A diagnostic kit based on pigeon adenovirus Hexon protein and its application

Abstract

The present invention discloses an anti-pigeon adenovirus Hexon protein monoclonal antibody, the anti-pigeon adenovirus Hexon protein monoclonal antibody is monoclonal antibody 1H5-8-14-5, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1H5-8-14-5 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The present invention also discloses a double antibody sandwich ELISA diagnostic kit for detecting pigeon adenovirus antigens, the kit comprising an enzyme labeling plate coated with an anti-Hexon protein polyclonal antibody, Hexon protein, HRP-labeled anti-pigeon adenovirus Hexon protein monoclonal antibody, a negative control, a color developing solution, a washing solution, and a stop solution. The kit of the present invention can effectively detect pigeon adenovirus, provides a new technical means for pigeon adenovirus prevention and control, and the detection result is accurate and specific, with good application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a diagnostic kit based on pigeon adenovirus Hexon protein and its application. Background Art

[0002] Pigeon adenovirus (PiAdV) is the primary pathogen of pigeon crop inflammation, a serious threat to pigeon health, particularly when it becomes prevalent in racing pigeons, causing significant losses. The virus is transmitted through feces, food contamination, and direct contact. PiAdV infection can cause pigeons to vomit, experience decreased digestion, produce green or white, watery feces, become listless, develop matted feathers, and lose weight. In severe cases, it can even lead to acute death. There are multiple types of PiAdV. Group I adenovirus (PIAdV-I) primarily affects young pigeons, while group II adenovirus (PIAdV-II) can infect pigeons of all ages. Currently, there is a lack of accurate and rapid methods to identify the type of adenovirus infecting infected pigeons, making targeted treatment difficult and causing significant economic losses to the pigeon industry.

[0003] Hexon, the primary antigenic protein of avian adenovirus, plays a crucial role in viral immunity and recognition. Its structure is complex and unique, consisting of conserved P1 and P2 regions and variable loops (Loop 1 through Loop 4). These regions not only give hexon its unique morphology but also enable it to form a homotrimeric structure, thereby stimulating the production of specific neutralizing antibodies. Within the hexon structure, the conserved P1 and P2 regions are located within the virus body, forming its stable base. The four highly variable loops (Loop 1, Loop 2, Loop 3, and Loop 4) form the apex. These regions, characterized by their high variability and complexity, together with the base, contribute to the unique tower-like structure of the hexon. Loop 1 is the most unique and important region of the hexon protein. It not only exhibits the highest sequence variability, but also possesses the longest length and a complex folded structure. This structural characteristic enables Loop 1 to harbor numerous antigenic determinants, enabling it to elicit a strong immune response. Therefore, by analyzing the sequence differences in the Loop1 region of the Hexon protein, adenoviruses of different serotypes can be effectively distinguished.

[0004] The present invention expresses the pigeon adenovirus Hexon protein in prokaryotes, and the recombinant protein with a quantitative of 1 mg / mL is fully mixed with an adjuvant in a volume ratio of 1:1 and emulsified, and then used to immunize pigeons, New Zealand white rabbits and BALB / c mice, respectively, to prepare polyclonal and monoclonal antibodies specific for the Hexon protein. Based on this material, an ELISA diagnostic kit based on the pigeon adenovirus Hexon protein was further developed. Summary of the Invention

[0005] In order to make up for the deficiencies of the existing technology and fill the shortage of pigeon adenovirus antigen diagnostic reagents on the market, the purpose of the present invention is to provide a recombinant pigeon adenovirus Hexon protein prepared based on a prokaryotic Escherichia coli expression system; provide rabbit and pigeon polyclonal antibodies and mouse monoclonal antibodies based on the recombinant protein; and further provide an ELISA detection kit for detecting pigeon adenovirus antigen developed based on the above materials.

[0006] In order to achieve the above objectives, the present invention is implemented through the following technical solutions.

[0007] In one aspect, the present invention discloses an anti-pigeon adenovirus Hexon protein monoclonal antibody. The anti-pigeon adenovirus Hexon protein monoclonal antibody is 1H5-8-14-5, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1H5-8-14-5 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0008] In another aspect, the present invention also discloses a double-antibody sandwich ELISA diagnostic kit for detecting pigeon adenovirus, the kit comprising an enzyme labeling plate coated with an anti-Hexon protein polyclonal antibody, Hexon protein, an HRP-labeled anti-pigeon adenovirus Hexon protein monoclonal antibody according to claim 1, a negative control, a color developing solution, a washing solution, and a stop solution.

[0009] Preferably, the anti-Hexon protein polyclonal antibody of the present invention is a rabbit anti-Hexon protein polyclonal antibody, which is diluted 1:1000 before being coated on an ELISA plate.

[0010] Preferably, the amino acid sequence of the Hexon protein of the present invention is shown as SEQ ID NO.1.

[0011] Preferably, the nucleotide sequence of the codon-optimized Hexon protein of the present invention is shown as SEQ ID NO.2.

[0012] Preferably, the concentration of the Hexon protein of the present invention is 0.5 μg / mL.

[0013] Preferably, the HRP-labeled anti-pigeon adenovirus Hexon protein monoclonal antibody of the present invention is used after being diluted 1:4000.

[0014] Preferably, the negative control of the present invention is PBST buffer.

[0015] In another aspect, the present invention also discloses the use of the monoclonal antibody 1H5-8-14-5 in the preparation of a pigeon adenovirus or pigeon adenovirus Hexon protein detection reagent.

[0016] The monoclonal antibody 1H5-8-14-5 prepared by the invention has high specificity and sensitivity and is suitable for detecting pigeon adenovirus or pigeon adenovirus Hexon protein.

[0017] The double-antibody sandwich ELISA diagnostic kit for pigeon adenovirus prepared by the present invention has good specificity and sensitivity and is suitable for detecting pigeon adenovirus or pigeon adenovirus Hexon protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of pET32a-Hexon construction.

[0019] Figure 2 SDS-PAGE electrophoresis of purified recombinant Hexon protein.

[0020] Figure 3 Western Blot identification of recombinant Hexon protein using His antibody.

[0021] Figure 4 The recombinant Hexon protein was combined with the positive serum of sick pigeons and identified by Western Blot.

[0022] Figure 5 Recombinant Hexon protein was identified by Western Blot with rabbit polyclonal antibody.

[0023] Figure 6 Western Blot identification of recombinant Hexon protein using pigeon antiserum.

[0024] Figure 7 Recombinant Hexon protein was identified by Western Blot with sera from immune mice.

[0025] Figure 8 Recombinant Hexon protein and hybridoma cell culture supernatant were identified by Western Blot.

[0026] Figure 9 Western Blot identification of HRP-labeled monoclonal antibody.

[0027] Figure 10 Determine the optimal dilution of capture antibody and enzyme-labeled antibody. Note: 1 to 6, from left to right, represent the capture antibody in a gradient dilution range of 1:1000 to 1:32000; A to D, from top to bottom, represent the enzyme-labeled antibody in a gradient dilution range of 1:1000 to 1:8000. The wells in the red box represent duplicate controls with the same sequence, and the wells in the yellow box represent negative controls with the same sequence.

[0028] Figure 11 Determination of the optimal blocking system. Note: 1: 5% gelatin; 3: 5% skim milk powder; 5: 5% BSA; 7: 5% calf serum; 2, 4, 6, and 8 are corresponding negative controls.

[0029] Figure 12 The optimal substrate incubation time was determined. Note: A1 to A3: substrate incubation for 30 min; B1 to B3: substrate incubation for 60 min; C1 to C3: substrate incubation for 90 min; D1 to D3: substrate incubation for 120 min; A4 to A6, B4 to B6, C4 to C6, and D4 to D6 are negative controls with corresponding substrate incubation times.

[0030] Figure 13 The optimal enzyme-labeled antibody incubation time was determined. Note: A1 to C1: 30 min incubation with enzyme-labeled antibody; A2 to C2: 60 min incubation with enzyme-labeled antibody; A3 to C3: 90 min incubation with enzyme-labeled antibody; A4 to C4: 120 min incubation with enzyme-labeled antibody; D1 to F1, D2 to F2, D3 to F3, and D4 to F4 are negative controls with the corresponding enzyme-labeled antibody incubation times.

[0031] Figure 14 The optimal color development time was determined. Note: A1 to C1: color development time 5 min; A2 to C2: color development time 10 min; A3 to C3: color development time 15 min; D1 to F1, D2 to F2, D3 to F3, and D4 to F4 are negative controls with corresponding color development times.

[0032] Figure 15 Kit sensitivity test. Note: 1 to 8 from right to left are serially diluted Hexon antigens, with dilution concentrations ranging from 16 ng / μL to 0.125 ng / μL; A to C are three replicate controls; D1 to F8 are negative controls for the corresponding diluted antigens.

[0033] Figure 16 Test results for positive samples. Note: 1 to 4, from left to right, are serially diluted positive samples, with dilution concentrations ranging from 1:5 to 1:40; A to C and D to F are three replicate controls for different positive samples; A5 to F8 are negative samples diluted to the corresponding concentrations. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0036] Example 1: Acquisition of target gene and construction of recombinant expression vector

[0037] The PiAdV-Hexon gene sequence was screened in the NCBI database, the nuclear localization signal peptide was removed, and the remaining 954bp nucleic acid sequence was optimized for prokaryotic Escherichia coli codon preference and then synthesized. After double digestion with BamHⅠ-HindⅢ restriction endonucleases, it was ligated with the prokaryotic expression vector pET-32a(+) to construct a recombinant expression plasmid. The recombinant plasmid obtained after identification was named pET32a-Hexon.

[0038] The amino acid sequence of the gene is shown in SEQ ID NO.1, the nucleotide sequence after codon optimization is shown in SEQ ID NO.2, and the schematic diagram of the pET32a-Hexon recombinant plasmid constructed after gene synthesis is shown in Figure 1 shown.

[0039] Example 2: Expression and purification of target protein

[0040] Transform pET32a-Hexon into Rosetta DE3 expression bacteria, perform IPTG-induced expression and protein purification, as follows:

[0041] ① Protein expression: Inoculate the recombinant expression bacteria into LB medium at a ratio of 1:100, add Amp antibiotics at a final concentration of 1 mM, shake the bacteria at 37°C, 220 rpm for 3-4 hours, then add IPTG at a final concentration of 1 mM to induce the bacteria, and induce the bacteria at 37°C, 220 rpm for 4 hours.

[0042] ② Bacterial disruption: Collect the bacterial solution induced overnight and centrifuge at 8000 rpm for 10 min to obtain bacterial pellet; resuspend and wash with an appropriate amount of sterile PBS solution, centrifuge at 12000 rpm and 4°C for 10 min, and discard the supernatant; resuspend the bacteria with a small amount of pre-cooled purified washing solution, and place them in an ultrasonic disruptor for disruption, with the conditions of supersonication for 3 seconds and pause for 5 seconds, and ultrasonication on ice for 15-20 minutes; finally, centrifuge at 12000 rpm and 4°C for 15 min to obtain the pellet.

[0043] ③ Protein denaturation and purification: Denature the precipitate with 8M urea and apply it to a pretreated nickel column. Allow to bind for 2 hours. Wash impurities with pre-cooled purification wash buffer four times, 10 minutes each time. Elute the target protein three times with pre-cooled purification eluent, 15 minutes each time. Collect and combine all eluates.

[0044] ④ Protein renaturation: Place the collected target protein in a dialysis bag and gradually reduce the urea content in the target protein to 0M according to the gradient renaturation method. Finally, dialyze it into PBS solution. The collected solution is the target protein.

[0045] Example 3: Identification, concentration and quantitative analysis of target protein

[0046] The purified Hexon protein was prepared and electrophoresed on 12% SDS-PAGE gel, stained with Coomassie brilliant blue, and destained. Figure 2 Western Blot identification was performed using His antibody as the primary antibody. The results are as follows: Figure 3 .

[0047] Based on the above identification, the protein was concentrated and desalted using a 10 kDa ultrafiltration tube, and the protein was quantified by the BCA method, and the final protein concentration was adjusted to 1 mg / mL.

[0048] Example 4: Immunogenicity detection of recombinant protein

[0049] The immunogenicity of recombinant Hexon protein was detected by Western Blot using pigeon adenovirus positive serum as primary antibody. The results showed that ( Figure 4 ), the recombinant protein Hexon can specifically bind to pigeon adenovirus positive serum.

[0050] Example 5: Preparation of polyclonal antibodies and monoclonal antibodies

[0051] (1) Preparation of anti-Hexon rabbit polyclonal antibody

[0052] New Zealand white rabbits weighing about 2.5 kg were adapted to the new breeding environment for 3-4 days, and blood was collected from the ear vein and immunized subcutaneously. On the 0th, 10th, 20th and 30th days of the experiment, 2 mL of Hexon immunogen emulsified with Freund's adjuvant was injected subcutaneously at multiple points on the back. Each immunized rabbit was injected with 1 mg of Hexon recombinant protein each time (composed of: 1 mL of 1 mg / mL recombinant Hexon protein and 1 mL of adjuvant, wherein the first immunization used Freund's complete adjuvant and the next three immunizations used Freund's incomplete adjuvant). 7 days after the last immunization, the experimental rabbits were anesthetized, the carotid artery was stripped, and the whole body blood was taken to kill them. After separating the serum, the rabbit polyclonal antibody against Hexon protein was purified using a Protein A column. The immunogenicity of the protein was detected by Western Blot using the prepared rabbit polyclonal antibody as the primary antibody. The results showed that the protein Hexon could be specifically bound by the rabbit polyclonal antibody. The results are as follows Figure 5 The titer of the prepared rabbit polyclonal antibody was 1:1280000, as shown in Table 1.

[0053] Table 1 Determination of the titer of rabbit polyclonal antibodies against Hexon protein

[0054]

[0055] (2) Preparation of pigeon polyclonal antibodies against Hexon protein

[0056] Eight pigeons were randomly divided into two groups: ①PBS negative control group; ②recombinant Hexon protein immunization group. Immunization was performed by chest muscle injection, twice on the 0th day and the 14th day respectively. Each pigeon in the PBS group was injected with 40μL sterile PBS solution, and each pigeon in the recombinant Hexon protein immunization group was injected with 40μL emulsified protein solution (composed of: 20μL 1mg / mL recombinant Hexon protein + 20μL Freund's adjuvant, wherein the first immunization used Freund's complete adjuvant and the second immunization used Freund's incomplete adjuvant). On the 28th day after immunization, the pigeon's blood was collected through the subwing vein and placed in a constant temperature environment of 4°C for 12 hours. Afterwards, the blood was centrifuged at 3000rpm for 10 minutes to separate the antiserum. The prepared pigeon antiserum was used as the primary antibody to detect the antigenicity of the protein by Western Blot method. The results showed that the protein Hexon can specifically bind to the pigeon antiserum. Figure 6 .

[0057] (3) Preparation of anti-Hexon protein mouse monoclonal antibody

[0058] Eight 6-8 week old female BALB / c mice were randomly divided into two groups, 4 mice per group: ① PBS negative control group; ② recombinant Hexon protein immunization group. On days 0, 14, and 28, 200 μL of Hexon immunogen emulsified with Freund's adjuvant (100 μL 1 mg / mL recombinant Hexon protein + 100 μL Freund's adjuvant, with complete Freund's adjuvant used for the first immunization and incomplete Freund's adjuvant used for the subsequent two immunizations) were injected subcutaneously at multiple points on the back. The mice with the best immune response were screened by Western Blot and indirect ELISA (see results). Figure 7 and Table 2), after the spleen was removed and fused with myeloma cells, multiple rounds of screening in HAT medium were performed to obtain the hybridoma cell line 1H5-8-14-5 that could stably secrete antibodies. Finally, monoclonal antibodies were prepared from ascites. Western Blot results showed that the protein Hexon could specifically bind to the prepared monoclonal antibodies. Figure 8 The prepared monoclonal antibody was further labeled using an HRP labeling kit, and the results were as shown in FIG. Figure 9 shown.

[0059] Table 2 Determination of Hexon protein mouse serum titer

[0060]

[0061] (4) Determination of the sequence of the variable region of the monoclonal antibody gene

[0062] The cultured hybridoma cells (1H5-8-14-5) were lysed and total RNA was extracted. After passing the test, the RNA was reverse transcribed into cDNA using RACE technology. The heavy and light chain variable region sequences were obtained by PCR amplification. The target fragments were ligated to the vector using ligase, and the ligation products were transformed into competent Escherichia coli cells. Then, single clones were picked for sequencing. Finally, the variable region sequences of the monoclonal antibody genes were obtained by analysis and annotation of the sequencing results (see Table 3).

[0063] Table 3 Monoclonal antibody (1H5-8-14-5) variable region amino acid sequence information

[0064]

[0065] Western blot analysis of Hexon protein or total protein extracted from pigeon adenovirus using the monoclonal antibody showed that the monoclonal antibody could specifically bind to Hexon protein and total protein extracted from pigeon adenovirus, indicating that the monoclonal antibody prepared by the present invention has good sensitivity and specificity.

[0066] Example 6: Development and application of ELISA diagnostic kit

[0067] 1. Optimal capture antibody and enzyme-labeled antibody dilution

[0068] To identify optimal capture and detection antibody dilutions for the detection of novel pigeon adenoviruses, a classic checkerboard assay was employed. The capture antibody (rabbit polyclonal antibody) was first diluted to a series of concentrations ranging from 1:1000 to 1:32000. The capture antibody was then added to each column of the ELISA plate at varying concentrations within this range, with 100 μL added to each well. The plate was incubated at 4°C for 16 hours. Following incubation, the plate was thoroughly washed with a specific wash buffer. Next, 300 μL of blocking buffer was added to each well, followed by blocking at 37°C for 2 hours. Following blocking, the plate was washed three times with wash buffer and placed at 4°C until ready for use. Subsequently, 100 μL of diluted Hexon antigen protein was added to each well and incubated at 37°C for 1 hour. Following incubation, the plate was washed three times with wash buffer. Next, the detection antibody (HRP-conjugated monoclonal antibody) was diluted into a series of concentration gradients with a concentration range set between 1:1000 and 1:8000, and 100 μL of solution was accurately added to each well. Subsequently, the ELISA plate was incubated at 37°C for 1 hour. After the incubation, the ELISA plate was washed with washing solution. Next, 200 μL of TMB colorimetric solution was added to each well and incubated in the dark at 37°C for 10 minutes to ensure that the color development reaction can proceed fully. Finally, in order to quickly terminate the color development reaction, 50 μL of stop solution was added to each well. After this series of operations were completed, we used a detection instrument to measure the optical density value (OD450) of each well at a wavelength of 450 nm. By comparing the P / N values ​​of different well positions, the optimal conditions ( Figure 10 and Table 4), it can be seen from the chart that the optimal capture antibody dilution is 1:1000, and the optimal enzyme-labeled antibody dilution is 1:4000.

[0069] Table 4 Determination of optimal capture antibody and enzyme-labeled antibody dilutions

[0070]

[0071] 2. Best closed system

[0072] After determining the optimal dilution of capture antibody and enzyme-labeled antibody, other conditions remained unchanged and four different blocking systems were set up, including 5% gelatin, 5% BSA, 5% skim milk powder and 5% calf serum as blocking solutions, and the blocking time was 30, 60, 90 and 120 min respectively. Figure 11 As shown in Tables 5 to 8, it can be seen from the charts that the best blocking system is 5% calf serum incubation for 2 hours.

[0073] Table 5 Confirmation of the optimal closed system for 30 min

[0074]

[0075] Table 6 Confirmation of the optimal closed system for 60 min

[0076]

[0077] Table 7 Confirmation of the optimal closed system for 90 min

[0078]

[0079] Table 8 Confirmation of the optimal closed system for 120 min

[0080]

[0081] 3. Optimal antigen (sample) incubation time

[0082] After determining the optimal dilution of capture antibody and enzyme-labeled antibody, different antigen incubation times were set, including incubation in a 37°C biochemical incubator for 30, 60, 90, and 120 min, respectively. Figure 12 As shown in Table 9, it can be seen from the chart that the optimal antigen (i.e. sample) incubation time is 60 min.

[0083] Table 9 Confirmation of optimal antigen (i.e. sample) incubation time

[0084]

[0085] 4. Optimal enzyme-labeled antibody incubation time

[0086] After determining the optimal dilution of capture antibody and enzyme-labeled antibody, other conditions remained unchanged and different enzyme-labeled antibody incubation times were set, including incubation in a 37°C biochemical incubator for 30 min, 60 min, 90 min, and 120 min, respectively. Figure 13 As shown in Table 10, it can be seen from the figure and the table that the optimal enzyme-labeled antibody incubation time is 90 min.

[0087] Table 10 Confirmation of the optimal enzyme-labeled antibody incubation time

[0088]

[0089] 5. Best color development time

[0090] After determining the optimal dilution of capture antibody and enzyme-labeled antibody, other conditions remained unchanged and different TMB development times were set, namely 5 min, 10 min and 15 min. Figure 14 As shown in Table 11, it can be seen from the chart that the optimal color development time is 15 minutes.

[0091] Table 11 Confirmation of optimal color development time

[0092]

[0093] 6. Sensitivity testing

[0094] Hexon protein was diluted to different concentrations: 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 ng / μL. The sensitivity was determined by determining whether the maximum dilution of the enzyme-labeled well was 2.1 times the OD value of the negative control well. The diluted serum was tested. The positive and negative values ​​were determined based on the critical value, such as Figure 15 As shown in Table 12, it can be seen from the chart that the minimum detection concentration of the diagnostic kit is 0.5 ng / μL.

[0095] Table 12 Sensitivity test results

[0096]

[0097] 7. Positive disease sample detection

[0098] In order to evaluate the specificity and sensitivity of the double antibody sandwich ELISA method for viral antigens, positive and negative disease samples were prepared, the samples were diluted to different concentrations, and the test results were analyzed to evaluate the performance of the ELISA double antibody sandwich method, such as Figure 16 As shown in Table 13, it can be seen from the figure and the table that the minimum serum detection sensitivity of this detection method is a dilution ratio of 1:5.

[0099] Table 13 Positive disease material detection results

[0100]

[0101] 8. Based on the above results, the preparation and detection of the ELISA kit are determined as follows:

[0102] 8.1 ELISA Plate Preparation: Dilute the capture antibody (rabbit polyclonal antibody prepared in Example 5) 1:1000 in antigen coating buffer and coat the plate. Add 100 μL / well of the plate and incubate at 4°C overnight (12-14 hours). Remove and discard the liquid in the wells. Wash three times with 300 μL / well of washing buffer. Add 300 μL / well of blocking buffer to the plate and incubate at 37°C for 2 hours. Remove and discard the blocking buffer. Wash three times with 300 μL / well of washing buffer.

[0103] 8.2 Detection

[0104] (1) Sample addition: According to the number of samples to be tested, take a detachable coated plate and place it flat on the table. Add the sample (which can be serum, protein or other liquid sample) diluted 1:5 with sample diluent, negative control (PBST buffer) and Hexon protein (concentration of 0.5 μg / mL) to each well and shake to mix.

[0105] (2) Incubate in a 37°C incubator for 60 minutes.

[0106] (3) Washing: Shake off the liquid in the wells, add washing solution, 300 μL / well, and wash three times.

[0107] (4) Enzyme-labeled antibody incubation: Add enzyme-labeled antibody (HRP-labeled monoclonal antibody (1H5-8-14-5) diluted 1:4000 with antibody diluent) at 100 μL / well and incubate at 37°C for 90 min.

[0108] (5) The washing method is the same as (2).

[0109] (6) Color development: Add TMB color development solution, 100 μL / well, and incubate in a 37°C incubator in the dark for 15 min.

[0110] (7) Add stop solution (50 μL / well) and shake gently to mix.

[0111] (8) Reading After adding the stop solution, immediately place the coated plate in a microplate reader and read the OD450nm value at a wavelength of 450nm.

[0112] (9) Result determination: When the OD450 of the test sample / the OD450 of the negative sample ≥ 2.1, it is determined to be positive; otherwise, it is determined to be negative.

[0113] Example 7: Reagent formulations mentioned in the above examples

[0114] Amp storage solution: 100 mg / mL Amp aqueous solution;

[0115] IPTG stock solution: 240 mg / mL IPTG in water;

[0116] PBS solution: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 dissolved in ddH2O to 1 L;

[0117] Purification washing solution: 20 mM Tris, 200 mM NaCl, 25 mM imidazole, 10% glycerol, dissolved in ddH2O, adjusted to pH 8.0, made up to 1 L, and filtered through a 0.45 μm filter to remove impurities;

[0118] Purification eluent: 20 mM Tris, 200 mM NaCl, 500 mM imidazole, 10% glycerol, dissolved in ddH2O, adjusted to pH 8.0, made up to 1 L, and filtered through a 0.45 μm filter to remove impurities;

[0119] Antigen coating buffer: 1.59 g Na2CO3, 2.93 g NaHCO3, dilute to 1 L with ddH2O;

[0120] ELISA wash buffer: 0.05% Tween-20 in PBS;

[0121] ELISA blocking solution: 5% calf serum in PBS;

[0122] ELISA antibody diluent (also called sample diluent): 1% BSA in PBS;

[0123] ELISA stop solution: 2M H2SO4.

[0124] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A monoclonal antibody against the Hexon protein of pigeon adenovirus, characterized in that: The anti-pigeon adenovirus Hexon protein monoclonal antibody is 1H5-8-14-5, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1H5-8-14-5 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. A double antibody sandwich ELISA diagnostic kit for detecting pigeon adenovirus, characterized in that: The kit comprises an ELISA plate coated with an anti-Hexon protein polyclonal antibody, Hexon protein, the HRP-labeled anti-pigeon adenovirus Hexon protein monoclonal antibody according to claim 1, a negative control, a color developing solution, a washing solution, and a stop solution.

3. The kit according to claim 2, wherein The anti-Hexon protein polyclonal antibody is a rabbit anti-Hexon protein polyclonal antibody. When used, the polyclonal antibody is diluted 1:1000 times and then coated on an ELISA plate.

4. The kit according to claim 2, wherein The amino acid sequence of the Hexon protein is shown in SEQ ID NO.

1.

5. The kit according to claim 2, wherein The concentration of the Hexon protein is 0.5 μg / mL.

6. The kit according to claim 2, wherein The HRP-labeled anti-pigeon adenovirus Hexon protein monoclonal antibody according to claim 1 is used after being diluted 1:4000 times.

7. The kit according to claim 2, characterized in that The negative control was PBST buffer.

8. Use of the monoclonal antibody 1H5-8-14-5 according to claim 1 in preparing a reagent for detecting pigeon adenovirus or pigeon adenovirus Hexon protein.

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