Monoclonal antibody combination for specific detection of type-5 adenovirus and application of monoclonal antibody combination

Abstract

The invention belongs to the technical field of virus detection, and particularly relates to a composition for specific detection of a monoclonal antibody for type-5 adenovirus and application of the composition. The antibody combination comprises 8B9 and 4A6 which respectively target different epitopes of HADV-5 Hexon protein, and is suitable for a double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) detection system. Wherein 8B9 is used as a coating antibody, 4A6 is used as a detection antibody marked by HRP, high-sensitivity and high-specificity detection of HADV-5 and replication-deficient recombinant adenovirus 5 can be realized through cooperation of 8B9 and 4A6, and cross reaction with other common adenovirus types is avoided. The invention also provides a variable region amino acid sequence and a nucleotide sequence of the antibody, and an ELISA detection method based on the antibody combination. The method is simple and convenient to operate, good in repeatability and suitable for detecting the type 5 adenovirus.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and specifically relates to a combination and application of monoclonal antibodies for specific detection of adenovirus type 5. Background Art

[0002] Adenovirus is an icosahedral double-stranded DNA virus without an envelope, which often invades multiple organs such as the respiratory system, digestive system, urinary system, and conjunctiva. Adenovirus is prevalent throughout the year in the population. Among the adenoviruses that have been found to infect humans, they can be divided into 7 subgroups from A to G, and more than 100 serotypes have been identified so far. Among them, adenovirus type 5 often causes respiratory infections as a pathogen, posing a great health threat to children and immunocompromised populations. In addition, adenovirus type 5 has also been widely used in gene therapy and vaccine development due to its efficient gene delivery ability. For example, after genetic engineering modification, HADV-5 deletes the genes in the E1B and E3 regions and becomes an oncolytic virus that selectively lyses p53-deficient tumor cells, such as the Ankerui replication-deficient recombinant adenovirus type 5 injection for the treatment of advanced nasopharyngeal carcinoma. As a viral vector, HADV-5 can efficiently deliver antigen genes, activate humoral immunity and cellular immunity. Its core is to use a replication-deficient recombinant adenovirus type 5 lacking E1 / E3 or E2 / E4 as a vector to deliver the target gene to human cells to stimulate an immune response or repair gene defects.

[0003] For the specific detection of adenovirus type 5, traditional PCR detection methods rely on nucleic acid amplification and have problems such as complex operation and high cost. Immunological detection methods based on monoclonal antibodies have the advantages of rapidity and sensitivity, but their establishment requires first obtaining antibodies that can specifically recognize HADV-5 and establishing a corresponding detection system. At the same time, adenovirus type 5 vectors are also widely used in the research, development, and production of vaccines, and accurate detection of the viral vector content is required during vaccine production to ensure safety and effectiveness. Therefore, it is very necessary to develop monoclonal antibodies that can be used for specific and accurate detection of HADV-5. Summary of the Invention

[0004] In view of the above-mentioned disadvantages and deficiencies of the prior art, the present invention uses the hybridoma technology to screen and obtain a paired monoclonal antibody combination that can be used for specific detection of adenovirus type 5, and develops an ELISA detection method for specific detection of the HADV-5 Hexon protein. After verification, it can be used for the detection of HADV-5 and replication-deficient recombinant adenovirus type 5, and can meet the needs of multiple scenarios such as clinical diagnosis, vaccine production, and gene therapy vector detection.

[0005] To achieve the above object, the main technical solutions adopted by the present invention include: In a first aspect, the present invention provides a combination of monoclonal antibodies for the specific detection of adenovirus type 5. The combination of monoclonal antibodies includes monoclonal antibody 8B9 and monoclonal antibody 4A6; The heavy chain variable region of monoclonal antibody 8B9 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown in SEQ ID NO.1-SEQ ID NO.3 respectively; The light chain variable region of monoclonal antibody 8B9 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown in SEQ ID NO.4-SEQ ID NO.6 respectively; The heavy chain variable region of monoclonal antibody 4A6 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown in SEQ ID NO.7-SEQ ID NO.9 respectively; The light chain variable region of monoclonal antibody 4A6 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown in SEQ ID NO.10-SEQ ID NO.12 respectively.

[0006] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 8B9 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 8B9 is shown in SEQ ID NO.14.

[0007] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 4A6 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 4A6 is shown in SEQ ID NO.16.

[0008] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 8B9 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 8B9 is shown in SEQ ID NO.18.

[0009] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4A6 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 4A6 is shown in SEQ ID NO.20.

[0010] In some embodiments, adenovirus type 5 includes HADV-5 and replication-deficient recombinant adenovirus type 5.

[0011] In a second aspect, the present invention provides the use of the combination of monoclonal antibodies in the preparation of a tool for detecting adenovirus type 5.

[0012] In some embodiments, the tool includes reagents, reagent kits, test strips, antibody chips, antibody probes or detectors; The reagent is a detection reagent constructed based on the double antibody sandwich ELISA detection method, using the 8B9 monoclonal antibody as the coating antibody and the 4A6 monoclonal antibody as the detection antibody.

[0013] The kit is a detection kit constructed based on the double antibody sandwich ELISA detection method, using the 8B9 monoclonal antibody as the coating antibody and the 4A6 monoclonal antibody as the detection antibody.

[0014] The test strip is a detection test strip constructed based on the double antibody sandwich ELISA detection method, using the 8B9 monoclonal antibody as the coating antibody and the 4A6 monoclonal antibody as the detection antibody.

[0015] The antibody chip is a detection antibody chip constructed based on the double antibody sandwich ELISA detection method, using the 8B9 monoclonal antibody as the coating antibody and the 4A6 monoclonal antibody as the detection antibody.

[0016] In some embodiments, the double antibody sandwich ELISA detection method includes the following steps: Step 1: Coating Use the 8B9 monoclonal antibody as the coating antibody, dissolve it in the coating buffer at a concentration of 1 - 2 μg / mL, add 50 μL to each well, and coat overnight at 4°C on the enzyme-linked immunosorbent assay (ELISA) plate; Step 2: Blocking Discard the coating solution, add 150 μL of blocking solution to each well, and incubate at 37°C for 1 - 2 hours; the blocking solution contains 1 - 2% bovine serum albumin (BSA) or gelatin; Step 3: Sample addition After washing the ELISA plate, add 50 μL of the HADV-5 adenovirus detection sample diluted 1000-fold to each well. The original virus titer of the sample is 1 × 10 12 to 2 × 10 12 VP / mL, and place the ELISA plate at 37°C for 35 minutes; Step 4: Plate washing Wash the ELISA plate 4 times with PBST buffer, soak and shake it well each time; Step 5: Add detection antibody Add the HRP-labeled 4A6 monoclonal antibody as the detection antibody, with a dilution ratio of 1:4000, add 50 μL to each well, and incubate at 37°C for 30 - 60 minutes; Step 6: Wash the plate again Wash the ELISA plate 4 times with PBST buffer to remove the unbound detection antibody; Step 7: Color development and detection Add 50 μL of TMB chromogenic solution to each well, develop color in the dark at room temperature for 10 - 15 minutes; then add 50 μL of TMB stop solution to terminate the reaction, and use an enzyme-linked immunosorbent assay reader to measure OD 450nm absorbance value; Determine whether there is specific antigen against adenovirus type 5 in the sample according to the absorbance.

[0017] In some embodiments, the coating concentration of the 8B9 monoclonal antibody is 1 μg / ml, the dilution factor of the HRP-labeled 4A6 monoclonal antibody is 4000-fold, the incubation condition for each step is 37 °C for 35 min, and the color development is carried out at room temperature for 10 min.

[0018] In addition, the double-antibody sandwich ELISA detection method is not used for the purpose of disease diagnosis.

[0019] The present invention provides a monoclonal antibody combination for detecting adenovirus type 5 with high specificity and good sensitivity and its application. Two monoclonal antibodies, 8B9 and 4A6, specifically recognizing the HADV-5 Hexon protein, are screened by hybridoma technology, and an efficient immunoassay method is constructed based on the principle of double-antibody sandwich ELISA. This antibody combination shows good type specificity in the cross-reactivity experiment, significantly reacts only with HADV-5, and has no obvious cross-reactivity with other common adenovirus types (such as HADV-2, HADV-3, HADV-6, HADV-7, etc.). At the same time, it does not non-specifically bind to host cell components (such as Hep-2 cell lysate), effectively avoiding false positive signals caused by residual cell components during virus purification.

[0020] Furthermore, by HRP-labeling the 4A6 monoclonal antibody and screening for paired antibodies, a double-antibody sandwich ELISA method suitable for the detection of HADV-5 and replication-defective recombinant adenovirus type 5 is established. This method has high sensitivity and can achieve stable and repetitive detection signal output in the virus titer range of 1×10 12 -2×10 12 VP / mL. In addition, this detection method is simple to operate and the result interpretation is rapid, and is applicable to various application scenarios such as the monitoring of virus content in clinical samples, vaccine production processes, and the quality control of gene therapy vectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 It is a diagram of the indirect ELISA identification result of the monoclonal antibody; Figure 2The figure shows the detection specificity results of the double-antibody sandwich ELISA; Figure 3 The detection sensitivity of the double-antibody sandwich ELISA method; Figure 4 The figure shows the monitoring results of the double-antibody sandwich ELISA method for replication-deficient recombinant adenovirus type 5. Specific implementation manners

[0023] The following further describes in detail the implementation manners of the present application in conjunction with the accompanying drawings and embodiments. The detailed descriptions and drawings of the following embodiments are used to exemplarily illustrate the principles of the present application, but cannot be used to limit the scope of the present application. The present application can be implemented in many different forms, not limited to the specific embodiments disclosed in the text, but including all technical solutions falling within the scope of the claims.

[0024] These embodiments are provided by the present application to make the present application thorough and complete, and to fully express the scope of the present application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangements of components and steps, the components of materials, numerical expressions and values described in these embodiments should be construed as merely exemplary, rather than as limitations.

[0025] Example 1 1. Mouse immunization First, HADV-5 purified by ultracentrifugation was diluted to 1x10 8 VP / ml and immunized 4-6-week-old Balb / C female mice at a dose of 200 μl / mouse. At the first immunization, it was mixed and emulsified with an equal volume of Freund's complete adjuvant and immunized subcutaneously at multiple points. For the subsequent second and third booster immunizations, the same dose of HADV-5 was mixed and emulsified with an equal volume of Freund's incomplete adjuvant, with a 2-week interval between each immunization. After the third immunization, the serum was collected to measure the titer, and the mice with higher titers were boosted intraperitoneally with HADV-5 Hexon protein (Abcam, ab123995) at 20 μg / mouse. Three days later, spleen cells were taken for fusion.

[0026] This process describes the mouse immunization protocol for preparing anti-HADV-5 antibodies, which is mainly used for the preparation of monoclonal antibodies. Through this immunization strategy, mice with high-titer anti-HADV-5 antibodies can be obtained, providing an ideal source of spleen cells for the subsequent preparation of specific monoclonal antibodies. HADV-5 is the whole virus obtained by isolation and culture.

[0027] 2. Screening of hybridoma cell lines All spleen cells of immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then cultured in HAT medium for screening. When the fused cells grew to half of the bottom of the well, clones that were positive for HADV-5 and HADV-5 Hexon protein but had a negative cell background were screened by indirect ELISA (HADV-5 adenovirus infects and replicates on Hep-2 sensitive cell lines. Although purified virus can be obtained by ultracentrifugation, it inevitably still contains cell components, which can cause interference in immunization and screening. Generally, the background is removed by coating the reverse screening antigen, such as the supernatant of broken Hep-2 cells, to obtain hybridoma cells that truly react with the target antigen). The positive cells were cloned to the monoclonal state by the limiting dilution method, and then the cell line was expanded and cryopreserved.

[0028] This process describes the screening of monoclonal hybridoma cell lines specific for HADV-5 and HADV-5 Hexon protein after the fusion of spleen cells of immunized mice and myeloma cells. This experiment can obtain hybridoma cell lines that stably secrete specific antibodies, and these cell lines can be used to produce high-quality monoclonal antibodies.

[0029] 3. Screening of positive clones by indirect ELISA: Coat HADV-5 Hexon protein and diluted purified HADV-5 virus (1 x 10 12 VP / ml) in a microplate. The coating buffer is carbonate buffer: 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L with pure water. The coating concentration of HADV-5 Hexon protein is 1 μg / mL, and the purified HADV-5 virus particles are coated after 500-fold dilution. At the same time, collect Hep-2 cells cultured in a T75 culture flask to 90% density. Treatment method: Resuspend with 1 ml of PBS and freeze-thaw 3 times repeatedly. After centrifugation at 12,000 rpm for 15 min, take the supernatant and coat it after 200-fold dilution at 4°C overnight. Block with 1% BSA, 150 μL per well, at 37°C for 2 hours. Wash the plate once with the washing solution and pat dry. Add 50 μL of the culture supernatant of hybridoma cells and react at 37°C for 30 min. Discard the liquid in the wells, wash the plate 4 times with PBST washing solution (0.01 M PBS, 0.1% Tween 20, pH 7.4), pat dry, and then add 50 μL of HRP-labeled goat anti-mouse IgG secondary antibody (Solarbio, SE131) diluted 5000-fold at 50 μL / well and react at 37°C for 30 min. Wash the plate 4 times again, pat dry, add 50 μL of TMB chromogenic solution (single-component chromogenic solution, MedKovan Biotech, 1001) per well and develop color at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (acidic, MedKovan Biotech, 1001SA) to terminate the reaction, and measure OD with an enzyme-linked immunosorbent assay (ELISA) reader. 450nm value. Select positive cell lines that react with both HADV-5 Hexon and HADV-5 virus for subsequent experiments.

[0030] In this experiment, the antigen recognition ability and specificity of antibodies secreted by hybridoma cells were systematically evaluated by indirect ELISA. By introducing the target antigen and the counter-screening antigen (Hep-2 lysate), non-specific signal interference possibly caused by residual host cell components during virus purification was effectively removed, ensuring that the finally screened cell lines producing high-specific monoclonal antibodies truly targeted HADV-5.

[0031] 4. Preparation of monoclonal antibody ascites After the screened monoclonal cell lines were expanded in culture, 0.2 mL (containing 2.5×10 6 cells) of female Balb / C mice pretreated with Freund's incomplete adjuvant were injected intraperitoneally. Approximately 10 days later, when the abdomen of the mice was significantly swollen, ascites was collected using a sterile syringe needle.

[0032] This experiment can obtain a large amount of high-purity and highly specific monoclonal antibodies.

[0033] 5. Affinity chromatography purification of monoclonal antibodies The ascites was centrifuged at 12,000 r / min for 5 minutes. The supernatant was diluted 10-fold with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), filtered through a 0.22-μm filter, and the filtered sample was pumped into a Protein L purification column equilibrated with binding buffer at a flow rate of 1 mL / min using a peristaltic pump. Connect the protein purifier, wash with binding buffer for 5 - 10 column volumes until the UV absorption peak is washed flat, and then elute with elution buffer (0.1 M glycine, pH 2.7). Collect the elution peak. The collected sample was adjusted to neutral with 1 M Tris-HCl, pH 9, loaded into a dialysis bag (MW: 8000 - 14000), and dialyzed in 20 mM PBS, pH 7.4 solution at 2 - 8 °C for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12,000 r / min for 5 minutes. The supernatant was the purified monoclonal antibody. The protein concentration of the purified monoclonal antibody was measured at a wavelength of 280 nm using an ultra-micro spectrophotometer and stored in aliquots.

[0034] This experiment isolated and purified high-purity and high-specific monoclonal antibodies from the prepared mouse ascites.

[0035] 6. Identification of monoclonal antibodies Referring to the indirect ELISA method described above, the purified viruses of four types, namely HADV-2, HADV-3, HADV-6, and HADV-7, were respectively diluted to the same concentration as HADV-5 (the purified virus was 1x10 12 VP / ml) for coating. Meanwhile, the supernatant of Hep-2 cells was coated as a control antigen. Specifically referring to the aforementioned ELISA method, the purified monoclonal antibody was diluted at a concentration of 1 μg / ml, and its reactivity with the adenoviruses of the above five types and Hep-2 cells was detected. Monoclonal antibody strains reactive with HADV-5 (including clones cross-reactive with the above five types of adenoviruses) were screened out for subsequent ELISA pairing tests.

[0036] Experimental results: Twenty-five purified monoclonal antibodies were diluted to a concentration of 1 μg / ml and identified by the indirect ELISA method. It was found that most monoclonal antibodies had cross-reactions with HADV-2, HADV-3, HADV-5, HADV-6, and HADV-7. Among them, only 2 monoclonal antibodies had specific reactions with HADV-5, and all monoclonal antibodies did not react with Hep-2 cells, indicating that the screened monoclonal antibodies reacted with adenoviruses. According to the detection readings, monoclonal antibodies with higher OD 450 nm readings were selected for subsequent coating and labeling pairing tests. The results showed that a total of 13 monoclonal antibodies had readings above 1. Therefore, these 13 monoclonal antibodies were selected for pairing screening.

[0037] See Figure 1 , most monoclonal antibodies showed certain OD 450 nm readings in different adenovirus types represented by different colors, indicating that they had cross-reactions with multiple adenoviruses. Among the numerous monoclonal antibodies, only 2 monoclonal antibodies showed significant reactivity with HADV-5 and no obvious cross-reactions with other adenovirus types. These two monoclonal antibodies were potential ideal candidates for specifically recognizing HADV-5. The blue columns represent the reactions with HADV-5. It can be seen that some monoclonal antibodies (such as the rightmost groups) had relatively high OD 450 nm readings with HADV-5. The green columns represent the reactions with Hep-2 cells, and the readings of all monoclonal antibodies here were close to zero, demonstrating the specificity of the monoclonal antibodies. Thirteen monoclonal antibodies with high readings will be used for subsequent coating and labeling pairing tests to determine the optimal antibody combination and improve the detection sensitivity and specificity.

[0038] 7. Double-antibody sandwich ELISA pairing HRP labeling of antibodies: Specifically: Dilute the antibody to be labeled with carbonate coupling buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water, pH 9.6) to a final concentration of 2 mg / mL. Dissolve 2 mg of HRP in 0.5 mL of ultrapure water, mix well with 0.5 mL of 0.06 M sodium periodate solution. Then add the diluted 1 mg antibody solution to the matching tube containing HRP, pipette and mix well, incubate at room temperature for 1 h, and mix regularly during incubation. Add 50 μL of 5 mg / ml sodium borohydride and mix for 15 min to terminate the labeling reaction. Finally, dialyze the labeled antibody overnight in 0.01 M PBS, pH 7.4 buffer, add glycerol in a 1:1 volume ratio, and store in aliquots at -20 °C.

[0039] The core purpose of this process is to prepare a highly potent and stable HRP-labeled monoclonal antibody as the detection antibody for subsequent use in the double antibody sandwich ELISA experiment. This labeled antibody can significantly enhance the sensitivity and specificity of the detection system.

[0040] Screening of paired antibodies: Coat the purified monoclonal antibodies at a concentration of 1 μg / mL with 50 μL / well of coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water, pH 9.6) overnight at 4 °C. The next day, discard the coating solution, block with 1 - 2% BSA or gelatin, 150 μL / well, incubate at 37 °C for 2 h, discard the blocking solution, add the purified HADV-2, HADV-3, HADV-5, HADV-6, HADV-7 viruses (titer 1x 10 12 VP / ml) diluted 1000-fold into the ELISA plate, 50 μL / well, incubate at 37 °C for 35 min, wash the plate 4 times with PBST wash solution, add the HRP-labeled monoclonal antibody diluted 1000-fold with PBS, 50 μL / well, incubate at 37 °C for 35 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB chromogenic solution, develop color at room temperature for 10 min, and finally add 50 μL of TMB stop solution (acidic, Beijing Meikewanda Biology, 1001SA) to terminate the reaction, and measure the OD 450 nm value with an ELISA reader.

[0041] Select the antibody combination with a higher detection value for HADV-5 and no reaction with other viruses as the optimal pair for the double antibody sandwich ELISA method for specific detection of HADV-5.

[0042] For the 13 monoclonal antibodies with relatively high titers, they were respectively coated and HRP-labeled to screen for paired combinations. Antibodies with high binding affinity have higher detection sensitivity when applied in detection. Purposefully selecting these high-titer antibodies can make the screening of paired monoclonal antibodies more efficient (the paired screening results of coating and labeling the 13 antibodies are not shown, only 2 combinations with relatively high detection values for HADV-5 samples are presented).

[0043] The screening process is shown in Table 1, Table 2 and Table 3.

[0044] Table 1: Detection OD values of different HRP-labeled monoclonal antibodies against various types of HADV viruses when the 7C9 monoclonal antibody is used as the coating antibody.

[0045]

[0046] Table 2: Detection OD values of different HRP-labeled monoclonal antibodies against various types of HADV viruses when the 8B9 monoclonal antibody is used as the coating antibody.

[0047]

[0048] Table 3: Detection OD values of 5F9 and 4A6 HRP-labeled monoclonal antibodies against various HADV viruses at different dilution factors.

[0049]

[0050] Among them, "*" represents the dilution factor.

[0051] Two combinations with relatively high detection values for HADV-5 were screened out. One is using the 8B9 monoclonal antibody as the coating antibody and the 4A6 monoclonal antibody as the HRP-labeled detection antibody, and the other is using the 8B9 monoclonal antibody as the coating antibody and the 5F9 monoclonal antibody as the HRP-labeled detection antibody. Furthermore, these two combinations were compared. Viruses of types HADV-1, HADV-3, HADV-5, HADV-6, HADV-7, HADV-10, and HADV-41 were diluted 500-fold, 5000-fold, 50000-fold, and 500000-fold respectively, and the detection results of these two combinations for the samples, especially HADV-5, were compared.

[0052] The results showed that the combination of coating with the 8B9 monoclonal antibody and HRP-labeling with the 4A6 monoclonal antibody had higher detection sensitivity for HADV-5 and did not cross-react with other HADVs, and thus was determined as the optimal paired monoclonal antibody.

[0053] 8. Specificity and sensitivity identification of the double-antibody sandwich ELISA for specific detection of HADV-5 After determining the combination of coated and labeled antibodies, the optimal concentration of monoclonal antibody coating and the dilution of HRP-labeled monoclonal antibody were further established.

[0054] The 8B9 monoclonal antibody was coated at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL with 50 μL / well of coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water, pH 9.6) overnight at 4°C. The next day, the coating solution was discarded, and blocked with 1-2% BSA, 150 μL / well, incubated at 37°C for 2 h. The blocking solution was discarded, and the positive protein HADV-5 Hexon and control samples were diluted to 100 ng / ml with PBS and added to the ELISA plate, 50 μL / well, incubated at 37°C for 35 min, and the plate was washed 4 times with PBST washing solution.

[0055] HRP-labeled 4A6 monoclonal antibody diluted 1000, 2000, 4000, and 8000 times with PBS was added, 50 μL / well, incubated at 37°C for 35 min, then the plate was washed 4 times again. After blotting dry, 50 μL / well of TMB chromogenic solution was added and developed at room temperature for 10 min. Finally, 50 μL of TMB stop solution (acidic, Beijing Meikewanda Biology, 1001SA) was added to terminate the reaction, and the OD 450 nm value was measured with an ELISA reader.

[0056] The condition with the highest positive reading and a negative control was taken as the optimal condition for the double antibody sandwich ELISA. The detection specificity and sensitivity of this method were evaluated under this optimal condition. Specifically: The 8B9 monoclonal antibody was coated at a concentration of 1 μg / ml with 50 μL / well of the above coating buffer overnight at 4°C. The coating solution was discarded, and each well was added with 150 μL of 2% BSA and incubated at 37°C for 2 h for blocking. The test sample was added, and after incubation, the plate was washed 4 times. Then, the HRP-labeled 4A6 monoclonal antibody was diluted 4000 times and added. The incubation condition for each step was 37°C for 35 min. Finally, it was developed at room temperature for 10 min, and 50 μL of TMB was added to terminate the reaction, and the detection value was read at the OD 450 nm wavelength.

[0057] When testing the specificity of the detection method, purified HADV-1, HADV-2, HADV-3, HADV-6, HADV-7, HADV-10, and HADV-41 viruses (titer 1 x 10 12They were detected after being diluted 500-fold (VP / ml). Verify whether this antibody combination only recognizes HADV-5 and does not cross-react with other adenovirus serotypes. When analyzing the sensitivity of the assay method, the purified HADV-5 Hexon protein was serially diluted 2-fold starting from a concentration of 1 μg / ml for detection, and the Fusion protein of respiratory syncytial virus (RSV) was used as a control.

[0058] Analysis of test results: The coating concentration of the 8B9 monoclonal antibody was determined to be 1 μg / ml, and the dilution factor of the HRP-labeled 4A6 monoclonal antibody was 4000-fold. The incubation conditions for each step were 37 °C for 35 min, and the color development was carried out at room temperature for 10 min, which were the optimal detection conditions. The detection specificity and sensitivity of this method were evaluated under these conditions. The results showed that this double-antibody sandwich ELISA method could only detect HADV-5 and did not react with viruses of HADV-1, HADV-2, HADV-3, HADV-6, HADV-7, HADV-10, and HADV-41 types (see Figure 2 ). Among them, HADV-5, HADV-1, HADV-2, and HADV-6 belong to subgroup C, HADV-3 and HADV-7 belong to subgroup B, HADV-10 belongs to subgroup D, and HADV-41 belongs to subgroup F. The results indicated that this detection method was specific for HADV-5. See Figure 3 . Using the purified HADV-5 Hexon protein to detect the sensitivity of this method, it was found that when the concentration was 3.9 ng / ml, the detection result was still weakly positive, indicating that the method had high detection sensitivity for the purified native protein of HADV-5.

[0059] 9. Detection and application of replication-defective recombinant adenovirus type 5 The replication-defective recombinant adenovirus type 5 was sourced from Beijing Wu Jia He Gene Technology Co., Ltd., and the susceptible cell line HEK-293A that it can infect was preserved by our company. Normally, HADV-5 virus can infect Hep-2 cells, but the replication-defective recombinant adenovirus type 5 (rADV) usually lacks the E1 gene and cannot replicate in ordinary cells. It can only replicate in cell lines that specifically express the E1 protein. 293A cells can provide the E1 protein required for replication for the replication-defective recombinant adenovirus type 5 lacking the E1 gene, so it can effectively infect and replicate. The stock solution of the replication-defective recombinant adenovirus type 5 (the stock solution titer was 4.2 x 10 8 TCID 50 / mL) was diluted 1000-fold and 10000-fold respectively, used to infect 293A cells, and the double-antibody sandwich ELISA method was used to monitor the content and replication of adenovirus type 5.

[0060] The experiment was carried out according to the aforementioned optimal detection conditions, namely: the coating concentration of the 8B9 monoclonal antibody was 1 μg / ml, the dilution factor of the 4A6 monoclonal antibody labeled with HRP was 4000-fold, the incubation conditions for each step were 37 °C for 35 min, and the color development was carried out at room temperature for 10 min. The test results are shown in Figure 4 , and the double-antibody sandwich ELISA method established using paired monoclonal antibodies can detect HADV-5 that can infect humans. Further, the packaged replication-deficient recombinant adenovirus type 5 was diluted 1000 and 10000 times, and the 293A sensitive cell line was infected respectively, and the replication-deficient recombinant adenovirus type 5 was detected by this method. It was found that the replication-deficient recombinant adenovirus type 5 could effectively infect 293A cells and the cytopathic effect (CPE) could be observed. The detection results of the double-antibody sandwich ELISA method showed that the OD reading gradually increased with the time after infection, indicating that the virus was continuously replicating, suggesting that this method could also effectively detect the replication-deficient recombinant adenovirus.

[0061] This experiment demonstrated the successful monitoring of the infection and replication dynamics of the replication-deficient recombinant adenovirus type 5 in specific host cells (HEK-293A) using the optimized double-antibody sandwich ELISA method. The cell control was the culture supernatant of uninfected 293A cells. 10 -3 and 10 -4 respectively represent the supernatants of cells infected with the virus diluted 10 3 and 10 4 times. The results showed that with the increase of the infection time, the virus content increased and the OD reading rose, confirming the effectiveness and sensitivity of this method for such viruses, indicating that the double-antibody sandwich ELISA method can not only detect HADV-5, but also effectively detect the replication of the replication-deficient recombinant adenovirus type 5. This is of great significance for the fields of virology research, development of new vaccine vectors and gene therapy.

[0062] 10. Gene sequence Cloning and sequencing of the variable region genes of monoclonal antibodies.

[0063] The total RNA of hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was reverse transcribed using RandomPrimers; universal primers for the variable regions of mouse antibodies were designed, and the VH and VL genes were amplified by 2 rounds of PCR. The Age1 and Bsiw1 restriction enzyme sites were introduced into the primers of the 3rd round of PCR. After the PCR products were gel-purified, they were ligated to the pUC19 vector, transformed into TOP10 strains, and single colonies were picked for sequencing after culturing at 37 °C for 14 h to obtain the gene sequences of the heavy and light chains of the monoclonal antibody.

[0064] The specific sequences are as follows: Among them, the amino acids of the heavy chain complementarity-determining regions of the 8B9 monoclonal antibody are as follows: CDR-H1 (SEQ ID NO.1): SYYMH; CDR-H2 (SEQ ID NO.2): YLNPVNDSTKYMEKFKG; CDR-H3 (SEQ ID NO.3): AIYESYYGY.

[0065] The amino acids of the light chain complementarity-determining regions of the 8B9 monoclonal antibody are as follows: CDR-L1 (SEQ ID NO.4): KASKRVSISGYSYMH; CDR-L2 (SEQ ID NO.5): LASNLLG; CDR-L3 (SEQ ID NO.6): QHGRLLPST.

[0066] The amino acids of the heavy chain complementarity-determining regions of the 4A6 monoclonal antibody are as follows: CDR-H1 (SEQ ID NO.7): GSVFK; CDR-H2 (SEQ ID NO.8): ELSPVIDSTSVLERAQQ; CDR-H3 (SEQ ID NO.9): MLKSVISV.

[0067] The amino acids of the light chain complementarity-determining regions of the 4A6 monoclonal antibody are as follows: CDR-L1 (SEQ ID NO.10): FARKSVLLSRVSYHI; CDR-L2 (SEQ ID NO.11): ETSVVG; CDR-L3 (SEQ ID NO.12): MHSGYLPST.

[0068] The amino acid sequence of the heavy chain variable region of the 8B9 monoclonal antibody (SEQ ID NO.13): EVKLEESGPELVKPGASVKMSCKASGYTFTSYYMHWVKQKPGQGLEWIGYLNPVNDSTKYMEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARAIYESYYGYWGQGTTLTVSA.

[0069] The amino acid sequence of the light chain variable region of the 8B9 monoclonal antibody (SEQ ID NO.14): DIQMNQSPASLAVSLGQRATISCKASKRVSISGYSYMHWYQQKPGQPPKLLIYLASNLLGGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHGRLLPSTFGGGTKLELKRTV.

[0070] Amino acid sequence of the heavy chain variable region of the 4A6 monoclonal antibody (SEQ ID NO.15): EVMLAESGPELVKPGASVKMSCKASGYTFTGSVFKWVKQKPGQGLEWIGELSPVIDSTSVLERAQQKATLTSDKSSSTAYMELSSLTSEDSAVYYCARMLKSVISVWGQGTTLTVSS.

[0071] Amino acid sequence of the light chain variable region of the 4A6 monoclonal antibody (SEQ ID NO.16): EIVLTQSPASLAVSLGQRATISCFARKSVLLSRVSYHIWYQQKPGQPPKLLIYETSVVGGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMHSGYLPSTFGGGTKLEIKRTV.

[0072] Nucleotide sequence of the heavy chain variable region of the 8B9 monoclonal antibody (SEQ ID NO.17): GAGGTGAAGCTGGAGGAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATGCACTGGGTGAAGCAGAAGCCCGGCCAGGGCCTGGAGTGGATCGGCTACCTGAACCCCGTGAACGACAGCACCAAGTACATGGAGAAGTTCAAGGGCAAGGCCACCCTGACCAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGGCCATCTACGAGAGCTACTACGGCTACTGGGGCCAGGGCACCACCCTGACCGTGAGCGCC.

[0073] The nucleotide sequence of the light chain variable region of the 8B9 monoclonal antibody is as follows (SEQ ID NO.18): GACATCCAGATGAACCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCAAGGCCAGCAAGAGGGTGAGCATCAGCGGCTACAGCTACATGCACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACCTGGCCAGCAACCTGCTGGGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCCAGCACGGCAGGCTGCTGCCCAGCACCTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG。

[0074] The nucleotide sequence of the heavy chain variable region of the 4A6 monoclonal antibody (SEQ ID NO.19): GAGGTGATGCTGGCCGAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCAGCGTGTTCAAGTGGGTGAAGCAGAAGCCCGGCCAGGGCCTGGAGTGGATCGGCGAGCTGAGCCCCGTGATCGACAGCACCAGCGTGCTGGAGAGGGCCCAGCAGAAGGCCACCCTGACCAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGATGCTGAAGAGCGTGATCAGCGTGTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

[0075] Nucleotide sequence of the light chain variable region of the 4A6 monoclonal antibody (SEQ ID NO.20): GAGATCGTGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCTTCGCCAGGAAGAGCGTGCTGCTGAGCAGGGTGAGCTACCACATCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACGAGACCAGCGTGGTGGGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCATGCACAGCGGCTACCTGCCCAGCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG。

[0076] In summary, the antibody combination of the present invention includes two monoclonal antibodies, 8B9 and 4A6, which target different epitopes of the HADV-5 Hexon protein respectively and are suitable for a double-antibody sandwich ELISA detection system. Among them, 8B9 is used as the coating antibody, and 4A6 is used as the HRP-labeled detection antibody. The two cooperate to achieve highly sensitive and highly specific detection of HADV-5 and replication-deficient recombinant adenovirus type 5, and do not cross-react with other common adenovirus types. The present invention also provides the amino acid sequences and nucleotide sequences of the variable regions of the above antibodies, as well as an ELISA detection method based on the antibody combination. This method is simple to operate and has good repeatability, and is suitable for the detection of adenovirus type 5.

[0077] So far, the embodiments of the present application have been described in detail. To avoid obscuring the concept of the present application, some details well known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0078] Although some specific embodiments of the present application have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration and not for limiting the scope of the present application. Those skilled in the art should understand that the above embodiments can be modified or partial technical features can be equivalently replaced without departing from the scope and spirit of the present application.

Claims

1. A combination of monoclonal antibodies for the specific detection of adenovirus type 5, characterized in that, The monoclonal antibody combination includes monoclonal antibody 8B9 and monoclonal antibody 4A6; The heavy chain variable region of the monoclonal antibody 8B9 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are respectively shown as SEQ ID NO.1-SEQ ID NO.3; The light chain variable region of the monoclonal antibody 8B9 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are respectively shown as SEQ ID NO.4-SEQ ID NO.6; The heavy chain variable region of the monoclonal antibody 4A6 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are respectively shown as SEQ ID NO.7-SEQ ID NO.9; The light chain variable region of the monoclonal antibody 4A6 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are respectively shown as SEQ ID NO.10-SEQ ID NO.

12.

2. The combination of monoclonal antibodies for specific detection of adenovirus type 5 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 8B9 is shown as SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 8B9 is shown as SEQ ID NO.

14.

3. The combination of monoclonal antibodies for specific detection of adenovirus type 5 according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4A6 is shown as SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 4A6 is shown as SEQ ID NO.

16.

4. The combination of monoclonal antibodies for the specific detection of adenovirus type 5 according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8B9 is shown as SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8B9 is shown as SEQ ID NO.

18.

5. The combination of monoclonal antibodies for specific detection of adenovirus type 5 according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4A6 is shown as SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4A6 is shown as SEQ ID NO.

20.

6. The combination of monoclonal antibodies for specific detection of adenovirus type 5 according to claim 5, characterized in that, The adenovirus type 5 includes HADV-5 and replication-deficient recombinant adenovirus type 5.

7. Use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for detecting adenovirus type 5.

8. The application according to claim 7, wherein The tool includes reagents, kits, test strips and antibody chips; The reagent is a detection reagent constructed based on the double antibody sandwich ELISA detection method, using monoclonal antibody 8B9 as the coating antibody and monoclonal antibody 4A6 as the detection antibody; The kit is a detection kit constructed based on the double antibody sandwich ELISA detection method, using monoclonal antibody 8B9 as the coating antibody and monoclonal antibody 4A6 as the detection antibody; The test strip is a detection test strip constructed based on the double antibody sandwich ELISA detection method, using monoclonal antibody 8B9 as the coating antibody and monoclonal antibody 4A6 as the detection antibody; The antibody chip is a detection antibody chip constructed based on the double antibody sandwich ELISA detection method, using monoclonal antibody 8B9 as the coating antibody and monoclonal antibody 4A6 as the detection antibody.

9. The application according to claim 8, characterized in that, The double antibody sandwich ELISA detection method includes the following steps: Step 1: Coating Use the 8B9 monoclonal antibody as the coating antibody, dissolve it in the coating buffer at a concentration of 1 - 2 μg / mL, add 50 μL to each well, and coat overnight at 4°C on the enzyme-linked immunosorbent assay (ELISA) plate; Step 2: Blocking Discard the coating solution, add 150 μL of blocking solution to each well, and incubate at 37°C for 1 - 2 hours; the blocking solution contains 1 - 2% bovine serum albumin (BSA) or gelatin; Step 3: Sample addition After washing the ELISA plate, add 50 μL of the HADV-5 adenovirus detection sample diluted 1000-fold to each well. The original virus titer of the sample is 1 × 10 12 to 2 × 10 12 VP / mL, and incubate the ELISA plate at 37°C for 35 minutes; Step 4: Wash the plate Wash the ELISA plate 4 times with PBST buffer, soak and shake well each time; Step 5: Add the detection antibody Add the HRP-labeled 4A6 monoclonal antibody as the detection antibody, with a dilution ratio of 1:4000, add 50 μL to each well, and incubate at 37°C for 30 - 60 minutes; Step 6: Wash the plate again Wash the ELISA plate 4 times with PBST buffer to remove the unbound detection antibody; Step 7: Color development and detection Add 50 μL of TMB chromogenic solution to each well and develop color at room temperature in the dark for 10 - 15 minutes; then add 50 μL of TMB stop solution to terminate the reaction, and measure the absorbance at OD 450 nm absorbance value; judge whether there is specific antigen against adenovirus type 5 in the sample according to the absorbance.

10. The application according to claim 9, wherein The coating concentration of the 8B9 monoclonal antibody is 1 μg / ml, the dilution factor of the HRP-labeled 4A6 monoclonal antibody is 4000-fold, the incubation condition for each step is 37°C for 35 minutes, and color development is carried out at room temperature for 10 minutes.

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