polymerase"). It can be seen that the Tongrenxin Taq DNA polymerase antibody can effectively block the activity of mainstream Taq enzymes on the market.
(5) High purity, no residual nucleic acid exonuclease or endonuclease.
According to the methods in Appendices B and C of "GB/T 35542-2017 Taq DNA Polymerase", the residual nucleic acid exonuclease and endonuclease of the Tongrenxin Taq DNA polymerase antibody were tested. The test results (Figure 5) indicate that there is no residual nucleic acid exonuclease or endonuclease in the Tongrenxin Taq DNA polymerase antibody; Tris-gly-SDS polyacrylamide gel electrophoresis of the Tongrenxin Taq DNA polymerase antibody shows that the antibody is of high purity.
(6) External evaluation of clinical samples.
This set of data comes from a "F*" customer, who has been engaged in molecular pathology research and development for many years. The laboratory used the Tongrenxin Taq DNA polymerase antibody for evaluation, and the evaluation results are detailed in Figure 6. The results show that under low copy template conditions (100 copies, 10 copies), the Tongrenxin Taq DNA polymerase antibody group performed well in both the FAM and ROX channels, with good amplification efficiency and linearity of the amplification curves.
