Anti-protease K monoclonal antibody and application thereof

Abstract

The invention discloses an anti-protease K monoclonal antibody and application thereof. A heavy chain variable region of the antibody comprises the following complementarity determining regions: VH-CDR1 as shown in SEQ NO.2, VH-CDR2 as shown in SEQ NO.4 and VH-CDR3 as shown in SEQ NO.6; the light chain variable region comprises the following complementarity determining regions: VL-CDR1 as shown in SEQ NO.9, VL-CDR2 as shown in SEQ NO.11 and VL-CDR3 as shown in SEQ NO.13, and the VH-CDR1-3 and the VL-CDR1-3 jointly form a protease K binding site. The anti-proteinase K monoclonal antibody can be applied to various nucleic acid detection technologies such as PCR, qPCR and reverse transcription PCR, the use process is simple, the anti-proteinase K monoclonal antibody is directly added to a reaction system, the activity of the proteinase K can be effectively inhibited without a pretreatment step, the active site of the proteinase K can be accurately targeted through the specific inhibition effect, the activity of the proteinase K can be specifically blocked, and meanwhile, the application range of the anti-proteinase K monoclonal antibody is widened. The activity of common nucleic acid polymerase (Taq, reverse transcriptase and the like) is not obviously influenced, complete neutralization can be realized at a low dosage, and excellent high efficiency is shown.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an anti-proteinase K monoclonal antibody and an application thereof. Background Art

[0002] Nucleic acid detection technology is an important technical means in modern molecular biology and medical diagnosis. During the nucleic acid detection process, the preservation and extraction of nucleic acid specimens are two key links to ensure the accuracy and sensitivity of the test results. Currently, commonly used nucleic acid specimen preservation technologies mainly include cryopreservation, desiccation, chemical preservation, solid-phase preservation, and biological preservation. Among them, biological preservation is widely used due to its advantages such as environmental friendliness and ease of use. Its principle is to hydrolyze nucleases by adding proteases (such as proteinase K), thereby protecting the integrity of nucleic acids.

[0003] In terms of nucleic acid extraction, although traditional methods such as phenol-chloroform extraction, column extraction, and magnetic bead extraction can obtain high-purity nucleic acids, they have problems such as complex operation and the use of harmful reagents. In contrast, extraction-free methods have attracted much attention due to their ease of operation. This type of method directly lyses cells to release nucleic acids without the need for purification steps. Currently, most commercial nucleic acid release agents use proteinase K as the main ingredient, which can not only preserve nucleic acid samples but also be directly used for downstream amplification.

[0004] However, proteinase K also has obvious limitations when used as an extraction-free reagent. This serine protease has broad substrate specificity. It can not only degrade nucleases, but also act on nucleic acid polymerases (such as Taq enzyme, reverse transcriptase, etc.) in the amplification system, resulting in inhibition of subsequent PCR reactions, decreased detection sensitivity, and even false negative results. Existing solutions such as dilution method, heat inactivation method and neutralization method each have their own defects: the dilution method cannot completely eliminate proteinase K activity and reduces detection sensitivity; the heat inactivation method increases the complexity of the operation; the neutralization method weakens the effect of proteinase K on polymerase by adding irrelevant proteins such as bovine serum albumin to the reaction system, but excessive irrelevant proteins will also inhibit the amplification of nucleases. Therefore, it is of great significance to develop a method that can specifically inhibit proteinase K activity without affecting the function of nucleic acid polymerase. Summary of the Invention

[0005] Based on this, it is necessary to provide an anti-proteinase K monoclonal antibody and its application to address the above technical problems.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides an anti-proteinase K monoclonal antibody, wherein the heavy chain variable region of the anti-proteinase K monoclonal antibody comprises the following complementarity determining regions: VH-CDR1 such as SEQ NO.2, VH-CDR2 such as SEQ NO.4 and VH-CDR3 such as SEQ NO.6; the light chain variable region comprises the following complementarity determining regions: VL-CDR1 such as SEQ NO.9, VL-CDR2 such as SEQ NO.11 and VL-CDR3 such as SEQ NO.13, and the VH-CDR1~3 and VL-CDR1~3 together form a proteinase K binding site.

[0007] Furthermore, the heavy chain variable region further comprises the following framework regions: VH-FR1 of SEQ NO.1, VH-FR2 of SEQ NO.3, VH-FR3 of SEQ NO.5 and VH-FR4 of SEQ NO.7.

[0008] Furthermore, the light chain variable region further comprises the following framework regions: VL-FR1 of SEQ NO.8, VL-FR2 of SEQ NO.10, VL-FR3 of SEQ NO.12 and VL-FR4 of SEQ NO.14.

[0009] The second aspect of the present invention provides the use of the above-mentioned anti-proteinase K monoclonal antibody in nucleic acid amplification.

[0010] Furthermore, the nucleic acid amplification is a nucleic acid amplification technology based on polymerase chain reaction technology such as real-time fluorescence quantitative PCR and conventional PCR.

[0011] Furthermore, the application includes the following steps:

[0012] (1) mixing a biological sample containing proteinase K with an anti-proteinase K monoclonal antibody to form a PCR reaction system; (2) performing amplification detection on the PCR reaction system; the molar concentration ratio of proteinase K to the anti-proteinase K monoclonal antibody in the PCR reaction system is less than 1:2.

[0013] The third aspect of the present invention provides a kit comprising the above-mentioned anti-proteinase K monoclonal antibody.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides an anti-proteinase K monoclonal antibody that can be applied to various nucleic acid detection technologies such as PCR, qPCR, and reverse transcription PCR. It has a simple use process and is directly added to the reaction system. It can achieve effective inhibition of proteinase K activity without the need for a pretreatment step. While achieving specific blocking of proteinase K activity, it has no significant effect on the activity of common nucleic acid polymerases (Taq, reverse transcriptase, etc.). Anti-proteinase K monoclonal antibodies with a dosage as low as a working concentration of 5 μg / mL can completely neutralize proteinase K activity. These characteristics improve the accuracy and sensitivity of nucleic acid detection and provide innovative solutions for the field of molecular detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0017] Figure 1 The figure is an SDS-PAGE electrophoresis diagram (non-reducing) of the anti-proteinase K monoclonal antibody of the present invention. In the figure, M is a protein marker, and the values ​​on the left are the molecular weights (in kDa) corresponding to the various bands of the protein marker. 8B9 is the anti-proteinase K monoclonal antibody of the present invention.

[0018] Figure 2 This is a diagram showing the effect of proteinase K on the PCR system containing proteinase K. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0020] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0021] Sources of main instruments and reagents:

[0022] Freund's complete adjuvant and Freund's incomplete adjuvant: Sigma-Aldrich;

[0023] 5-week-old BALB / c mice and 10-week-old BALB / c mice were obtained from Guangdong Provincial Laboratory Animal Center.

[0024] 1640 culture medium: Corning Life Sciences Co., Ltd.;

[0025] HAT, HT supplement, and fetal bovine serum: Thermo Fisher Scientific;

[0026] SP20 myeloma cells: preserved by the applicant;

[0027] 50% PEG1450: Roche Diagnostics GmbH;

[0028] Proteinase K: Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.;

[0029] HRP-goat anti-mouse IgG: Beijing Biolong Immunotechnology Co., Ltd.;

[0030] Protein A filler: Hangzhou Newlong Biotechnology Co., Ltd.

[0031] 5×Superstart Premix: Zhuhai Baorui Biotechnology Co., Ltd.;

[0032] Influenza A virus cDNA: deposited by the applicant;

[0033] 5×Fast Direct RT Buffer IV (DG): Zhuhai Baorui Biotechnology Co., Ltd.;

[0034] 12.5×Fast Direct Part Ampli / UNG Mix IV (with dNTPs) (DG): Zhuhai Baorui Biotechnology Co., Ltd.;

[0035] 50×Fast Direct Part RTase IV (DG): Zhuhai Baorui Biotechnology Co., Ltd.;

[0036] 25× Primer-Probe Mix: Zhuhai Baorui Biotechnology Co., Ltd.;

[0037] 4× lyophilized protective agent: Zhuhai Baorui Biotechnology Co., Ltd.;

[0038] Primers & probes: Sangon Biotech (Shanghai) Co., Ltd.

[0039] Real-time PCR instrument: Shanghai Hongshi Medical Technology Co., Ltd., model SLAN-96S

[0040] Example 1

[0041] This embodiment provides a method for preparing an anti-proteinase K monoclonal antibody, which comprises the following steps:

[0042] S1. Animal Immunization

[0043] Proteinase K (1 mg / mL) was emulsified with an equal volume of adjuvant and immunized via multiple subcutaneous injections into the back of 5-week-old BALB / c mice. Each mouse received 200 μL of the emulsified antigen (containing 100 μg of proteinase K antigen) per injection. Immunizations were repeated 14 days apart for a total of five immunizations.

[0044] S2. Fusion of spleen lymphocytes from immunized mice with myeloma cells

[0045] 1. Booster immunization: Three days before cell fusion, the immunized mice were boosted with 50 μg proteinase K via intraperitoneal injection.

[0046] 2. Feeder cell preparation: One day before fusion, prepare feeder cell suspension and plate into 96-well culture plates at 100 μL / well;

[0047] 3. Killing and disinfecting mice: Kill mice by removing their eyes and drawing blood, then dislocating their necks. Immediately disinfect the mouse carcasses by immersing them in 75% ethanol solution for 5 minutes.

[0048] 4. Preparation of spleen cell suspension: Remove the spleen, grind it on a gauze, rinse with PBS to prepare a single cell suspension, and count the cells under a microscope;

[0049] 5. Centrifugal washing: Centrifuge at 1200 rpm for 5 minutes and discard the supernatant;

[0050] 6. Adjust the cell ratio: Mix splenocytes and SP20 myeloma cells at a ratio of 5:1, add 1640 medium to a final volume of 30 mL, and mix thoroughly.

[0051] 7. Second centrifugation: Centrifuge at 1200 rpm for 10 minutes and discard the supernatant;

[0052] 8. Resuspend cells: Gently tap the bottom of the centrifuge tube to loosen the cell pellet;

[0053] 9. Cell fusion: Add 1 mL of 50% PEG1450 solution along the tube wall within 1 minute at 37°C.

[0054] 10.Terminate fusion: Slowly add 25 mL of 1640 medium to terminate the fusion reaction;

[0055] 11. Centrifugation after fusion: Centrifuge at 900 rpm for 7 minutes and discard the supernatant;

[0056] 12. Cell plating: Resuspend cells in 120 mL of HAT medium and plate 100 μL / well into the prepared feeder cell plate.

[0057] 13. Culture: Culture in a 37°C 5% CO2 incubator;

[0058] 14. Medium change: On the 8th day, the medium was changed with HT medium;

[0059] 15. Antibody detection: On the 10th day of culture, the culture supernatant was collected and the specific antibody titer was detected by ELISA.

[0060] S3. ELISA screening of hybridoma cells

[0061] The indirect ELISA method was used to detect specific antibodies in the hybridoma cell culture supernatant. The specific steps are as follows:

[0062] 1. Antigen coating: Dilute proteinase K to 1 μg / ml in carbonate buffer (pH 9.6), add 100 μL / well to a 96-well ELISA plate, and coat overnight at 4°C.

[0063] 2. Wash: Wash 6 times with PBST buffer;

[0064] 3. Blocking: Add 200 μL of 1% BSA blocking solution to each well and incubate at 37°C for 120 minutes;

[0065] 4. Washing: Wash 5 times with PBST buffer;

[0066] 5. Primary antibody incubation: Add 100 μL of the cell culture supernatant to each well and incubate at 37°C for 45 minutes;

[0067] 6. Washing: Wash 5 times with PBST buffer;

[0068] 7. Secondary antibody incubation: Add 100 μL of 10,000-fold diluted HRP-goat anti-mouse IgG in 1% BSA to each well; incubate at 37°C for 30 minutes;

[0069] 8. Washing: Wash 5 times with PBST buffer;

[0070] 9. Color development: Add 100 μL of TMB substrate solution to each well and incubate at 37°C in the dark for 15 minutes;

[0071] 10. Termination of reaction and detection: Add 50 μL of 2 M sulfuric acid to each well to terminate the reaction, and immediately measure the absorbance at 450 nm (OD450 nm) using a microplate reader.

[0072] S4. Hybridoma cell cloning

[0073] 1. Preparation of cell suspension: Select the hybridoma cell wells that are strongly positive by ELISA and dilute them with 1640 complete medium to a cell concentration of 5 cells / mL;

[0074] 2. Limiting dilution cloning: The diluted cell suspension was dispensed into 96-well cell culture plates at a rate of 200 μL / well and cultured in a 37°C 5% CO2 incubator.

[0075] 3. Monoclonal screening: When the cells in each microwell proliferate to approximately 100 cells / well, the culture supernatant is collected for ELISA detection;

[0076] 4. Subclone screening: Perform clone screening again until all cell wells show a positive reaction;

[0077] 5. Expansion culture: The stable monoclonal hybridoma cells obtained by screening are sequentially transferred into 24-well cell culture plates for culture, and then expanded into cell culture flasks for culture.

[0078] S5. Cell Cryopreservation

[0079] 1. Cell collection: After the cells enter the logarithmic growth phase, blow off the cells;

[0080] 2. Centrifugation: Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant;

[0081] 3. Aliquot: suspend cells in cell freezing solution and dispense into cryopreservation tubes;

[0082] 4. Cryopreservation: The first stage is to equilibrate at 4°C for 40 minutes, the second stage is to pre-freeze at -20°C for 30 minutes, the third stage is to transfer to a -80°C ultra-low temperature freezer for overnight storage, and finally the next day to transfer to liquid nitrogen for long-term storage.

[0083] S6. Monoclonal Antibody Preparation

[0084] 1. Cell recovery: Remove frozen cells from liquid nitrogen and thaw in a 37°C water bath;

[0085] 2. Centrifugation washing: Transfer the cells to a centrifuge tube containing 1640 complete medium, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant;

[0086] 3. Cell culture: Resuspend cells in fresh 1640 complete medium, transfer to culture flasks, and culture in a 37°C, 5% CO2 incubator.

[0087] 4. Cell passaging: When cells reach the logarithmic phase, gently pipette the cells off the tube, transfer them to a centrifuge tube, centrifuge at 1,000 rpm for 5 minutes, and discard the supernatant.

[0088] 5. Cell counting and dilution: Resuspend the cells in 1640 culture medium and count them on a hemocytometer, then adjust the concentration to 1×10 6 / mL;

[0089] 6. Mouse sensitization: 10-week-old BALB / c mice were selected and pre-sensitized by intraperitoneal injection of 0.5 mL of paraffin oil;

[0090] 7. Injection of monoclonal cells: 7 days after sensitization, inject 1 mL of 1640 culture medium suspension containing monoclonal cells (prepared in step 5) into the mouse peritoneal cavity.

[0091] 8. Ascites collection: About 10 days after injection, observe the ascites formation in the mice and collect the ascites aseptically;

[0092] 9. Ascites treatment: Centrifuge the ascites at 4°C and 12,000 rpm for 10 minutes, collect the middle layer of clarified liquid, aliquot and store at -20°C until use.

[0093] S7. Monoclonal antibody purification (Protein A affinity chromatography)

[0094] 1. Ascites pretreatment: Centrifuge the ascites at 4°C, 12,000 rpm for 15 minutes to remove precipitates and impurities, and collect the supernatant.

[0095] 2. Ammonium sulfate precipitation: Mix the ascites supernatant with saturated ammonium sulfate solution in a 1:1 ratio and let it stand at room temperature for 30 minutes. Centrifuge at 12,000 rpm at 4°C for 30 minutes, discard the supernatant, and collect the precipitate.

[0096] 3. Dissolve and filter the precipitate: Dissolve the precipitate in binding buffer (1× PBS, pH 7.4) and filter through neutral filter paper to remove insoluble matter.

[0097] 4. Protein A column affinity purification: The filtrate was slowly loaded onto a Protein A chromatography column to allow the antibody to bind to the filler, rinsed with binding buffer (1× PBS, pH 7.4) to remove unbound foreign proteins, eluted with 0.1 M citrate buffer (pH 3.0), and neutralized with Tris-HCl (pH 8.8) buffer to avoid acid denaturation of the antibody;

[0098] 5. Dialysis and concentration: Place the eluate into a dialysis bag and dialyze against 1× PBS (pH 7.4) at 4°C, changing the buffer several times to remove residual salt and acidic buffer;

[0099] 6. Antibody detection: The antibody concentration was 8.2 mg / mL using a UV spectrophotometer and Coomassie brilliant blue staining was performed to assess the antibody purity, which showed that the antibody purity was greater than 90%. Figure 1 As shown, it meets the requirements of subsequent experiments.

[0100] S8. Determination of the ratio of proteinase K to monoclonal antibody

[0101] 1. Take equal volumes of 1 μg / mL proteinase K and mix with 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL anti-proteinase K monoclonal antibody (equivalent to a molar concentration ratio of proteinase K to anti-proteinase K monoclonal antibody of 1:1, 1:2, 1:3, 1:4, and 1:5) for later use; set up a control group in which equal volumes of ultrapure water were used instead of antibody and proteinase K. The specific groupings are shown in Table 1 below.

[0102] Table 1: Experimental group settings for different concentrations of anti-proteinase K monoclonal antibodies

[0103]

[0104] 2. Configure the real-time PCR reaction system. The specific components are shown in Table 2 below, and the primers and probes used are shown in Table 3 below.

[0105] Table 2: Real-time PCR reaction system composition

[0106]

[0107]

[0108] Table 3: Influenza A virus primer and probe sequences

[0109]

[0110] 3. Real-time PCR amplification detection: The real-time PCR reaction system was placed in a real-time PCR instrument for amplification detection. The amplification program was as follows: (a) 56°C for 60 min; (b) 95°C for 5 min; (c) 45 cycles of denaturation at 95°C for 5 sec and annealing / extension at 60°C for 40 sec (FAM channel fluorescence was collected).

[0111] 4. The results are as follows Figure 2 As shown in the figure, after adding anti-proteinase K monoclonal antibody to the PCR system, as the concentration of anti-proteinase K monoclonal antibody increased, the Ct value and fluorescence signal intensity gradually approached the 108 group (without proteinase K and antibody), indicating that anti-proteinase K monoclonal antibody can effectively inhibit the activity of proteinase K. When the molar concentration ratio of proteinase K to antibody is ≤1:3, proteinase K activity can be completely inhibited.

[0112] S9. Monoclonal antibody sequence: The cells secreting the proteinase K monoclonal antibody (8B9) described in the present invention were sent to Nanjing Mingyan Biotechnology Co., Ltd. for sequencing, and the light and heavy chain amino acid sequences of the antibody were measured as shown in Table 4, wherein the heavy chain of the proteinase K monoclonal antibody is VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3 and VH-FR4, and their amino acid sequences are shown in SEQ NO.1 to SEQ NO.7, respectively; the light chain is VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3 and VL-FR4, and their amino acid sequences are shown in SEQ NO.9 to SEQ NO.14, respectively. Table 4 shows the amino acid sequences of each domain of the heavy and light chains of the monoclonal antibody.

[0113] Table 4: Amino acid sequences of the heavy and light chain domains of proteinase K monoclonal antibody

[0114]

[0115]

[0116] Example 2 Application of Proteinase K Monoclonal Antibody

[0117] This embodiment provides a method for using an anti-proteinase K monoclonal antibody in PCR amplification, which comprises the following steps:

[0118] S1. Sample collection and storage: Dilute proteinase K to 10 μg / mL with physiological saline and dispense into sample tubes (3 mL / tube); take a sampling swab, collect an upper respiratory tract specimen (nasopharyngeal swab), break off the swab head and place it in a centrifuge tube containing proteinase K, screw the lid, mix it upside down so that the solution submerges the surface of the swab, and store it at room temperature (25°C) for 10 days. The liquid in the control group sample tube is physiological saline solution without proteinase K. Among them, control group 1 repeatedly squeezes the sampling swab in physiological saline to release the virus, then takes 1 mL and immediately freezes it at -80°C for 10 days (positive control). Control group 2 stores the remaining sample at 25°C under the same conditions for 10 days (degradation control). The specific groups are shown in Table 6.

[0119] S2. A real-time PCR reaction system was prepared. The specific components are shown in Table 5 below. The primers and probes used were the same as those shown in Table 3 of Example 1. The final concentration of the anti-proteinase K monoclonal antibody in the PCR reaction system in this example was 200 μg / mL.

[0120] Table 5: PCR reaction system composition

[0121]

[0122] S3. Freeze-dry: Freeze-dry the PCR reaction system prepared in S2 at -50°C for 300 min, -30°C for 720 min, and 25°C for 300 min. After lyophilization, place the mixture in an aluminum foil bag, add desiccant, vacuum-seal, and store at room temperature.

[0123] S4. Preparation of detection system:

[0124] Experimental group: Add 25 μL of the liquid in the S1 storage tube to the S3 freeze-dried detection system to dissolve the freeze-dried powder. Set up two replicates for each reaction.

[0125] Control group: The nucleic acid of control group 1 and control group 2 was detected by Qiagen QIAamp Nucleic acid was extracted using the ViralRNA Mini Kit. The nucleic acid was dissolved in 140 μL of the eluent provided by the nucleic acid extraction kit. 25 μL of the eluent was then used to dissolve the S3 freeze-dried reaction wells. Each reaction was repeated in duplicate.

[0126] Table 6: Comparison of detection systems between the experimental group and the control group

[0127]

[0128]

[0129] S5. PCR amplification. The amplification procedure included the following steps: (a) reverse transcription reaction: 50°C for 15 min; (b) pre-denaturation: 95°C for 5 min; (c) cyclic amplification for 45 cycles: denaturation: 95°C for 5 sec; annealing / extension: 60°C for 40 sec, and simultaneous acquisition of FAM channel fluorescence signals.

[0130] S6. Three samples were tested using the experimental and control groups 1 and 2 detection systems, respectively. The results are shown in Table 7. The results demonstrate that Proteinase K effectively protects RNA stability at room temperature. After 10 days at room temperature, RNA in samples stored in Proteinase K solution showed little impact on PCR detection, whereas RNA in samples stored in saline solution showed significant degradation.

[0131] Table 7: Results of the Flu A Sample Preservation Evaluation Experiment

[0132]

[0133]

[0134] The application of proteinase K monoclonal antibody in PCR amplification in this embodiment is not limited to real-time PCR, but can also be used in any other method of nucleic acid amplification based on polymerase chain reaction, including but not limited to conventional PCR, reverse transcription PCR, nested PCR, high-resolution melting curve PCR, melting curve fluorescence PCR, MeltArray, etc.

[0135] Obviously, the embodiments described above are only some of the embodiments of the present application, rather than all of the embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions described in the aforementioned specific embodiments, or to make equivalent replacements for some of the technical features therein. Any equivalent structure made using the contents of the present application specification and the accompanying drawings, directly or indirectly used in other related technical fields, is also within the scope of patent protection of the present application.

Claims

1. An anti-proteinase K monoclonal antibody, characterized in that The heavy chain variable region comprises the following complementary determining regions: VH-CDR1 of SEQ NO. 2, VH-CDR2 of SEQ NO. 4, and VH-CDR3 of SEQ NO. 6; the light chain variable region comprises the following complementary determining regions: VL-CDR1 of SEQ NO. 9, VL-CDR2 of SEQ NO. 11, and VL-CDR3 of SEQ NO.

13. VH-CDR1~3 and VL-CDR1~3 together form the proteinase K binding site.

2. The anti-proteinase K monoclonal antibody according to claim 1, characterized in that The heavy chain variable region further comprises the following framework regions: VH-FR1 such as SEQ NO.1, VH-FR2 such as SEQ NO.3, VH-FR3 such as SEQ NO.5 and VH-FR4 such as SEQ NO.

7.

3. The anti-proteinase K monoclonal antibody according to claim 2, characterized in that The light chain variable region further comprises the following framework regions: VL-FR1 such as SEQ NO.8, VL-FR2 such as SEQ NO.10, VL-FR3 such as SEQ NO.12 and VL-FR4 such as SEQ NO.

14.

4. Use of the anti-proteinase K monoclonal antibody according to any one of claims 1 to 3 in nucleic acid amplification.

5. The use according to claim 4, characterized in that The nucleic acid amplification is a nucleic acid amplification technology based on polymerase chain reaction technology such as real-time fluorescence quantitative PCR and ordinary PCR.

6. The use according to claim 5, characterized in that It includes the following steps: (1) mixing a biological sample containing proteinase K with an anti-proteinase K monoclonal antibody to form a PCR reaction system; (2) performing amplification detection on the PCR reaction system; the molar concentration ratio of proteinase K to the anti-proteinase K monoclonal antibody in the PCR reaction system is less than 1:

2.

7. A kit, characterized in that The invention comprises any one of the anti-proteinase K monoclonal antibodies according to claims 1 to 3.

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