A combination and application of monoclonal antibodies for specific detection of adenovirus type 5

Abstract

The present invention belongs to the field of virus detection technology, and specifically relates to a combination and application of monoclonal antibodies for the specific detection of type 5 adenovirus. The antibody combination includes 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 a coating antibody, and 4A6 is used as an HRP-labeled detection antibody. The combination of the two can achieve high sensitivity and high specificity detection of HADV‑5 and replication-deficient recombinant type 5 adenovirus, and does not cross-react with other common adenovirus types. The present invention also provides the variable region amino acid sequence and nucleotide sequence of the above-mentioned antibody, as well as an ELISA detection method based on the antibody combination. The method is simple to operate, has good repeatability, and is suitable for the detection of type 5 adenovirus.

Description

Technical Field

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

[0002] Adenoviruses are non-enveloped, icosahedral, double-stranded DNA viruses that frequently invade multiple organs, including the respiratory, digestive, urinary, and conjunctival systems. Adenoviruses are prevalent in the human population year-round. Adenoviruses that have been found to infect humans can be divided into seven subgroups, A and B, with over 100 serotypes identified. Adenovirus type 5, among them, frequently causes respiratory infections, posing a significant health threat to children and immunocompromised individuals. Furthermore, due to its efficient gene delivery capabilities, adenovirus type 5 is widely used in gene therapy and vaccine development. For example, HADV-5 has been genetically engineered to delete the E1B and E3 regions, resulting in an oncolytic virus that selectively lyses p53-deficient tumor cells. This is exemplified by the Ankorui replication-deficient recombinant adenovirus type 5 injection used to treat advanced nasopharyngeal carcinoma. As a viral vector, HADV-5 can efficiently deliver antigen genes and activate humoral immunity and cellular immunity. Its core is to use a replication-deficient recombinant adenovirus type 5 that lacks E1 / E3 or E2 / E4 as a vector to deliver target genes to human cells to stimulate immune responses or repair genetic defects.

[0003] For the specific detection of adenovirus type 5, the traditional PCR detection method relies on nucleic acid amplification, which has problems such as complex operation and high cost. The immunological detection method based on monoclonal antibodies has the advantages of being fast and sensitive, but its 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 the content of viral vectors must be accurately detected during vaccine production to ensure safety and effectiveness. Therefore, it is very necessary to develop monoclonal antibodies that can be used for the specific and accurate detection of HADV-5. Summary of the Invention

[0004] In view of the above-mentioned shortcomings and deficiencies of the existing technology, the present invention uses hybridoma technology to screen and obtain a paired monoclonal antibody combination that can be used for the specific detection of adenovirus type 5, and develops an ELISA detection method for the specific detection of HADV-5 Hexon protein. It has been verified that 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] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0006] In a first aspect, the present invention provides a combination of monoclonal antibodies for specific detection of adenovirus type 5, the combination of monoclonal antibodies comprising 8B9 monoclonal antibody and 4A6 monoclonal antibody;

[0007] The heavy chain variable region of the 8B9 monoclonal antibody includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively;

[0008] The light chain variable region of the 8B9 monoclonal antibody includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively;

[0009] The heavy chain variable region of the 4A6 monoclonal antibody includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 7 to SEQ ID NO. 9, respectively;

[0010] The light chain variable region of the 4A6 monoclonal antibody includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 10 to SEQ ID NO. 12, respectively.

[0011] In some embodiments, the amino acid sequence of the heavy chain variable region of the 8B9 monoclonal antibody is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the 8B9 monoclonal antibody is shown in SEQ ID NO.14.

[0012] In some embodiments, the amino acid sequence of the heavy chain variable region of the 4A6 monoclonal antibody is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the 4A6 monoclonal antibody is shown in SEQ ID NO.16.

[0013] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the 8B9 monoclonal antibody is shown as SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the 8B9 monoclonal antibody is shown as SEQ ID NO.18.

[0014] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the 4A6 monoclonal antibody is shown as SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the 4A6 monoclonal antibody is shown as SEQ ID NO. 20.

[0015] In some embodiments, adenovirus type 5 includes HADV-5 and replication-deficient recombinant adenovirus type 5.

[0016] In a second aspect, the present invention provides a method for preparing a tool for detecting adenovirus type 5 using a combination of monoclonal antibodies.

[0017] In some embodiments, the tool comprises a reagent, a kit, a test strip, an antibody chip, an antibody probe, or a detector;

[0018] The reagent is a detection reagent constructed based on the double antibody sandwich ELISA detection method, using 8B9 monoclonal antibody as the coating antibody and 4A6 monoclonal antibody as the detection antibody.

[0019] The kit is a detection kit constructed based on the double-antibody sandwich ELISA detection method, using 8B9 monoclonal antibody as the coating antibody and 4A6 monoclonal antibody as the detection antibody.

[0020] The test strip is constructed based on the double antibody sandwich ELISA detection method, using 8B9 monoclonal antibody as the coating antibody and 4A6 monoclonal antibody as the detection antibody.

[0021] The antibody chip is a detection antibody chip constructed based on the double antibody sandwich ELISA detection method, using 8B9 monoclonal antibody as the coating antibody and 4A6 monoclonal antibody as the detection antibody.

[0022] In some embodiments, the double antibody sandwich ELISA detection method comprises the following steps:

[0023] Step 1: Coating

[0024] Use 8B9 monoclonal antibody as the coating antibody, dissolve it in coating buffer at a concentration of 1-2 μg / mL, add 50 μL to each well, and coat the plate at 4°C overnight;

[0025] Step 2: Closure

[0026] Discard the coating solution and add 150 μL of blocking solution to each well. Incubate at 37°C for 1–2 hours. The blocking solution contains 1–2% BSA or gelatin.

[0027] Step 3: Add sample

[0028] After washing the ELISA plate, 50 μL of 1000-fold diluted HADV-5 adenovirus test sample was added to each well. The original virus titer of the sample was 1 × 10 12 to 2 × 10 12 VP / mL, and incubate the ELISA plate at 37°C for 35 minutes;

[0029] Step 4: Wash the plate 4 times with PBST buffer, soaking and shaking thoroughly each time;

[0030] Step 5: Add detection antibody

[0031] HRP-labeled 4A6 monoclonal antibody was added as the detection antibody at a dilution ratio of 1:4000, 50 μL was added to each well, and incubated at 37°C for 30–60 minutes;

[0032] Step 6: Wash the plate again with PBST buffer 4 times to remove unbound detection antibody;

[0033] Step 7: Color development and detection

[0034] Add 50 μL of TMB colorimetric solution to each well and develop the 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 OD value using a microplate reader. 450 nm absorbance value; determine whether the sample contains specific antigens for adenovirus type 5 based on the absorbance.

[0035] In some embodiments, the coating concentration of 8B9 monoclonal antibody is 1 ug / ml, the dilution factor of HRP-labeled 4A6 monoclonal antibody is 4000-fold, and the incubation conditions for each step are 37° C., 35 min, and color development at room temperature for 10 min.

[0036] In addition, the double antibody sandwich ELISA detection method is not used for disease diagnosis purposes.

[0037] The present invention provides a highly specific and sensitive monoclonal antibody combination for detecting adenovirus type 5 and its application. Two monoclonal antibodies, 8B9 and 4A6, that specifically recognize the HADV-5 Hexon protein were screened using hybridoma technology, and a highly efficient immunoassay method was constructed based on the double-antibody sandwich ELISA principle. This antibody combination demonstrated good type specificity in cross-reactivity experiments, reacting significantly only with HADV-5 and showing no significant cross-reactivity with other common adenovirus types (such as HADV-2, HADV-3, HADV-6, and HADV-7). Furthermore, it exhibited no nonspecific binding to host cell components (such as Hep-2 cell lysate), effectively avoiding false-positive signals caused by residual cellular components during the virus purification process.

[0038] Furthermore, a double-antibody sandwich ELISA method suitable for the detection of HADV-5 and replication-deficient recombinant adenovirus type 5 was established by HRP-labeling the 4A6 monoclonal antibody and performing paired antibody screening. This method has high sensitivity and can detect HADV-5 at a virus titer of 1×10 12 -2×10 12 Stable and reproducible detection signal output is achieved within the VP / mL range. Furthermore, this assay is simple to operate and provides rapid result interpretation, making it suitable for a variety of applications, including clinical samples, viral content monitoring during vaccine production, and quality control of gene therapy vectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only 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.

[0040] Figure 1 This is the result of indirect ELISA identification of monoclonal antibodies;

[0041] Figure 2 This is a graph showing the detection specificity results of the double-antibody sandwich ELISA;

[0042] Figure 3 is the detection sensitivity of the double antibody sandwich ELISA method;

[0043] Figure 4 This is a graph showing the monitoring results of the replication-defective recombinant adenovirus type 5 using the double-antibody sandwich ELISA method. DETAILED DESCRIPTION

[0044] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are intended to illustrate the principles of the present application, but are not intended to limit the scope of the present application. The present application may be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but rather includes all technical solutions within the scope of the claims.

[0045] The present application provides these embodiments to make this application thorough and complete, and to fully express the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values ​​set forth in these embodiments should be interpreted as merely exemplary, and not as limiting.

[0046] Example 1

[0047] 1. Mouse immunization

[0048] First, HADV-5 purified by ultracentrifugation was diluted to 1x10 84-6-week-old Balb / C female mice were immunized with VP / ml at a dose of 200 μl / mouse. For the first immunization, HADV-5 was mixed and emulsified with an equal volume of Freund's complete adjuvant and administered subcutaneously at multiple sites. Subsequent booster immunizations were administered two weeks apart using the same dose of HADV-5 mixed with an equal volume of Freund's incomplete adjuvant. Serum was collected after the third immunization for titer determination. Mice with the highest titer were then intraperitoneally injected with 20 μg / mouse of HADV-5 Hexon protein (Abcam, ab123995). Three days later, spleen cells were harvested for fusion.

[0049] This process describes a mouse immunization protocol for producing anti-HADV-5 antibodies, primarily for the preparation of monoclonal antibodies. This immunization strategy can generate mice with high-titer anti-HADV-5 antibodies, providing an ideal source of splenocytes for subsequent production of specific monoclonal antibodies. HADV-5 is a whole virus isolated and cultured.

[0050] 2. Screening of hybridoma cell lines

[0051] All spleen cells from immunized mice are fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then cultured in HAT medium for screening. When the fused cells reach 1 / 2 of the bottom of the well, clones that are positive for HADV-5 and HADV-5 Hexon proteins but negative for cellular background are screened using indirect ELISA. (HADV-5 adenovirus infects and replicates on the sensitive Hep-2 cell line. Purified virus is obtained by ultracentrifugation, but it inevitably still contains cellular components that can interfere with immunization and screening. Background is generally removed by coating with a counter-screening antigen, such as the supernatant of Hep-2 cell fragments, to obtain hybridoma cells that truly react with the target antigen.) Positive cells are cloned to a monoclonal state by limiting dilution, and the cell line is then expanded and cryopreserved.

[0052] This study describes the process from fusion of spleen cells from immunized mice with myeloma cells to the screening of monoclonal hybridoma cell lines specific for HADV-5 and HADV-5 Hexon proteins. This experiment yields hybridoma cell lines that stably secrete specific antibodies, which can be used to produce high-quality monoclonal antibodies.

[0053] 3. Screening of positive clones by indirect ELISA:

[0054] The microtiter plates were coated with HADV-5 Hexon protein and diluted purified HADV-5 virus (1 x 10 12VP / ml). Coating buffer: carbonate buffer: 1.59g sodium carbonate, 2.93g sodium bicarbonate, diluted to 1L of pure water. HADV-5 Hexon protein coating concentration: 1μg / mL. Purified HADV-5 viral particles were diluted 500-fold for coating. Hep-2 cells cultured to 90% confluency were collected from a T75 culture flask. Treatment: Resuspend in 1ml PBS and freeze-thaw three times. Centrifuge at 12,000 rpm for 15 minutes. The supernatant was diluted 200-fold for coating and incubated at 4°C overnight. Block with 1% BSA (150μL per well) at 37°C for 2 hours. Wash the plate once with wash buffer and pat dry. Add 50μL of hybridoma cell culture supernatant and incubate at 37°C for 30 minutes. The liquid in the wells was shaken off, and the plate was washed four times with PBST washing solution (0.01M PBS, 0.1% Tween 20, pH 7.4). After patting dry, 50 μL / well of 5000-fold diluted HRP-labeled goat anti-mouse IgG secondary antibody (Beijing Solebao, SE131) was added. The reaction was carried out at 37°C for 30 min. The plate was washed four times again, and after patting dry, 50 μL / well of TMB color development solution (single-component color development solution, Beijing Meikewande Biological, 1001) was added for color development at room temperature for 10 min. Finally, 50 μL TMB stop solution (acidic, Beijing Meikewande Biological, 1001SA) was added to terminate the reaction, and the OD was measured using a microplate reader. 450 The positive cell lines that reacted with both HADV-5 Hexon and HADV-5 virus were selected for subsequent experiments.

[0055] This study systematically evaluated the antigen recognition and specificity of antibodies secreted by hybridoma cells using an indirect ELISA method. By introducing the target antigen and a counter-screening antigen (Hep-2 lysate), nonspecific signal interference, potentially caused by residual host cell components during the virus purification process, was effectively eliminated, ensuring that the final screening cell line was a true, highly specific monoclonal antibody producing cell line targeting HADV-5.

[0056] 4. Preparation of Monoclonal Antibody Ascites

[0057] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×10 6 Female Balb / C mice (100 cells) were pretreated with Freund's incomplete adjuvant. About 10 days later, when the abdomen of the mice was obviously swollen, ascites was collected using a sterile syringe needle.

[0058] This experiment can produce large quantities of high-purity and highly specific monoclonal antibodies.

[0059] 5. Affinity chromatography purification of monoclonal antibodies

[0060] Ascites was centrifuged at 12,000 rpm for 5 minutes. The supernatant was diluted 10-fold with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4) and filtered through a 0.22 μm filter. The filtered sample was pumped through a Protein L purification column equilibrated with binding buffer at a flow rate of 1 ml / min using a peristaltic pump. The column was then connected to a protein purifier and washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled out. The column was then eluted with elution buffer (0.1 M glycine, pH 2.7). The eluted peak was collected and adjusted to neutral with 1 M Tris-HCl, pH 9. The sample was placed in a dialysis bag (MW: 8,000-14,000) and dialyzed against 20 mM PBS, pH 7.4, at 2-8°C for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody. The protein concentration of the purified monoclonal antibody was measured using an ultra-micro spectrophotometer at a wavelength of 280 nm and the antibody was aliquoted for storage.

[0061] In this experiment, high-purity and high-specificity monoclonal antibodies were isolated and purified from the prepared mouse ascites.

[0062] 6. Identification of Monoclonal Antibodies

[0063] Referring to the above-mentioned indirect ELISA method, the purified viruses of HADV-2, HADV-3, HADV-6, and HADV-7 were diluted to the same concentration as HADV-5 (the purified virus was 1 x 10 12 VP / ml) for coating, and Hep-2 cell supernatant was coated as a control antigen. Referring to the above-mentioned ELISA method, the purified monoclonal antibody was diluted to a concentration of 1 μg / ml, and its reactivity with the above five types of adenovirus and Hep-2 cells was tested. Monoclonal antibody strains reactive with HADV-5 (including clones cross-reactive with all five types of adenovirus) were screened for subsequent ELISA pairing tests.

[0064] Experimental results: The 25 purified monoclonal antibodies were diluted to a concentration of 1ug / ml and identified by indirect ELISA. 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 two monoclonal antibodies reacted specifically with HADV-5, and all monoclonal antibodies did not react with Hep-2 cells, indicating that the screened monoclonal antibodies reacted with adenovirus. According to the detection reading, OD 450 Monoclonal antibodies with higher nm readings were subjected to subsequent coating and labeling pairing tests. The results showed that a total of 13 monoclonal antibodies had readings above 1, so these 13 monoclonal antibodies were selected for pairing screening.

[0065] See also Figure 1 Most monoclonal antibodies showed a certain OD in different adenovirus types represented by different colors. 450 The nm readings indicate that they cross-react with a variety of adenoviruses. Among the numerous monoclonal antibodies, only two showed significant reactivity with HADV-5, while there was no significant cross-reaction with other adenovirus types. These two monoclonal antibodies are potential ideal candidates for specific recognition of HADV-5. The blue columns represent the reaction with HADV-5. It can be seen that some monoclonal antibodies (such as the groups on the far right) have higher OD with HADV-5. 450 The green column represents the reaction with Hep-2 cells. All monoclonal antibodies showed a reading close to zero here, demonstrating their specificity. The 13 monoclonal antibodies with high readings will be used in subsequent coating and labeling pairing experiments to identify the optimal antibody combination and improve detection sensitivity and specificity.

[0066] 7. Double Antibody Sandwich ELISA Pairing

[0067] HRP labeling of antibodies:

[0068] Specifically, dilute the labeled antibody to a final concentration of 2 mg / mL in carbonate coupling buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6). Dissolve 2 mg HRP in 0.5 mL ultrapure water and mix thoroughly with 0.5 mL of 0.06 M sodium periodate solution. Add 1 mg of the diluted antibody solution to the HRP tube and pipette to mix thoroughly. Incubate at room temperature for 1 hour, mixing regularly during the incubation period. Terminate the labeling reaction by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 minutes. Finally, dialyze the labeled antibody overnight against 0.01 M PBS, pH 7.4. Add glycerol in a 1:1 ratio and store in aliquots at -20°C.

[0069] The core purpose of this process is to prepare a high-titer and stable HRP-labeled monoclonal antibody as a detection antibody for subsequent use as a detection antibody in a double-antibody sandwich ELISA experiment. This labeled antibody can significantly enhance the sensitivity and specificity of the detection system.

[0070] Screening of paired antibodies: Purified monoclonal antibodies were coated at a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6) 50 μL / well for overnight coating at 4°C. The next day, the coating solution was discarded and the cells were blocked with 1-2% BSA or gelatin at 150 μL / well and incubated at 37°C for 2 h. The blocking solution was discarded and purified HADV-2, HADV-3, HADV-5, HADV-6, and HADV-7 viruses (titer of 1x 10 12 VP / ml) was diluted 1000-fold and added to the ELISA plate, 50 μL / well, and incubated at 37°C for 35 min. The plate was washed 4 times with PBST, and HRP-labeled monoclonal antibody diluted 1000-fold with PBS was added, 50 μL / well, and incubated at 37°C for 35 min. The plate was washed 4 times, and then patted dry and TMB color development solution was added at 50 μL / well. The color was developed at room temperature for 10 min. Finally, 50 μL TMB stop solution (acidic, Beijing Meikewande Biological, 1001SA) was added to terminate the reaction, and the OD was measured with a microplate reader. 450 nm value.

[0071] The antibody combination with a high detection value for HADV-5 virus but no reaction with other types of virus was selected as the optimal pairing for the double antibody sandwich ELISA method for specific detection of HADV-5.

[0072] Thirteen monoclonal antibodies with high titers were individually coated and HRP-labeled to screen for paired combinations. High-binding antibodies have higher sensitivity when used in detection, and the targeted selection of these high-titer antibodies can make the screening of paired monoclonal antibodies more efficient (the results of the paired screening of the 13 antibody coating and labeling methods are not shown; only the two combinations with the highest detection values ​​for HADV-5 samples are shown).

[0073] The screening process is shown in Tables 1, 2, and 3.

[0074] Table 1: OD values ​​of different HRP-labeled monoclonal antibodies for detection of various types of HADV viruses when 7C9 monoclonal antibody was used as the coating antibody.

[0075]

[0076] Table 2: OD values ​​of different HRP-labeled monoclonal antibodies for detection of various types of HADV viruses when 8B9 monoclonal antibody was used as the coating antibody.

[0077]

[0078] Table 3: OD values ​​of 5F9 and 4A6 HRP-labeled monoclonal antibodies for detection of various HADV viruses at different dilutions.

[0079]

[0080] Wherein, “*” indicates the dilution multiple.

[0081] Two combinations were screened out with higher detection values ​​for HADV-5. One was using 8B9 monoclonal antibody as coating antibody and 4A6 monoclonal antibody as HRP-labeled detection antibody, and the other was using 8B9 monoclonal antibody as coating antibody and 5F9 monoclonal antibody as HRP-labeled detection antibody.

[0082] The two combinations were further compared by diluting HADV-1, HADV-3, HADV-5, HADV-6, HADV-7, HADV-10, and HADV-41 viruses by 500 times, 5000 times, 50,000 times, and 500,000 times, respectively, and comparing the detection results of the two combinations on samples, especially HADV-5.

[0083] The results showed that the combination of 8B9 monoclonal antibody coating and 4A6 monoclonal antibody HRP labeling had higher detection sensitivity for HADV-5 and had no cross-reaction with other HADVs, thus being determined as the best paired monoclonal antibody.

[0084] 8. Specificity and sensitivity evaluation of double antibody sandwich ELISA for specific detection of HADV-5

[0085] After determining the coating and labeling antibody combination, the optimal monoclonal antibody coating concentration and HRP-labeled monoclonal antibody dilution were further determined.

[0086] 8B9 monoclonal antibody was coated at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL using coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6) at 50 μL / well for overnight coating at 4°C. The next day, the coating solution was discarded and the plates were blocked with 1-2% BSA at 150 μL / well and 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 at 50 μL / well. The plates were incubated at 37°C for 35 min and washed four times with PBST.

[0087] HRP-labeled 4A6 monoclonal antibody diluted 1000, 2000, 4000, and 8000 times with PBS was added at 50 μL / well and incubated at 37°C for 35 min. The plate was then washed 4 times, patted dry, and TMB color development solution was added at 50 μL / well. The color was developed at room temperature for 10 min. Finally, 50 μL TMB stop solution (acidic, Beijing Meikewande Biological, 1001SA) was added to terminate the reaction. The OD was measured using a microplate reader.450 nm value.

[0088] The condition with the highest positive reading and negative control was taken as the optimal condition for double antibody sandwich ELISA. The detection specificity and sensitivity of this method were evaluated under this optimal condition. Specifically: 8B9 monoclonal antibody was coated at a concentration of 1ug / ml and 50ul / well at 4℃ overnight using the above-mentioned coating buffer, the coating solution was discarded, 150ul of 2% BSA was added to each well and incubated at 37℃ for 2h for blocking, the sample to be tested was added, and the plate was washed 4 times after incubation, and then HRP-labeled 4A6 monoclonal antibody was added with a dilution of 4000 times. The incubation conditions for each step were 37℃ for 35min, and finally the color was developed at room temperature for 10min, and 50μL TMB was added to terminate the reaction. 450 The detection value is read at nm wavelength.

[0089] When testing the specificity of the method, purified HADV-1, HADV-2, HADV-3, HADV-6, HADV-7, HADV-10, and HADV-41 viruses (titer of 1 x 10 12 VP / ml) were tested in 500-fold dilutions. This was done to verify that the antibody combination recognized only HADV-5 and did not cross-react with other adenovirus types. To assess the sensitivity of the assay, purified HADV-5 Hexon protein was tested in two-fold dilutions starting at 1 μg / ml, with the respiratory syncytial virus (RSV) Fusion protein used as a control.

[0090] Analysis of test results: The coating concentration of 8B9 monoclonal antibody was determined to be 1ug / ml, the dilution ratio of HRP-labeled 4A6 monoclonal antibody was 4000 times, and the incubation conditions for each step were 37°C for 35min and color development at room temperature for 10min, which were the optimal detection conditions. The detection specificity and sensitivity of the method were evaluated under these conditions. The results showed that the double antibody sandwich ELISA method could only detect HADV-5, but did not react with HADV-1, HADV-2, HADV-3, HADV-6, HADV-7, HADV-10, and HADV-41 viruses (see Figure 2 ), among which HADV-5 belongs to subgroup C with HADV-1, HADV-2, and HADV-6, HADV-3 and HADV-7 belong to subgroup B, HADV-10 belongs to subgroup D, and HADV-41 belongs to subgroup F. The results show that this detection method is specific for HADV-5. Figure 3The sensitivity of this method was tested using purified HADV-5 Hexon protein. It was found that at a concentration of 3.9 ng / ml, the test result was still weakly positive, indicating that the method has a high detection sensitivity for HADV-5 purified natural protein.

[0091] 9. Detection and application of replication-defective recombinant adenovirus type 5

[0092] The replication-deficient recombinant adenovirus type 5 is sourced from Beijing Wujiahe Gene Technology Co., Ltd., and the infectible sensitive cell line HEK-293A is maintained by our company. Under normal circumstances, HADV-5 can infect Hep-2 cells, but replication-deficient 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 of the replication-deficient recombinant adenovirus type 5 lacking the E1 gene, allowing it to infect and replicate effectively. The replication-deficient recombinant adenovirus type 5 stock solution (stock solution titer of 4.2 x10 8 TCID 50 / mL) were diluted 1000-fold and 10,000-fold respectively, and used to infect 293A cells. The content and replication of adenovirus type 5 were monitored using the double antibody sandwich ELISA method.

[0093] The test was conducted according to the aforementioned optimal detection conditions, namely: the coating concentration of 8B9 monoclonal antibody was 1ug / ml, the dilution factor of HRP-labeled 4A6 monoclonal antibody was 4000 times, each step of incubation was 37°C for 35min, and the color development was performed at room temperature for 10min. Figure 4 The double-antibody sandwich ELISA method established using paired monoclonal antibodies can detect HADV-5, which can infect humans. Furthermore, the packaged replication-deficient recombinant adenovirus type 5 was diluted 1,000 and 10,000 times and used to infect the 293A sensitive cell line, respectively. This method was used to detect the replication-deficient recombinant adenovirus type 5. It was found that the replication-deficient recombinant adenovirus type 5 was able to effectively infect 293A cells and a cytopathic effect (CPE) was observed. The double-antibody sandwich ELISA test results showed that the OD reading gradually increased with time after infection, indicating that the virus was continuously replicating, indicating that this method can also effectively detect replication-deficient recombinant adenoviruses.

[0094] This study demonstrated the successful monitoring of the infection and replication dynamics of a replication-deficient recombinant adenovirus type 5 in specific host cells (HEK-293A) using an optimized double-antibody sandwich ELISA method. The cell control was the culture supernatant of uninfected 293A cells. -3 and 10 -4 Represents virus 10 3 and 104 The supernatant of infected cells was diluted 1 / 4 times. The results showed that with increasing infection time, the viral content and OD readings increased, confirming the effectiveness and sensitivity of this method for this type of virus. This demonstrates that the double-antibody sandwich ELISA method can not only detect HADV-5, but also effectively detect the replication of replication-defective recombinant adenovirus type 5. This has important implications for virology research, the development of new vaccine vectors, and gene therapy.

[0095] 10. Gene sequence

[0096] Monoclonal antibody variable region gene cloning and sequencing.

[0097] Total RNA from hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the mouse antibody variable regions were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the third-round PCR primers. The PCR products were gel-cleaved and purified, then ligated into the pUC19 vector and transformed into the TOP10 strain. After incubation at 37°C for 14 hours, single colonies were picked and sequenced to obtain the gene sequences of the monoclonal antibody light and heavy chains.

[0098] The specific sequence is as follows:

[0099] The amino acids of the heavy chain complementary determining region 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.

[0100] The amino acids of the light chain complementary 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.

[0101] The amino acids of the heavy chain complementary 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.

[0102] The amino acids of the light chain complementary 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.

[0103] The amino acid sequence of the heavy chain variable region of the 8B9 monoclonal antibody (SEQ ID NO. 13): EVKLEESGPELVKPGASVKMSCKASGYTFTSYYMHWVKQKPGQGLEWIGYLNPVNDSTKYMEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARAIYESYYGYWGQGTTLTVSA.

[0104] The amino acid sequence of the light chain variable region of the 8B9 monoclonal antibody (SEQ ID NO. 14): DIQMNQSPASLAVSLGQRATISCKASKRVSISGYSYMHWYQQKPGQPPKLLIYLASNLLGGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHGRLLPSTFGGGTKLELKRTV.

[0105] The amino acid sequence of the heavy chain variable region of the 4A6 monoclonal antibody (SEQ ID NO. 15): EVMLAESGPELVKPGASVKMSCKASGYTFTGSVFKWVKQKPGQGLEWIGELSPVIDSTSVLERAQQKATLTSDKSSSTAYMELSSLTSEDSAVYYCARMLKSVISVWGQGTTLTVSS.

[0106] The amino acid sequence of the light chain variable region of the 4A6 monoclonal antibody (SEQ ID NO. 16): EIVLTQSPASLAVSLGQRATISCFARKSVLLSRVSYHIWYQQKPGQPPKLLIYETSVVGGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMHSGYLPSTFGGGTKLEIKRTV.

[0107] Nucleotide sequence of the heavy chain variable region of the 8B9 monoclonal antibody (SEQ ID NO.17): GAGGTGAAGCTGGAGGAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATGCACTGGGTGAAGCAGAAGCCCGGCCAGGGCCTGGAGTGGATCGGCTACCTGAACCCCGTGAACGACAGCACCAAGTACATGGAGAAGTTCAAGGGCAAGGCCACCCTGACCAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGGCCATCTACGAGAGCTACTACGGCTACTGGGGCCAGGGCACCACCCTGACCGTGAGCGCC。

[0108] Nucleotide sequence of the light chain variable region of the 8B9 monoclonal antibody (SEQ ID NO.18): GACATCCAGATGAACCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCAAGGCCAGCAAGAGGGTGAGCATCAGCGGCTACAGCTACATGCACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACCTGGCCAGCAACCTGCTGGGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCCAGCACGGCAGGCTGCTGCCCAGCACCTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG。

[0109] Nucleotide sequence of the heavy chain variable region of the 4A6 monoclonal antibody (SEQ ID NO.19): GAGGTGATGCTGGCCGAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCAGCGTGTTCAAGTGGGTGAAGCAGAAGCCCGGCCAGGGCCTGGAGTGGATCGGCGAGCTGAGCCCCGTGATCGACAGCACCAGCGTGCTGGAGAGGGCCCAGCAGAAGGCCACCCTGACCAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGATGCTGAAGAGCGTGATCAGCGTGTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

[0110] Nucleotide sequence of the light chain variable region of the 4A6 monoclonal antibody (SEQ ID NO.20): GAGATCGTGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCTTCGCCAGGAAGAGCGTGCTGCTGAGCAGGGTGAGCTACCACATCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACGAGACCAGCGTGGTGGGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCATGCACAGCGGCTACCTGCCCAGCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG。

[0111] 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 the 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 combination of the two can achieve high sensitivity and high specificity detection of HADV-5 and replication-deficient recombinant adenovirus type 5, and does not cross-react with other common adenovirus types. The present invention also provides the variable region amino acid sequence and nucleotide sequence of the above-mentioned antibody, as well as an ELISA detection method based on the antibody combination. The method is simple to operate, has good repeatability, and is suitable for the detection of type 5 adenovirus.

[0112] So far, the various 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. Based on the above description, those skilled in the art can fully understand how to implement the technical solutions disclosed herein.

[0113] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents without departing from the scope and spirit of the present application.

Claims

1. A combination of monoclonal antibodies for specific detection of adenovirus type 5, characterized in that: The monoclonal antibody combination includes 8B9 monoclonal antibody and 4A6 monoclonal antibody; The heavy chain variable region of the 8B9 monoclonal antibody includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively; The light chain variable region of the 8B9 monoclonal antibody includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively; The heavy chain variable region of the 4A6 monoclonal antibody includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively; The light chain variable region of the 4A6 monoclonal antibody includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively.

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 8B9 monoclonal antibody is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the 8B9 monoclonal antibody is shown in SEQ ID NO.

14.

3. 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 4A6 monoclonal antibody is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the 4A6 monoclonal antibody is shown in SEQ ID NO.

16.

4. The combination of monoclonal antibodies for specific detection of adenovirus type 5 according to claim 2, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the 8B9 monoclonal antibody is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the 8B9 monoclonal antibody is shown in SEQ ID NO.

18.

5. The combination of monoclonal antibodies for specific detection of adenovirus type 5 according to claim 3, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the 4A6 monoclonal antibody is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the 4A6 monoclonal antibody is shown in SEQ ID NO.

20.

6. The combination of monoclonal antibodies for specific detection of adenovirus type 5 according to claim 1, characterized in that: The adenovirus type 5 includes HADV-5 and replication-deficient recombinant adenovirus type 5.

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

8. The use according to claim 7, characterized in that The tools include reagents, kits, test strips and antibody chips; The reagent is a detection reagent constructed based on the double antibody sandwich ELISA detection method, using 8B9 monoclonal antibody as the coating antibody and 4A6 monoclonal antibody as the detection antibody; The kit is a detection kit constructed based on the double antibody sandwich ELISA detection method, using 8B9 monoclonal antibody as the coating antibody and 4A6 monoclonal antibody as the detection antibody; The test strip is a test strip constructed based on the double antibody sandwich ELISA detection method, using 8B9 monoclonal antibody as the coating antibody and 4A6 monoclonal antibody as the detection antibody; The antibody chip is a detection antibody chip constructed based on a double-antibody sandwich ELISA detection method, using 8B9 monoclonal antibody as a coating antibody and 4A6 monoclonal antibody as a detection antibody.

9. The use according to claim 8, characterized in that The double antibody sandwich ELISA detection method comprises the following steps: Step 1: Coating Use 8B9 monoclonal antibody as coating antibody, dissolve it in coating buffer at a concentration of 1-2 μg / mL, add 50 μL to each well, and coat the plate at 4°C overnight; Step 2: Closure 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% BSA or gelatin; Step 3: Add sample After washing the ELISA plate, 50 μL of 1000-fold diluted HADV-5 adenovirus test sample was added to each well. The original virus titer of the sample was 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 4 times with PBST buffer, soaking and shaking thoroughly each time; Step 5: Add detection antibody HRP-labeled 4A6 monoclonal antibody was added as the detection antibody at a dilution ratio of 1:4000, 50 μL was added to each well, and incubated at 37°C for 30–60 minutes; Step 6: Wash the plate again with PBST buffer 4 times to remove unbound detection antibody; Step 7: Color development and detection Add 50 μL of TMB colorimetric solution to each well and develop the 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 OD value using a microplate reader. 450 nm absorbance value; determine whether the sample contains specific antigens for adenovirus type 5 based on the absorbance.

10. The use according to claim 9, characterized in that The coating concentration of the 8B9 monoclonal antibody was 1 ug / ml, the dilution ratio of the HRP-labeled 4A6 monoclonal antibody was 4000 times, and the incubation conditions for each step were 37° C., 35 min, and color development was performed at room temperature for 10 min.

Images