Anti-varicella-pox-virus neutralizing monoclonal antibody as well as preparation method and application thereof
By preparing anti-varicella virus neutralizing monoclonal antibodies gE-7D9 and gE-11F3, the problem of difficult to develop antibodies that efficiently bind to the shingles virus gE protein in the prior art is solved, and the efficient neutralization and detection methods for the varicella virus gE protein are established, providing an effective means for the quality control of shingles vaccines.
Patent Information
- Application Number
- CN202510319542.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-06-20
AI Technical Summary
The prior art is difficult to develop antibodies that efficiently bind the gE protein of shingles virus, resulting in ineffective detection methods for shingles vaccines.
Anti-varicella virus neutralizing monoclonal antibodies gE-7D9 and gE-11F3 were prepared, and monoclonal antibodies of IgG1 subclass were obtained through animal immunity, cell fusion, positive cell line screening, cell strain building and ascites preparation and purification, neutralization activity identification and other steps.
It has achieved efficient neutralization of varicella virus gE protein, established a dual-antibody sandwich ELISA method, which can accurately detect gE protein content, and provides a basis for quality control of shingles vaccines.
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Abstract
Description
Technical Field
[0001] This application relates to the technical field of biological detection, and specifically relates to an anti-varicella virus neutralizing monoclonal antibody, a preparation method thereof, and an application thereof. Background Art
[0002] Herpes zoster is widely prevalent in spring and winter, and the prevalence rate increases year by year and shows a trend of getting younger. Most patients are concentrated over 50 years old, and the proportion of female patients is greater than that of male patients. Therefore, age is considered the most important risk factor for herpes zoster infection. Although the mortality rate after illness is low, patients often suffer great physical pain. Investigations show that at least 50% of the elderly have had or are having herpes zoster, and the incidence rate of post-herpetic neuralgia in people over 50 years old is as high as 80%. Herpes zoster brings serious physical and mental pain and property losses to patients and their families.
[0003] The viruses VZV gE, gB, and gH that cause the disease can all induce the body to produce neutralizing antibodies. gE has the largest molecular weight, has neutralization-related epitopes, has the highest content on the virus envelope, is the main viral antigen, and is also the main candidate antigen for preparing virus subunit vaccines.
[0004] Therefore, it is necessary to develop antibodies that can efficiently bind to the gE protein, and develop related detection reagents based on the antibodies to provide an effective detection method for the development and application of herpes zoster vaccines. Summary of the Invention
[0005] In order to solve the above problems, this application provides an anti-varicella virus neutralizing monoclonal antibody, a preparation method thereof, and an application thereof.
[0006] In a first aspect, this application provides an anti-varicella virus neutralizing monoclonal antibody, and the anti-varicella virus neutralizing monoclonal antibody is monoclonal antibody gE-7D9 or monoclonal antibody gE-11F3; The heavy chain amino acid sequence of the monoclonal antibody gE-7D9 is as shown in SEQ ID NO: 1, and the light chain amino acid sequence is as shown in SEQ ID NO: 2; The heavy chain amino acid sequence of the monoclonal antibody gE-11F3 is as shown in SEQ ID NO: 3, and the light chain amino acid sequence is as shown in SEQ ID NO: 4.
[0007] The heavy chain amino acid sequence (461aa) of the monoclonal antibody gE-7D9 provided by this application: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 is as shown in SEQ ID NO: 1, and the light chain amino acid sequence (239aa): Signalpeptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon is as shown in SEQ ID NO: 2; The heavy chain amino acid sequence (462aa) of the monoclonal antibody gE-11F3: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon is as shown in SEQ ID NO: 3, and the light chain amino acid sequence (238aa): Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon is as shown in SEQ ID NO: 4 In a second aspect, this application provides a nucleic acid molecule that encodes the above-mentioned anti-varicella virus neutralizing monoclonal antibody.
[0008] In a third aspect, this application provides a biological material that contains the above nucleic acid molecule, and the biological material is selected from any one of an expression cassette, a vector, or a host cell.
[0009] In a fourth aspect, this application provides a method for preparing the above-mentioned anti-varicella virus neutralizing monoclonal antibody, which is prepared by using the above nucleic acid molecule; or prepared by using the above biological material.
[0010] In a fifth aspect, this application provides a method for preparing the above-mentioned anti-varicella virus neutralizing monoclonal antibody, which specifically includes the following steps in sequence: Animal immunization, cell fusion, screening of positive cell lines, cell line establishment, preparation and purification of ascites, and neutralizing activity identification; the purified antibody is identified as the IgG1 subclass by subclass identification.
[0011] In a sixth aspect, this application provides an antibody conjugate, which is obtained by conjugating the above-mentioned anti-varicella virus neutralizing monoclonal antibody with a labeling agent, and the labeling agent is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.
[0012] In a seventh aspect, this application provides a detection kit that contains the above-mentioned anti-varicella virus neutralizing monoclonal antibody, or contains the above antibody conjugate.
[0013] In an eighth aspect, the present application provides a method for detecting the content of gE protein by double-antibody sandwich ELISA for non-diagnostic and non-therapeutic purposes, which uses the above-mentioned anti-varicella virus neutralizing monoclonal antibody for detection, or uses the above-mentioned antibody conjugate for detection, or uses the above-mentioned detection kit for detection.
[0014] Preferably, the detection method uses gE-11F3 as the coating antibody and gE-7D9-HRP as the enzyme-labeled antibody for checkerboard testing.
[0015] Preferably, the linear range of the detection method is 7.8 ng / ml - 500 ng / ml; the limit of quantification is 7.8 ng / ml.
[0016] The present application relates to a method for preparing monoclonal antibodies that are neutralizing, specific, and bind to the gE protein of varicella-zoster virus with high affinity. The present application performs checkerboard titration and self-prepares monoclonal antibodies gE-7D9 and gE-11F3 labeled with horseradish peroxidase; the two monoclonal antibody cell lines are of the IgG1 subclass.
[0017] The monoclonal antibodies gE-7D9 and gE-11F3 involved in the present application have the ability to neutralize live varicella virus. After neutralizing the varicella virus at 500 - 1000 pfu / ml and infecting MRC-5 cells, the reduction rate of cytopathic plaques reaches more than 90%, indicating that the monoclonal antibody has the effect of neutralizing the virus.
[0018] The present application uses two neutralizing antibodies to establish a double-antibody sandwich ELISA method for detecting the concentration of varicella virus gE protein. In this kit method, both the coating antibody and the labeled antibody are neutralizing monoclonal antibodies. The linear range of this method is 500 ng / ml - 7.8 ng / ml, and the limit of quantification is 7.8 ng / ml.
[0019] In summary, the technical solution of the present application has the following effects: The present application uses a neutralizing monoclonal antibody as the coating and the labeled neutralizing monoclonal antibody as the capture antibody to establish a double-antibody sandwich ELISA method, which can measure the content of the effective components of the vaccine and provide a basis for the quality control of herpes zoster vaccines.
[0020] The double-antibody sandwich ELISA method provided by the present application for detecting varicella virus gE protein has good precision and accuracy. Detailed implementation manners
[0021] The following further describes the present application in detail with reference to embodiments, and these embodiments should not be construed as limiting the scope claimed by the present application. Embodiment
[0022] (1)Animal immunization Five female BALB / c mice aged 6 - 8 weeks were immunized with the recombinant varicella-zoster virus E protein prepared by the company. For the first immunization, the above antigen was emulsified evenly with Freund's complete adjuvant at a ratio of 1:1, and immunized subcutaneously at five points with a protein amount of 50 μg per mouse; immunization was carried out once every two weeks. For the second and third immunizations, the virus purified solution was emulsified with Freund's incomplete adjuvant at a ratio of 1:1 and injected subcutaneously at five points with a protein amount of 50 μg per mouse; for the fourth immunization, no adjuvant was used and it was injected intraperitoneally with a protein amount of 50 μg per mouse; three days before fusion, a booster immunization was carried out without using adjuvant and injected intraperitoneally at 150 μg per mouse. After the immunization was completed, blood was collected for antibody titer detection.
[0023] (2)Cell fusion Five mice to be fused that had been boosted three days in advance were taken, and their spleens were aseptically removed, ground, and filtered through a 200-mesh sieve to release splenocytes into a petri dish. Splenocytes and SP2 / 0 cells were mixed at a ratio of 5:1 in a 50-ml centrifuge tube and centrifuged at 1000×g for 5 min. The supernatant was discarded, and the cell pellet at the bottom of the centrifuge tube was gently flicked to disperse the cells and mixed evenly. 1 ml of pre-warmed 50% PEG4000 at 37°C was slowly added along the wall of the tube within 1 min, and the centrifuge tube was slowly rotated while adding. After standing for 90 s, pre-warmed serum-free 1640 medium was added to 40 ml to terminate the fusion, and then centrifuged at 1000×g for 5 min. The cell pellet was resuspended with the prepared HAT selective medium, aliquoted into a 96-well cell culture plate, 200 μl per well, and cultured in a cell culture incubator at 37°C and 5% CO2. The supernatant of the wells containing single cells was taken for screening after 10 - 12 days.
[0024] (3)Screening of positive cell lines. The screening methods were indirect ELISA coated with gE protein and indirect immunofluorescence method using a varicella-zoster virus fluorescent antigen plate.
[0025] Indirect ELISA screening First, varicella-zoster virus gE protein was selected and coated at 1 μg / ml, 100 μl per well. After adding the blocking solution and blocking for 2 h, about 100 μl of the supernatant of the monoclonal cell well was added and incubated at 37°C for 60 min. After washing the plate, goat anti-mouse IgG secondary antibody (sigma) at 1:10000 was added and incubated at 37°C for 60 min, then the plate was washed and developed for reading. The positive cell lines were further verified by the indirect immunofluorescence method.
[0026] Indirect immunofluorescence method: First, seed MRC-5 cells into a 96-well cell culture plate at a density of 20,000 cells per well. After growing for 24 hours, discard the cell culture medium. Then, add live varicella-zoster virus solution to MEM medium with 2% bovine serum at a volume ratio of 1:100, 200 μl per well. After the cells show cytopathic effect, which is approximately 24 hours later, discard the virus solution, add 80% virus fixative, fix at 4°C for 30 minutes, dry by baking, and store at -20°C. The antigen plate is then prepared.
[0027] After taking out the antigen plate and restoring it to room temperature, add the supernatant of the monoclonal cell wells to be tested into the 96-well antigen plate, 50 μl per well, incubate at 37°C for 1 hour, wash the plate 3 times, add FITC-labeled IgG secondary antibody (purchased from sigma), dilute it 50 times with 1:8000 Evans blue solution, incubate at 37°C for 1 hour, wash the plate 3 times, and then add 50 μl per well of 80% glycerol, and observe under a fluorescence microscope.
[0028] (4)Cell line establishment and ascites preparation Amplify and cryopreserve the cell lines that are positive in the two screening results. When a part of the cells are amplified to a cell number of 1×10^6, inject them into the abdomen of mice to prepare ascites, 0.5 ml per mouse. After approximately 10 - 12 days, take out the ascites and purify it using a protein A affinity chromatography column (purchased from Cytiva company), and simultaneously perform subclass identification.
[0029] The purified antibody is identified as IgG1 subclass through subclass identification.
[0030] (5)Neutralizing activity identification Prepare MRC-5 cells that cover the bottom of the wells in advance. Neutralize 150 μl of each sample before and after purification of the ascites of 7D9 and 11F3 with the virus diluted to 500 - 1000 PFU / ml for 60 minutes. Take another 150 μl of the dilution and add an equal amount of virus as a virus control. At the same time, add 100 μl of the diluted virus per well to 2 wells of the cells in a 6-well plate for titer detection. Add 100 μl of the neutralized solution per well to the 6-well plate, with 2 replicate wells. Add the dilution as a cell control.
[0031] The results of the neutralizing activity identification are shown in Table 1. The results indicate that the monoclonal antibodies gE-7D9 and gE-11F3 involved in this application have the ability to neutralize live varicella virus. After neutralizing with varicella virus at 500 - 1000 pfu / ml and infecting MRC-5 cells, the reduction rate of cytopathic plaques reaches more than 90%, indicating that the monoclonal antibody has the effect of neutralizing the virus.
[0032] Table 1 Results of neutralizing activity identification
[0033] 6. Methodology establishment Study on the usage concentration of the antibody: A checkerboard test was performed using the 11F3-coated antibody (1, 3, 5 μg / ml) and the 7D9-HRP labeled antibody (0.1, 0.2, 0.5 μg / ml).
[0034] The results of the checkerboard test for the antibody usage concentrations are shown in Table 2. Result analysis: When the coated antibody was at 3 μg / ml and the labeled antibody concentration was 0.2 μg / ml, the calculated accuracy values of the reference samples in the range of 7.81 - 500 ng / ml were all between 80% - 120%. The working concentration of 3 μg / ml for the coated antibody and 0.1 μg / ml for the labeled antibody was selected.
[0035] Table 2 Checkerboard test results of antibody usage concentrations
[0036] 7 Methodological verification: 7.1 Method specificity detection: The relevant indicators during the test process are shown in Table 3: Table 3 Relevant indicators during the method specificity detection test process
[0037] Acceptance criteria: Samples with an OD value ≥ 2.1 times the blank control are considered positive, and those less than the determination criteria are considered negative, without interfering with antigen detection.
[0038] The above results show that the specificity of this detection method is good.
[0039] 7.2 ELISA method precision detection: 7.2.1 Repeatability: Experimental process: Select a batch of protein and detect it six times repeatedly to calculate the RSD.
[0040] Acceptance criteria: The RSD of the protein concentration of the samples repeatedly detected in the same batch of samples ≤ 20%.
[0041] The results of the repeatability test are shown in Table 4: The RSD of the repeatability test results ≤ 20%, meeting the standard.
[0042] Table 4 Results of the repeatability test
[0043] 7.2.2 Intermediate precision: Experimental process: Select three batches of samples and detect them by 2 experimental operators at the same time, and count the RSD Acceptance criteria: For samples of the same batch by different experimental personnel, the RSD of the protein content ≤ 20% The results of the intermediate precision test are shown in Table 5: the RSD of the intermediate precision test results ≤ 20%, meeting the standard.
[0044] Table 5 Results of the intermediate precision test
[0045] 7.3 Accuracy: Test procedure: In this application, the standard product and different samples are tested according to the same dilution concentration and the operation steps of the double antibody sandwich ELISA method. After the test, the different concentrations of the standard product and the samples are compared in parallel.
[0046] Acceptance criteria: Parallelism passes.
[0047] Test results: The ELISA results are run through the software softmax pro7.2 of MD company according to the requirements of the pharmacopoeia for the parallelism detection of two curves, and the parallelism passes, proving that the accuracy of this method is good.
[0048] 7.4 Linear range and detection limit Test procedure: Summarize the results of 6 tests to obtain the linearity, detection range and detection limit of the standard curve.
[0049] Acceptance criteria: The correlation coefficient R2 of the standard curve ≥ 0.99, and the detection ranges and detection limits of each standard curve are consistent.
[0050] Comparing the standard curves, it is found that: The linear range and detection limit are shown in Table 6: Table 6 Results of the linear range and detection limit
[0051] The results of 5 tests show that the detected R2 is all above 0.99, the detection range is 500 ng / ml - 7.8 ng / ml, and the detection limit is 7.8 ng / ml.
[0052] 7.5 Applicability: Test procedure: Compare the detection results of the intermediate product samples at different stages of the newly established method in this application with those of the commercially available kits.
[0053] Acceptance criteria: RSD ≤ 20% The applicability results are shown in Table 7: The detection results of the intermediate product samples at different stages of the newly established method in this application and the commercially available kits are all ≤ 20%, proving that the method in this application is applicable to the detection of samples at all stages of production and there is no difference from the detection values of the commercially available kits.
[0054] Table 7 Applicability results
[0055] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.
Claims
1. An anti-varicella virus neutralizing monoclonal antibody, characterized in that: The anti-varicella virus neutralizing monoclonal antibody is monoclonal antibody gE-7D9 or monoclonal antibody gE-11F3; The heavy chain amino acid sequence of the monoclonal antibody gE-7D9 is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2; The heavy chain amino acid sequence of the monoclonal antibody gE-11F3 is shown in SEQ ID NO:3, and the light chain amino acid sequence is shown in SEQ ID NO:
4.
2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-varicella virus neutralizing monoclonal antibody according to claim 1.
3. A biomaterial, characterized in that: The biological material contains the nucleic acid molecule according to claim 2, and the biological material is selected from any one of an expression cassette, a vector or a host cell.
4. The method for preparing the anti-varicella virus neutralizing monoclonal antibody according to claim 1, characterized in that: Prepared using the nucleic acid molecule of claim 2; or prepared using the biological material of claim 3.
5. The method for preparing the anti-varicella virus neutralizing monoclonal antibody according to claim 1, characterized in that: Specifically, the following steps are performed in sequence: Animal immunization, cell fusion, positive cell line screening, cell line establishment and ascites preparation and purification, neutralization activity identification; the purified antibody was identified as IgG1 subclass.
6. An antibody conjugate, characterized in that: The antibody conjugate is obtained by conjugating the anti-varicella virus neutralizing monoclonal antibody according to claim 1 with a marker, and the marker is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.
7. A detection kit, characterized in that: The method comprises the anti-varicella virus neutralizing monoclonal antibody according to claim 1, or the antibody conjugate according to claim 6.
8. A method for detecting gE protein content by double antibody sandwich ELISA for non-disease diagnosis and treatment purposes, characterized in that: The method comprises using the anti-varicella virus neutralizing monoclonal antibody of claim 1 for detection, using the antibody conjugate of claim 4 for detection, or using the detection kit of claim 6 for detection.
9. The method for detecting gE protein content by double antibody sandwich ELISA according to claim 7, characterized in that: The checkerboard test was performed using gE-11F3 as the coating antibody and gE-7D9-HRP as the enzyme-labeled antibody.
10. The method for detecting gE protein content by double antibody sandwich ELISA according to claim 1, characterized in that: The linear range of the detection method is 7.8 ng / ml-500 ng / ml; the limit of quantification is 7.8 ng / ml.