Anti-Taq enzyme monoclonal antibody, preparation method thereof and application of anti-Taq enzyme monoclonal antibody in hot start PCR (Polymerase Chain Reaction)
By preparing high-affinity anti-Taq enzyme monoclonal antibodies, the shortcomings of antibody methods in the existing hot-start PCR technology are solved, and the high specific binding and high sensitivity hot-start PCR effect is achieved, which is suitable for multiple PCR and complex sample detection.
Patent Information
- Application Number
- CN202510651122.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-20
- Publication Date
- 2025-08-26
AI Technical Summary
In the existing hot-start PCR technology, antibody methods have problems such as insufficient affinity, poor specificity, low thermal activation efficiency and poor stability, resulting in insufficient non-specific amplification and detection sensitivity at low temperatures, especially in trace detection of pathogens.
A monoclonal antibody against Taq enzyme was prepared, designed by specific amino acid sequences, and using mouse immunity, cell fusion and purification techniques to obtain high affinity and high specificity antibodies for forming complexes with Taq enzymes and releasing enzyme activity only at high temperatures.
It realizes high specific binding, effectively inhibits non-specific amplification at low temperature, detects low abundance templates with high sensitivity, is good stability, is easy to operate, and is suitable for multiple PCR and complex sample detection.
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Figure CN120535637A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to an anti-Taq enzyme monoclonal antibody, a preparation method thereof and an application thereof in hot-start PCR. Background Art
[0002] Polymerase chain reaction (PCR) is a core technology in the field of molecular biology, widely used in scenarios such as gene amplification, pathogen detection, and genetic disease diagnosis. However, during the low-temperature annealing stage, traditional PCR is prone to the production of nonspecific amplification products due to nonspecific binding of primers to templates and residual activity of the Taq enzyme at low temperatures, which seriously affects detection sensitivity and accuracy. Hot Start PCR (Hot Start PCR) fundamentally solves the problem of nonspecific amplification by inhibiting the activity of the Taq enzyme at low temperatures and only releasing enzyme activity at high temperatures (such as 95°C), making it a key technology for high-precision PCR.
[0003] At present, hot start PCR technology mainly includes the following categories:
[0004] 1. Physical method (wax sealing method)
[0005] Principle: The Taq enzyme is isolated from the reaction system by a paraffin or oil layer. The wax layer melts at high temperature, and the enzyme and substrate mix to start the reaction.
[0006] Disadvantages: The operation is cumbersome, requires an additional heating step, is easily contaminated, and the wax layer may remain and affect enzyme activity, so it is gradually being eliminated.
[0007] 2. Chemical modification
[0008] Aptamer binding: The nucleic acid aptamer specifically binds to the active center of the Taq enzyme, and the aptamer structure changes at high temperature, releasing the enzyme.
[0009] Chemical group modification: The active site of the Taq enzyme is chemically modified (such as phosphorylation, alkylation), and the modified group falls off at high temperature to restore enzyme activity.
[0010] defect:
[0011] Aptamers are expensive and time-consuming to prepare, and their binding affinity to enzymes is limited. Chemical modification can disrupt enzyme structure, leading to loss of activity. Chemical modification methods generally inhibit Taq enzyme activity by less than 90% at low temperatures, leading to the appearance of spurious bands when amplifying low-abundance templates.
[0012] 3. Antibody Method
[0013] Core mechanism: Antibodies bind to the active center or key domain of the Taq enzyme, blocking the binding of substrates (dNTPs, primers) to the enzyme, inhibiting enzyme activity at low temperatures and releasing the enzyme after antibody denaturation at high temperatures.
[0014] Limitations of existing antibodies:
[0015] ① Insufficient affinity: The dissociation constant (KD) of some commercially available antibodies (such as sheep and rabbit polyclonal antibodies) is less than 10 -7 -10 -8 M is prone to partial dissociation at low temperatures, resulting in residual enzyme activity and poor inhibition effect. 3 The detection limit of existing methods is difficult to meet the demand, and it is necessary to rely on high-affinity antibodies to improve the inhibition effect.
[0016] Poor specificity: Polyclonal antibodies contain multiple subtypes, which may bind to inactive sites of the Taq enzyme, affecting hot start efficiency. Some antibodies also exhibit species cross-reactivity, affecting multiplex PCR or complex sample detection.
[0017] ③Poor thermal activation efficiency: Some antibodies are still partially bound to the Taq enzyme after high-temperature dissociation, resulting in incomplete recovery of enzyme activity and affecting amplification efficiency.
[0018] ④Poor stability: Although mouse monoclonal antibodies have high specificity, some products are prone to aggregation during long-term storage, resulting in a decrease in binding ability.
[0019] ⑤ The purification process of some antibodies is insufficient, and there is contamination by foreign enzymes, which affects the amplification efficiency. Summary of the Invention
[0020] The technical problem to be solved by the present invention is to address the deficiencies in the above-mentioned prior art and provide an anti-Taq enzyme monoclonal antibody, a preparation method thereof and an application thereof in hot-start PCR.
[0021] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: In the first aspect of the present invention, an anti-Taq enzyme monoclonal antibody is provided, wherein the heavy chain variable region sequence in the amino acid sequence is:
[0022] QVTLKESGPSILKPSQTLSLTCSFSGFSLSTSDMGVGWIRQPSGKGLEW LAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCAR RANTTGYFDVWGAGTTVTVSS;
[0023] The light chain variable region sequence in its amino acid sequence is:
[0024] DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLI YAATSLADGVPSRFSGSGSGTKFSFKISSLQAEDFVNYYCQQLYNPQLTFGS GTKLEIK.
[0025] The second aspect of the present invention provides a method for preparing an anti-Taq enzyme monoclonal antibody, comprising the following steps:
[0026] S1. Immunization of animals: Select mice and use wild-type recombinant Taq enzyme protein as antigen, emulsified with Freund's adjuvant and injected subcutaneously. Immunize 2-6 times with an interval of 1-4 weeks, and measure the serum titer.
[0027] S2. Cell fusion: spleen cells from the mouse with the highest titer were fused with NS-1 myeloma cells to screen hybridoma cells;
[0028] S3. Screening and cloning: Screen positive clones that secrete anti-Taq enzyme antibodies and obtain stable cell lines through 3-4 subcloning;
[0029] S4. Antibody purification: purify the IgG antibody in the ascites or cell supernatant to obtain the anti-Taq enzyme monoclonal antibody.
[0030] Preferably, step S1 specifically comprises: selecting 6-8 week old Balb / c mice, using wild-type recombinant Taq enzyme protein as antigen, emulsifying it with Freund's adjuvant and then injecting it subcutaneously, immunizing them 4 times with an interval of 2 weeks, and measuring the serum titer by indirect ELISA.
[0031] Preferably, step S2 specifically comprises: taking spleen cells from Balb / c mice with the highest titer and fusing them with NS-1 myeloma cells, and screening hybridoma cells using HAT medium.
[0032] Preferably, step S3 specifically comprises: screening positive clones secreting anti-Taq enzyme antibodies by indirect ELISA, and obtaining stable cell lines through 3-4 subcloning.
[0033] Preferably, step S4 specifically comprises: purifying the IgG antibody in the ascites or cell supernatant by Protein G affinity chromatography, and verifying the purity by SDS-PAGE to be ≥95%, to obtain the anti-Taq enzyme monoclonal antibody.
[0034] The third aspect of the present invention provides a use of the anti-Taq enzyme monoclonal antibody described above in hot-start PCR.
[0035] Preferably, the application method is:
[0036] 1) Mixing the antibody with Taq enzyme and incubating to form an antibody-Taq enzyme complex;
[0037] 2) When in use, the antibody-Taq enzyme complex is added to the hot start PCR system for PCR amplification.
[0038] Preferably, the molar ratio of the antibody to the Taq enzyme in step 1) is 1:1.
[0039] Preferably, step 1) is specifically:
[0040] Mix the antibody and Taq enzyme at a molar ratio of 1:1 and incubate at 4°C for 30 minutes or at room temperature for 10 minutes to form an antibody-Taq enzyme complex;
[0041] In step 2), the final concentration of the antibody-Taq enzyme complex in the hot start PCR system is 0.1-1 μM.
[0042] The beneficial effects of the present invention are:
[0043] The present invention provides an anti-Taq enzyme monoclonal antibody, a preparation method thereof, and an application thereof in hot-start PCR. The anti-Taq enzyme monoclonal antibody has the following advantages:
[0044] 1. High specificity: The binding constant (KD) of this anti-Taq enzyme monoclonal antibody to Taq enzyme is 1.8×10 -13 M, can effectively inhibit nonspecific amplification at low temperatures;
[0045] 2. High sensitivity: When used in hot-start PCR, this anti-Taq monoclonal antibody can detect specific products at template concentrations as low as 1 copy / μL;
[0046] 3. Good stability: After storage at 2-8°C for 12 months, the anti-Taq monoclonal antibody still maintains >90% of its activity;
[0047] 4. Easy to operate: This anti-Taq enzyme monoclonal antibody does not require chemical modification or complex treatment and can be directly mixed with Taq enzyme for use. BRIEF DESCRIPTION OF THE DRAWINGS
[0048] Figure 1 The results of the affinity test of anti-Taq enzyme monoclonal antibody using SPR method;
[0049] Figure 2 This is the SDS-PAGE electrophoresis diagram of anti-Taq enzyme monoclonal antibody;
[0050] Figure 3 The results of antibody titer detection by indirect ELISA;
[0051] Figure 4 The comparison results of the amplification performance of Taq enzyme blocked by the anti-Taq enzyme monoclonal antibody prepared in Example 1 and the Taq enzyme blocked by the control antibody at different concentration gradients of DNA templates;
[0052] Figure 5 This is the test result of the binding constant (KD) between the anti-Taq enzyme monoclonal antibody prepared in Example 1 and Taq enzyme. DETAILED DESCRIPTION
[0053] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.
[0054] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.
[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. In the following examples, where specific conditions are not specified, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products.
[0056] Example 1
[0057] An anti-Taq enzyme monoclonal antibody, the preparation method of which comprises the following steps:
[0058] S1. Immunization of animals: 6-8 week old Balb / c mice were selected and wild-type recombinant Taq enzyme protein was used as antigen. After emulsification with Freund's adjuvant, it was injected subcutaneously. A total of 4 immunizations were performed with an interval of 2 weeks. The serum titer was determined by indirect ELISA (titer>1:10 5 is qualified);
[0059] S2. Cell fusion: The spleen cells of the Balb / c mice with the highest titer were fused with NS-1 myeloma cells, and the hybridoma cells were screened using HAT medium;
[0060] S3. Screening and cloning: Screen positive clones that secrete anti-Taq enzyme antibodies and obtain stable cell lines through 3-4 subcloning;
[0061] S4. Antibody purification: Protein G affinity chromatography is used to purify the IgG antibody in the ascites or cell supernatant. The purity is verified by SDS-PAGE to be ≥95%, thereby obtaining the anti-Taq enzyme monoclonal antibody.
[0062] Reference Figure 1, which is the result of SPR method to detect the affinity of anti-Taq enzyme monoclonal antibody. It can be seen that the affinity of anti-Taq enzyme monoclonal antibody to Taq enzyme is very high, and the binding constant (KD) can reach 1.8×10 -13 M.
[0063] Reference Figure 2 , is the SDS-PAGE electrophoresis diagram of anti-Taq enzyme monoclonal antibody.
[0064] Reference Figure 3 , is the result of indirect ELISA test on antibody titer, the titer is 1:10 6 .
[0065] The amino acid sequence of the heavy chain variable region of the anti-Taq enzyme monoclonal antibody is:
[0066] QVTLKESGPSILKPSQTLSLTCSFSGFSLSTSDMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCARRANTTGYFDVWGAGTTVTVSS(SEQ ID NO:1);
[0067] The light chain variable region sequence in its amino acid sequence is:
[0068] DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLI YAATSLADGVPSRFSGSGSGTKFSFKISSLQAEDFVNYYCQQLYNPQLTFGS GTKLEIK (SEQ ID NO: 2).
[0069] Example 2
[0070] The anti-Taq enzyme monoclonal antibody prepared in Example 1 was used in hot start PCR, and the application method was as follows:
[0071] 1) Mix the antibody and Taq enzyme at a molar ratio of 1:1 and incubate at 4°C for 30 minutes or at room temperature for 10 minutes to form an antibody-Taq enzyme complex;
[0072] 2) Antibody-Taq enzyme complex was added to the conventional PCR system (final concentration 0.1-1 μM), and the initial step was set at 95°C for 5 minutes to dissociate the antibody.
[0073] 1. Verification of antibody inhibition effect: In a PCR system containing 0.5 μM antibody, no amplification was observed after incubation at low temperature (25°C) for 30 minutes. After preheating at 95°C, the amplification efficiency was equivalent to that of the group without antibody (Ct value difference <0.5).
[0074] 2. Sensitivity test: Ten low-concentration DNA samples (1-5 copies / μL) were tested. Compared with the traditional method, the detection rate of this antibody increased from 60% to 100%, and there were no non-specific bands.
[0075] Reference Figure 4 The results show a comparison of the amplification performance of different DNA template concentration gradients, with the Taq enzyme blocked by the anti-Taq enzyme monoclonal antibody prepared in Example 1 (i.e., the antibody-Taq enzyme complex in Step 1) and the Taq enzyme blocked by the control antibody [Taq enzyme blocked by an antibody from T Company, specifically, the hot start Taq enzyme antibody (Cat. No.: TCP-101) from Toyobo Company]. It can be seen that under the same conditions, the Ct value of the blue curve is lower and the slope of the exponential phase is steeper, indicating that the Taq enzyme blocked by this antibody has higher amplification efficiency and better sensitivity.
[0076] Reference Figure 5 , which is the binding constant (KD) test result between the anti-Taq enzyme monoclonal antibody prepared in Example 1 and Taq enzyme.
[0077] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.
Claims
1. An anti-Taq enzyme monoclonal antibody, characterized in that The heavy chain variable region sequence in its amino acid sequence is: QVTLKESGPSILKPSQTLSLTCSFSGFSLSTSDMGVGWIRQPSGKGLEW LAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCAR RANTTGYFDVWGAGTTVTVSS; The light chain variable region sequence in its amino acid sequence is: DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLI YAATSLADGVPSRFSGSGSGTKFSFKISSLQAEDFVNYYCQQLYNPQLTFGS GTKLEIK.
2. A method for preparing an anti-Taq enzyme monoclonal antibody according to claim 1, characterized in that: The following steps are involved: S1. Immunization of animals: Select mice and use wild-type recombinant Taq enzyme protein as antigen, emulsified with Freund's adjuvant and injected subcutaneously. Immunize 2-6 times with an interval of 1-4 weeks, and measure the serum titer. S2. Cell fusion: spleen cells from the mouse with the highest titer were fused with NS-1 myeloma cells to screen hybridoma cells; S3. Screening and cloning: Screen positive clones that secrete anti-Taq enzyme antibodies and obtain stable cell lines through 3-4 subcloning; S4. Antibody purification: purify the IgG antibody in the ascites or cell supernatant to obtain the anti-Taq enzyme monoclonal antibody.
3. The method for preparing an anti-Taq enzyme monoclonal antibody according to claim 2, characterized in that: Step S1 specifically comprises: selecting 6-8 week old Balb / c mice, using wild-type recombinant Taq enzyme protein as antigen, emulsifying it with Freund's adjuvant and then injecting it subcutaneously, for a total of 4 immunizations with an interval of 2 weeks, and measuring the serum titer by indirect ELISA.
4. The method for preparing an anti-Taq enzyme monoclonal antibody according to claim 3, characterized in that: Step S2 specifically includes: taking spleen cells from Balb / c mice with the highest titer and fusing them with NS-1 myeloma cells, and screening hybridoma cells using HAT medium.
5. The method for preparing an anti-Taq enzyme monoclonal antibody according to claim 2, characterized in that: Step S3 specifically includes: screening positive clones secreting anti-Taq enzyme antibodies by indirect ELISA method, and obtaining stable cell lines through 3-4 subcloning.
6. The method for preparing an anti-Taq enzyme monoclonal antibody according to claim 2, characterized in that: Step S4 specifically comprises: purifying the IgG antibody in the ascites or cell supernatant by Protein G affinity chromatography, and verifying the purity by SDS-PAGE to be ≥95%, thereby obtaining the anti-Taq enzyme monoclonal antibody.
7. Use of the anti-Taq enzyme monoclonal antibody according to claim 1 in hot-start PCR.
8. The use according to claim 7, characterized in that The application method is: 1) Mixing the antibody with Taq enzyme and incubating them to form an antibody-Taq enzyme complex; 2) When in use, the antibody-Taq enzyme complex is added to the hot start PCR system for PCR amplification.
9. The use according to claim 8, characterized in that The molar ratio of the antibody to the Taq enzyme in step 1) is 1:
1.
10. The use according to claim 9, characterized in that Step 1) is specifically as follows: Mix the antibody and Taq enzyme at a molar ratio of 1:1 and incubate at 4°C for 30 minutes or at room temperature for 10 minutes to form an antibody-Taq enzyme complex; In step 2), the final concentration of the antibody-Taq enzyme complex in the hot start PCR system is 0.1-1 μM.