Background

The perfect combination of Taq DNA polymerase and PCR technology has made PCR a practical tool, widely applied not only in scientific research, but also in pathogen detection, genetic testing, companion diagnostics for cancer, as well as forensic science, environmental monitoring, and food safety.   As the technology advances, PCR is evolving toward greater convenience, sensitivity, and cost-efficiency.   To meet the demand for efficiency and convenience, fully premixed PCR reagents are gradually replacing stepwise reagents and becoming mainstream. During large-scale outbreaks such as COVID-19 and African swine fever, to ensure accurate results, testing organizations have placed increasing emphasis on the stability of premixed reagents.

Antibody-based hot-start modified Taq DNA polymerase, as a mature commercial product, has been widely adopted in practical applications for its ability to reduce non-specific amplification in PCR.   However, in conventional probe-based detection systems, the 5’→3’ exonuclease activity of Taq polymerase can degrade critical components like primers and probes, reducing amplification efficiency and compromising reagent stability. With increasing market competition and the push for international expansion, reagent manufacturers are compelled to further improve the stability of their products, requiring that fully premixed reagents maintain excellent performance after being stressed for 14 days at 4°C or 7 days at 37°C.

As a result, companies both domestically and internationally have continued to explore and develop more efficient Taq enzyme antibodies, aiming to fully block both the 5’→3’ polymerase activity and 5’→3’ exonuclease activity of Taq polymerase. Since the COVID-19 outbreak, various manufacturers have launched dual-function blocking antibody products, but none are ideal. Either the blocking efficiency for both 5’→3’ polymerase and exonuclease activity fail to exceed 90% simultaneously, or the antibody dissociates from Taq enzyme above 55°C, losing its blocking function and compromising the stability of premixed reagents.

To discover a more ideal Taq antibody and further enhance the performance of PCR reagents, Hzymes employed B cell cloning technology, sorting magnetically enriched B cells using the Beacon single-cell optofluidic system to isolate those that meet screening criteria, then amplifying cDNA by PCR to construct linear expression cassettes for transient transfection, and verifying expression supernatants via ELISA screening. The selected antibody genes were then sequenced and validated for binding affinity and blocking function through plasmid-based expression.   During the process of identifying and screening the blocking effects of different antibodies and antibody combinations,   the Hzymes R& D team was surprised to discover a certain degree of synergy among different Taq antibodies.

It is generally believed that selecting two highly specific antibodies—one blocking 5’→3’ polymerase activity and the other 5’→3’ exonuclease activity—and mixing them in suitable proportions would achieve full dual-function blocking of Taq enzyme. However, practical results have not been entirely satisfactory.   Yet, when we added a third antibody to a mixture of two antibodies that partially but not completely block polymerase and exonuclease activity respectively,   the dual-function blocking efficiency of Taq enzyme was significantly improved.

Figure 1. Schematic of Hzymes’ fully-blocking Taq antibody mechanism (gray: 5’→3’ exonuclease domain; cyan: 5’→3’ polymerase domain; yellow: 3’→5’ exonuclease domain).

The fully-blocking Taq antibody developed by Hzymes was obtained through extensive screening and consists of a combination of three antibodies in an optimized ratio (Figure 1). Compared with commercial products using single or dual antibody combinations for Taq dual-function blocking, this product can simultaneously block over 99% of both 5’→3’ polymerase and 5’→3’ exonuclease activity of Taq enzyme at 70°C. It offers higher blocking efficiency and greater stability.   It prevents non-specific amplification caused by mismatched templates or primer-dimers,   and avoids degradation of probes or other critical materials, which can lead to non-specific signals and reduced amplification efficiency,   Additionally, it enables rapid enzyme activation, releasing over 95% activity with a 30-second heat shock at 95°C, ensuring high amplification sensitivity.

Superior Product Performance

Fully Blocked

Simultaneously and completely blocks both polymerase activity and 5’→3’ exonuclease activity, with blocking efficiency exceeding 99%.

In assays measuring 5’→3’ exonuclease activity and 5’→3’ polymerase activity, Hzymes’ fully blocking Taq DNA polymerase antibody can fully inhibit both activities at 70°C. The blocking efficiency not only surpasses conventional antibodies (Figure 2 Supplier T antibody) but also significantly exceeds that of commercially available dual-function blocking antibodies for Taq polymerase and exonuclease activities (Figure 2 Supplier V and Supplier Y antibodies).

Product Description

Whole-Block Taq antibody V1 is a meticulously developed monoclonal antibody cocktail reagent by our company. This reagent strictly blocks both the 5’→3’ polymerase activity and the 5’→3’ exonuclease activity of Taq DNA polymerase. An upgrade to the Dual-Block Taq antibodyV1 (HMD0311) has resulted in the Whole-Block Taq antibody V1. This new product is composed of three antibodies in an optimized ratio. It exhibits higher blocking efficiency and stronger blocking stability towards the 5’→3’ polymerase activity and 5’→3’ exonuclease activity of the Taq, making it suitable for multiplex primer-probe fully pre-mixed reagents. It prevents nonspecific amplification caused by mismatched primers, while also avoiding nonspecific signals and loss of amplification efficiency due to degradation of primers, probes, or other critical reagents. In this way, it doubly ensures the stability of amplification reagents. This product can fully release the activities of Taq DNA polymerase after heating at 95°C for only 30 seconds. Using this product to block Taq DNA polymerase significantly enhances the stability of fully pre-mixed reagents and the detection rate of low templates.

Figure 2. Efficiency of Hzymes fully blocking Taq antibody against Taq polymerase 5’→3’ exonuclease activity and 5’→3’ polymerase activity.

Performance Data

Wide Compatibility

Compatible with different types of Taq DNA polymerase, with blocking efficiency over 99%.

The Taq antibody binds to Taq enzyme through specific antigen epitopes, and mutations in Taq enzyme that occur at these epitope sites may reduce antibody affinity. Hzymes’ fully blocking Taq DNA polymerase antibody maintains high affinity for different types of Taq enzymes (wild-type and mutants), and effectively blocks all types with efficiency greater than 99%.

Figure 3. Blocking efficiency of Hzymes fully blocking Taq antibody on 5’→3’ exonuclease and 5’→3’ polymerase activities of different Taq enzyme types.

Fast Activation

Releases over 95% enzyme activity within 30 seconds of heat activation, ensuring high amplification efficiency.   Compared to chemical modifications, antibody-blocked Taq enzyme offers faster enzyme activation without damaging the enzyme.   Hzymes’ fully blocking antibody releases more than 95% enzyme activity after 30 seconds heat activation at 95°C,   maintaining activation efficiency comparable to Supplier T antibody, despite significantly higher blocking efficiency.

Figure 4. Enzyme activity release rates of Hzymes fully blocking Taq antibody versus Supplier T antibody.

Strict Quality Control

Strict quality control, quality assurance.

Hzymes strictly controls antibody product quality, in addition to routine tests such as concentration, purity, blocking efficiency, and functional assays, focuses on detecting residual nucleases and host nucleic acids to ensure no contamination of downstream reagents.

Customer Cases

The hot-start Taq enzyme modified with Hzymes' fully blocking antibody has helped customers optimize and solve various challenges across different applications, with the following specific case studies illustrating its effectiveness.

1.   Exceptional Stability in Fully Premixed Reagents

Compared with similar dual-function blocking antibodies on the market,   Hzymes’ fully blocking Taq antibody can block both 5’→3’ polymerase and exonuclease activity simultaneously at higher temperatures,   with superior blocking efficiency and enhanced stability. It effectively prevents degradation of key components such as primers and probes, resulting in greater reagent stability.

Figure 5. Stability testing of Hzymes’ fully blocking antibody-modified Taq enzyme premixed with multiplex primers and probes (bacterial gene amplification).

Taq enzymes modified with Hzymes' fully blocking antibody and with a competing dual-function antibody (Supplier V) were premixed with multiplex primers and probes, and subjected to 28 days of accelerated stress at 37°C.   Premixed reagents stored at -20°C were used as controls and tested in parallel.   Results showed that the premixed reagents containing Hzymes’ fully blocking antibody exhibited no significant difference in performance between stressed and control conditions.   In contrast, Supplier V’s antibody-modified reagents showed a significant drop in amplification efficiency after stress treatment. In contrast, Supplier V’s antibody-modified reagents showed a significant drop in amplification efficiency after stress treatment.

2. Outstanding Multiplex Amplification Specificity

Hzymes’ fully blocking antibody effectively inhibits the 5’→3’ polymerase activity of Taq enzyme, completely blocking both polymerase and exonuclease activities below 70°C, which significantly reduces non-specific amplification products. Compared to unmodified Taq enzyme (Figure 6, top), Taq enzyme modified with the fully blocking antibody (Figure 6, bottom) significantly reduced non-specific amplification products in forensic multiplex PCR assays (highlighted in red boxes).

Figure 6. Capillary electrophoresis profiles of PCR products using Hzymes' fully blocking antibody in forensic detection systems.

References

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[3] Sharkey DJ, Scalice ER, Christy KG Jr, Atwood SM, Daiss JL. Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction. Biotechnology (N Y). 1994; 12 (5): 506-509.

[4] Kim Y, Eom SH, Wang J, Lee DS, Suh SW, Steitz TA. Crystal structure of Thermus aquaticus DNA polymerase. Nature. 1995; 376 (6541): 612-616.

[5] Lin Y, Jayasena SD. Inhibition of multiple thermostable DNA polymerases by a heterodimeric aptamer. J Mol Biol. 1997; 271 (1): 100-111.

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