Diagnostic kit based on pigeon adenovirus Hexon protein and application

Abstract

The invention discloses an anti-pigeon adenovirus Hexon protein monoclonal antibody, the anti-pigeon adenovirus Hexon protein monoclonal antibody is a monoclonal antibody 1H5-8-14-5, and amino acid sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody 1H5-8-14-5 are respectively as shown in SEQ ID NO.3 and SEQ ID NO.4. The invention further discloses a preparation method of the anti-pigeon adenovirus Hexon protein monoclonal antibody. The invention also discloses a double-antibody sandwich ELISA diagnostic kit for detecting the pigeon adenovirus antigen. The kit comprises an elisa plate coated with an anti-Hexon protein polyclonal antibody, Hexon protein, an HRP labeled anti-pigeon adenovirus Hexon protein monoclonal antibody, a negative control, a developing solution, a washing solution and a stop solution. The kit disclosed by the invention can effectively detect the pigeon adenovirus, provides a new technical means for prevention and control of the pigeon adenovirus, is accurate in detection result and high in specificity, and has a relatively good application value.

Description

Technical Field

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

[0002] Pigeon adenovirus (PiAdV) is the main pathogen of pigeon crop inflammation, which seriously threatens the health of pigeons, especially when it is prevalent in racing pigeons, causing more serious losses. The virus is transmitted through feces, food contamination and direct contact. After PiAdV infection, pigeons will vomit, digestion will be weakened, excrete green or white watery feces, become listless, have messy feathers, lose weight, and even cause acute death in severe cases. There are many types of PiAdV, among which group I adenovirus (Pigeon adenovirus type I, PIAdV-Ⅰ) mainly attacks young pigeons, while group II adenovirus (Pigeon adenovirus type Ⅱ, PIAdV-Ⅱ) can infect pigeons of different ages. At present, there is a lack of accurate and quick methods worldwide to confirm the type of adenovirus infected by sick pigeons, which makes targeted treatment more difficult and causes huge economic losses to the pigeon industry.

[0003] As the main antigen protein of avian adenovirus, Hexon plays a vital role in viral immunity and recognition mechanisms. Its structure is complex and unique, consisting of the conserved P1 and P2 regions and the variable Loop1 to Loop4 loops. These regions not only give Hexon a unique morphology, but also enable it to form a homotrimeric structure, thus stimulating the body to produce specific neutralizing antibodies. In the structure of Hexon, the conserved P1 and P2 regions are located in the virus body, forming its stable base part. The four highly variable loop regions of Loop1, Loop2, Loop3 and Loop4 form the spire region. These regions are highly variable and complex, and together with the base part, they form the unique tower structure of Hexon. Among them, the Loop1 loop region is the most unique and important part of the Hexon protein. It not only has the highest sequence variability, but also has the longest length and a complex folding structure. This structural feature makes the Loop1 loop region carry a large number of antigenic determinants, which can trigger a strong immune response in the body. 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 quantification of 1 mg / mL is fully mixed with the adjuvant in a volume ratio of 1:1 and emulsified, and then used to immunize pigeons, New Zealand white rabbits and BALB / c mice, and polyclonal and monoclonal antibodies specific to the Hexon protein are prepared respectively. Based on this material, an ELISA diagnostic kit based on the pigeon adenovirus Hexon protein is further developed. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art 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; to provide rabbit, pigeon polyclonal antibodies and mouse monoclonal antibodies based on the recombinant protein; and to 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] On the one hand, the present invention discloses an anti-pigeon adenovirus Hexon protein monoclonal antibody, wherein 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] On the other hand, the present invention also discloses a double antibody sandwich ELISA diagnostic kit for detecting pigeon adenovirus, the kit comprising an ELISA plate coated with an anti-Hexon protein polyclonal antibody, Hexon protein, the anti-pigeon adenovirus Hexon protein monoclonal antibody according to claim 1 labeled with HRP, 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 and coated on an ELISA plate when used.

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

[0011] Preferably, the nucleotide sequence of the codon-optimized Hexon protein of the present invention is as shown in 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 invention has good specificity and sensitivity, and is suitable for the detection of 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] Fig. 9 Western Blot identification of HRP-labeled monoclonal antibody.

[0027] Fig.10 The best capture antibody and enzyme-labeled antibody dilutions are determined. Note: 1 to 6 from left to right are capture antibodies with gradient dilutions, with dilution concentrations ranging from 1:1000 to 1:32000; A to D from top to bottom are enzyme-labeled antibodies with gradient dilutions, with dilution concentrations ranging from 1:1000 to 1:8000; the wells in the red frame in the figure are duplicate controls with the same sequence, and the wells in the yellow frame are negative controls with the same sequence.

[0028] Fig.11 The best blocking system was determined. Note: 1: 5% gelatin; 3: 5% skim milk powder; 5: 5% BSA; 7: 5% calf serum; 2, 4, 6, 8 are the corresponding negative controls.

[0029] Fig.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, D4 to D6 are negative controls with corresponding substrate incubation times.

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

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

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

[0033] Fig.16 Positive disease sample test results. Note: 1 to 4 from left to right are gradient dilutions of positive disease samples, with dilution concentrations ranging from 1:5 to 1:40; A to C and D to F are three replicate controls of different positive disease 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 those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related 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 commercially available.

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

[0037] The PiAdV-Hexon gene sequence was screened and searched in the NCBI database, the nuclear localization signal peptide was removed, and the remaining 954bp nucleic acid sequence was synthesized after optimizing the codon preference of prokaryotic Escherichia coli. The recombinant expression plasmid was constructed after double digestion with BamHⅠ-HindⅢ restriction endonucleases and ligation with the prokaryotic expression vector pET-32a(+). 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 h, then add IPTG at a final concentration of 1 mM for induction, and induce at 37°C, 220 rpm for 4 h.

[0042] ② Bacterial cell disruption: Collect the bacterial solution of the induced overnight culture, centrifuge at 8000rpm for 10min to obtain the bacterial precipitate; resuspend and wash with an appropriate amount of sterile PBS solution, centrifuge at 12000rpm, 4℃ for 10min, and discard the supernatant; resuspend the bacteria with a small amount of pre-cooled purified washing solution, put them into an ultrasonic disruptor for disruption, and ultrasonicate for 3s and stop for 5s, and ultrasonicate on ice for 15-20min; finally, centrifuge at 12000rpm, 4℃ for 15min to obtain the precipitate.

[0043] ③ Protein denaturation and purification: Denature the obtained precipitate with 8M urea, add it to the pretreated nickel column, and bind it for 2 hours; wash the impurities with precooled purification washing solution 4 times, 10 minutes each time; elute the target protein with precooled purification eluent 3 times, 15 minutes each time. Finally, collect and combine all the 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 0 M according to the gradient renaturation method, and 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 sampled and electrophoresed on 12% SDS-PAGE gel, stained with Coomassie Brilliant Blue, and destained. Figure 2 Western Blot 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 using the BCA method, and the protein concentration was finally adjusted to 1 mg / mL.

[0048] Example 4: Immunogenicity detection of recombinant protein

[0049] The immunogenicity of the recombinant Hexon protein was detected by Western Blot using pigeon adenovirus positive serum as the 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 1 mg / mL recombinant Hexon protein and 1 mL 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 prepared rabbit polyclonal antibody was used as the primary antibody to detect the immunogenicity of the protein by the Western Blot method. The results showed that the protein Hexon could be specifically bound by the rabbit polyclonal antibody. The results are shown in Figure 5 As shown; the titer was further determined by indirect ELISA method, and the titer of the prepared rabbit polyclonal antibody was 1:1280000, and the results are 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: ① PBS negative control group; ② recombinant 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 40 μL of sterile PBS solution, and each pigeon in the recombinant Hexon protein immunization group was injected with 40 μL of emulsified protein solution (composed of: 20 μL 1 mg / mL recombinant Hexon protein + 20 μL 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, centrifugation was performed at 3000 rpm 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 the 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 female BALB / c mice aged 6-8 weeks were randomly divided into two groups, 4 mice / group: ① PBS negative control group; ② recombinant Hexon protein immunization group. 200 μL of Hexon immunogen emulsified with Freund's adjuvant (composed of: 100 μL 1 mg / mL recombinant Hexon protein + 100 μL Freund's adjuvant, Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the next two immunizations) was injected subcutaneously at multiple points on the back at 0, 14, and 28 days. The mice with the best immune effect were screened by Western Blot and indirect ELISA (results are shown in Figure 7 and Table 2), after the spleen was removed and fused with myeloma cells, multiple rounds of screening were performed in HAT medium to obtain the hybridoma cell line 1H5-8-14-5 that can stably secrete antibodies, and finally monoclonal antibodies were prepared through ascites. Western Blot results showed that the protein Hexon can specifically bind to the prepared monoclonal antibodies, such as Figure 8 The prepared monoclonal antibody was further labeled using an HRP labeling kit, and the results were as shown in Fig. 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 qualified detection, the RNA was reverse transcribed into cDNA using RACE technology; the heavy chain and light chain variable region sequences were obtained by PCR amplification; the target fragments were connected to the vector using ligase, and the ligation products were transformed into Escherichia coli competent cells, and then single clones were picked for sequencing; finally, the variable region sequences of the monoclonal antibody gene were obtained by analysis and annotation of the sequencing results, as shown in Table 3.

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

[0064]

[0065] The monoclonal antibody was used to perform western blot detection on Hexon protein or total protein extracted from pigeon adenovirus, and the results 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] In order to screen the best capture antibody and detection antibody dilution for the detection of new pigeon adenovirus, the classic experimental design of the chessboard method was used. During the experiment, the capture antibody (rabbit polyclonal antibody) was first diluted into a series of concentration gradients, ranging from 1:1000 to 1:32000. Subsequently, the capture antibody was added to each column of the ELISA plate according to the different concentrations in this range, and 100 μL of solution was added to each well. The ELISA plate was incubated at 4°C for 16 hours. After the incubation, the ELISA plate was thoroughly washed with a specific washing solution. Then, 300 μL of blocking solution was added to each well, and then blocked at a constant temperature of 37°C for 2 hours. After the blocking was completed, it was washed again with washing solution three times, and the ELISA plate was placed at 4°C for standby use. Subsequently, 100 μL of diluted Hexon antigen protein was added to each well and incubated at 37°C for 1 hour. After the incubation, it was washed three times with washing solution. Next, the detection antibody (HRP-conjugated monoclonal antibody) was diluted into a series of concentration gradients, with the concentration range set between 1:1000 and 1:8000, and 100 μL of solution was accurately added to each well. Subsequently, the ELISA plate continued to be 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 at 37°C in the dark 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 wells, the optimal conditions ( Fig.10 and Table 4), it can be seen from the chart that the best capture antibody dilution is 1:1000, and the best 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 capture antibody and enzyme-labeled antibody dilutions, four different blocking systems were set up with other conditions unchanged, 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. Fig.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 best closed system for 30 min

[0074]

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

[0076]

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

[0078]

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

[0080]

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

[0082] After determining the optimal capture antibody and enzyme-labeled antibody dilutions, other conditions remained unchanged and different antigen incubation times were set, including incubation in a 37°C biochemical incubator for 30, 60, 90, and 120 min, respectively. Fig.12 As shown in Table 9, it can be seen from the chart that the best antigen (ie 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 capture antibody and enzyme-labeled antibody dilutions, 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. Fig.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 capture antibody and enzyme-labeled antibody dilutions, other conditions remained unchanged and different TMB development times were set, namely 5 min, 10 min, and 15 min. Fig.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 the 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, and the sensitivity was judged by 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 judged according to the critical value, such as Fig.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 detection

[0098] 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 Fig.16 As shown in Figure and Table 13, it can be seen from the figure and 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 Preparation of ELISA plate: Dilute the capture antibody (rabbit polyclonal antibody prepared in Example 5) with antigen coating buffer at 1:1000, and coat it on the ELISA plate; add the ELISA plate, 100 μL / well, and incubate at 4°C overnight (12-14 hours). Take out, discard the liquid in the well, and wash 3 times with washing solution, 300 μL / well. Add blocking solution to the ELISA plate, 300 μL / well, and incubate at 37°C for 2 hours. Take out and discard the blocking solution, and wash 3 times with washing solution, 300 μL / well.

[0103] 8.2 Detection

[0104] (1) Sample addition: According to the number of samples to be tested, take the 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 min.

[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), 100 μL / well, and incubate at 37°C incubator 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 gently shake 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 storage solution: 240 mg / mL IPTG aqueous solution;

[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: 20mM Tris, 200mM NaCl, 25mM imidazole, 10% glycerol, dissolved in ddH2O, pH adjusted to 8.0, fixed to 1L, filtered through a 0.45μm filter to remove impurities;

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

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

[0120] ELISA washing solution: 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A monoclonal antibody against pigeon adenovirus Hexon protein, 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, characterized in that 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, characterized in that The amino acid sequence of the Hexon protein is shown in SEQ ID NO.

1.

5. The kit according to claim 2, characterized in that The nucleotide sequence of the codon-optimized Hexon protein is shown in SEQ ID NO.

2.

6. The kit according to claim 2, characterized in that The concentration of the Hexon protein is 0.5 μg / mL.

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

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

9. Use of the monoclonal antibody 1H5-8-14-5 as claimed in claim 1 in preparing a pigeon adenovirus or pigeon adenovirus Hexon protein detection reagent.

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