Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) kit capable of identifying highly pathogenic and non-pathogenic serum type 4 fowl adenoviruses and application of double-antibody sandwich ELISA kit

Abstract

The invention discloses a double-antibody sandwich ELISA (Enzyme Linked Immunosorbent Assay) kit capable of identifying highly pathogenic and non-pathogenic serum type 4 fowl adenoviruses and application thereof. The kit comprises an anti-serum type 4 fowl adenovirus monoclonal antibody 6F11 and an anti-highly pathogenic serum type 4 fowl adenovirus monoclonal antibody 2C5. The kit prepared by the invention can accurately identify and diagnose HHS caused by highly pathogenic serum type 4 fowl adenovirus, eliminates interference of non-pathogenic serum type 4 fowl adenovirus on HHS diagnosis in clinical detection, has the advantages of strong specificity, high sensitivity, good repeatability, low cost, simplicity and convenience in operation and the like, can realize rapid detection of a large batch of samples, and has a wide application prospect. And a technical support is provided for prevention and control of HP-FAdV-4.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a double-antibody sandwich ELISA kit capable of distinguishing highly pathogenic and non-pathogenic serotype 4 avian adenoviruses and an application thereof. Background Art

[0002] Avian adenovirus (FAdV) belongs to the genus Avianadenovirus in the family Adenoviridae. Based on restriction endonuclease digestion patterns and serum cross-neutralization assays, FAdV is currently classified into five species (FAdV-A, B, C, D, and E) and 12 serotypes (FAdV-1-7, 8a, 8b, 9-11). FAdV was first reported in South Africa in 1949 and subsequently spread widely worldwide, posing a continuing threat to the poultry industry. FAdV infection typically causes hepatitis-pericardial effusion syndrome (HHS), inclusion body hepatitis (IBH), and gizzard erosion (GE). Among the different FAdV serotypes, serotypes FAdV-2, 8a, 8b, and 11 primarily induce IBH, while FAdV-4 is the primary causative agent of HHS. Based on their pathogenicity, FAdV-4 can be divided into highly pathogenic FAdV-4 (HP-FAdV-4) and non-pathogenic FAdV-4 (LP-FAdV-4). Highly pathogenic serotype 4 avian adenovirus (HP-FAdV-4) is prevalent in China, primarily infecting 3- to 5-week-old broilers and laying hens. HP-FAdV-4 causes HHS, which has a high mortality rate and causes significant economic losses. Although several vaccines against HP-FAdV-4 have been approved for marketing, there are currently no specific diagnostic tools for HP-FAdV-4.

[0003] The genome size of FAdV-4 is about 45kb, encoding three major surface structural proteins, including Hexon, Penton and Fiber. Among them, Hexon protein has important genus, species and serotype specific antigenic epitopes, and is closely related to the high pathogenicity of the virus. Therefore, it is very necessary to develop an effective, reliable and accurate double-antibody sandwich ELISA method for the diagnosis and control of HP-FAdV-4 in China by relying on specific monoclonal antibodies against the highly pathogenic FAdV-4 Hexon protein. However, due to the high homology of amino acids between HP-FAdV-4 and LP-FAdV-4, the existing methods reported for detecting FAdV-4, such as multi-enzyme isothermal rapid amplification (MIRA), loop-mediated isothermal amplification pyrococcus (LAMP-PfAgo) detection method and LAMP-CRISPR / Cas12a detection method, cannot effectively distinguish HP-FAdV-4 from LP-FAdV-4. Summary of the Invention

[0004] Purpose of the invention: In view of the current lack of simple and rapid detection reagents and methods for identifying and distinguishing highly pathogenic strains of serotype 4 avian adenovirus from non-pathogenic strains, which has affected the accurate diagnosis of hepatitis-pericardial effusion syndrome (HHS) caused by the prevalent highly pathogenic serotype 4 avian adenovirus, the present invention provides a double-antibody sandwich ELISA kit that can distinguish highly pathogenic and non-pathogenic serotype 4 avian adenoviruses. The kit prepared by the present invention can accurately distinguish and diagnose HHS caused by highly pathogenic serotype 4 avian adenovirus, eliminate the interference of non-pathogenic serotype 4 avian adenoviruses on the diagnosis of HHS in clinical detection, has the advantages of strong specificity, high sensitivity, good repeatability, low cost and simple operation, and can realize rapid detection of large quantities of samples, providing technical support for the prevention and control of HP-FAdV-4.

[0005] The present invention also provides application of the highly pathogenic serotype 4 avian adenovirus double antibody sandwich ELISA detection kit.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses an application of a combination of an anti-serum type 4 avian adenovirus monoclonal antibody 6F11 and 2C5 in the preparation of a kit for detecting highly pathogenic serotype 4 avian adenovirus. The anti-serum type 4 avian adenovirus monoclonal antibody 6F11 is secreted by hybridoma cell 6F11, the deposit number of the hybridoma cell line 6F11 is CGMCC NO.45830, and the deposit date is March 21, 2024. The anti-highly pathogenic serotype 4 avian adenovirus monoclonal antibody 2C5 is secreted by hybridoma cell line 2C5, the deposit number of the hybridoma cell line 2C5 is CGMCC NO.45829, and the deposit date is March 21, 2024.

[0007] The highly pathogenic serotype 4 avian adenovirus double antibody sandwich ELISA detection kit of the present invention comprises the anti-serotype 4 avian adenovirus monoclonal antibody 6F11 and the anti-highly pathogenic serotype 4 avian adenovirus monoclonal antibody 2C5. The anti-highly pathogenic serotype 4 avian adenovirus monoclonal antibody 2C5 is secreted by the hybridoma cell line 2C5. The deposit number of the hybridoma cell line 2C5 is CGMCC NO.45829, and the deposit date is March 21, 2024.

[0008] Among them, the monoclonal antibody 6F11 can specifically recognize the Hexon protein of FAdV-4.

[0009] Among them, the monoclonal antibody 2C5 only reacts with HP-FAdV-4, but has no antigen-antibody reaction with LP-FAdV-4 and other common avian viruses.

[0010] The kit includes a pre-coated ELISA plate and an enzyme-labeled antibody. The pre-coated ELISA plate is an ELISA plate coated with the anti-highly pathogenic serum type 4 avian adenovirus monoclonal antibody 2C5, and the enzyme-labeled antibody is the anti-serum type 4 avian adenovirus monoclonal antibody 6F11 labeled with horseradish peroxidase.

[0011] Preferably, the coating amount of the monoclonal antibody 2C5 is 8 μg / mL, and the concentration of the enzyme-labeled antibody 6F11 is 0.6 μg / mL.

[0012] The kit further comprises a positive standard, a negative standard, an antibody coating solution, a sample diluent, a color developer, a stop solution and a washing solution.

[0013] Preferably, the positive standard is HP-FAdV-4 recombinant Hexon protein with a concentration of 12.5 ng / mL; the negative standard is LMH cell culture supernatant; the antibody coating solution is 0.05 mol / L carbonate buffer with a pH of 9.6; the sample diluent is a PBS buffer with a pH of 0.01 mM and a pH of 7.4; the solvent of the washing solution is 0.01 mM PBS buffer with a pH of 7.4, the solute is Tween-20, and the volume percentage concentration of Tween-20 in the washing solution is 0.05%; and the stop solution is a 2M aqueous sulfuric acid solution.

[0014] The invention relates to an application of the highly pathogenic serotype 4 avian adenovirus double antibody sandwich ELISA detection kit in preparing reagent materials for detecting highly pathogenic serotype 4 avian adenovirus in clinical samples.

[0015] Among them, the highly pathogenic serotype 4 avian adenovirus in the clinical sample includes representative strains of serotype 4 highly pathogenic virus strains SD2015, AHFY15, JH13, and JSCZ15.

[0016] Furthermore, the serotype 4 highly pathogenic virus strain (representative strains are SD2015, AHFY15, JH13, JSCZ15, etc.) is the main pathogen causing HHS in domestic chickens and the 188th amino acid of its Hexon protein is the virulence protein R.

[0017] Preferably, the double-antibody sandwich ELISA kit for detecting highly pathogenic serotype 4 fowl adenovirus of the present invention comprises: an enzyme labeling plate coated with a mouse anti-HP-FAdV-4 Hexon protein monoclonal antibody (2C5) and an HRP-labeled mouse anti-FAdV-4 Hexon protein monoclonal antibody (6F11).

[0018] Furthermore, the mouse anti-HP-FAdV-4 Hexon protein monoclonal antibody is secreted by the hybridoma cell line 2C5. The monoclonal antibody 2C5 secreted by the hybridoma cell line 2C5 only reacts with HP-FAdV-4, and has no antigen-antibody reaction with LP-FAdV-4 and other common avian viruses. Specific information about the hybridoma cell line 2C5 can be found in the applicant's previously disclosed patent CN202410802456.3.

[0019] Furthermore, the HRP-labeled mouse anti-FAdV-4 Hexon protein monoclonal antibody is secreted by the hybridoma cell line 6F11. The monoclonal antibody 6F11 secreted by the hybridoma cell line 6F11 can specifically recognize the Hexon protein of FAdV-4. The preservation number of the hybridoma cell line is CGMCC NO.45830, and the preservation date is March 21, 2024.

[0020] Furthermore, in the present invention, the kit further comprises a positive standard, a negative standard, an antibody coating solution, a color developer, a stop solution and a washing solution.

[0021] When using the kit of the present invention to detect HP-FAdV-4, preferably, the following steps are followed:

[0022] (1) Dilute mouse anti-HP-FAdV-4 Hexon protein monoclonal antibody (2C5) with antibody coating solution, add 100 μL / well to the ELISA plate, and coat at 4°C for 12 h.

[0023] (2) Wash the plate twice with washing solution for 2 min each time, add blocking solution, 250 μL per well, and block in a 37°C water bath for 2 h;

[0024] (3) Wash the ELISA plate three times with washing solution for 2 minutes each time. Add the antigen sample to be tested to the ELISA plate, set up parallel samples, and incubate at 37°C for 1 hour. At the same time, set up positive and negative controls.

[0025] (4) Wash the plate three times with washing buffer for 2 min each time, add HRP-labeled monoclonal antibody (6F11) diluted in PBS, and incubate at 37°C for 1 h;

[0026] (6) Wash the plate three times with washing solution for 2 minutes each time, add TMB substrate color development solution, and develop in a dark room for 15 minutes;

[0027] (7) Add the stop solution and read the value. The critical value of positive and negative is 0.086. The reading value of the positive standard is greater than 1.0, and the reading value of the negative standard is less than 0.086. The result is considered valid. At this time, if the OD450nm value of the sample is ≥0.086, it is judged as positive; if the OD450nm value is <0.086, it is judged as negative.

[0028] The formula of the washing solution PBST is KH2PO4 0.2g / L, Na2HPO4·12H2O 2.9g / L, sodium chloride 8.0g / L, KCl 0.2g / L, 0.05% Tween-20, and the rest is deionized water, and the pH is adjusted to 7.4;

[0029] The formula of the antibody coating solution is NaHCO3 2.93g / L, Na2CO3 1.59g / L, and the rest is deionized water, and the pH is adjusted to 9.6;

[0030] The blocking solution is PBS containing 5% skim milk;

[0031] The stop solution is a 2M aqueous solution of sulfuric acid;

[0032] The positive control is HP-FAdV-4Hexon recombinant protein at a concentration of 12.5 ng / mL;

[0033] The negative control is LMH cell culture supernatant;

[0034] Preferably, in the present invention, the coating concentration of the anti-highly pathogenic serum type 4 Hexon protein monoclonal antibody 2C5 is 8 μg / mL, and the concentration of the enzyme-labeled antibody HRP-6F11 is 0.6 μg / mL.

[0035] The present invention uses the monoclonal antibody 2C5, which targets the 188th amino acid R (188R) of the HP-FAdV-4 hexon protein and can specifically recognize HP-FAdV-4, as the capture antibody and the horseradish peroxidase (HRP)-conjugated monoclonal antibody 6F11 as the detection antibody to develop a double-antibody sandwich ELISA to effectively distinguish highly pathogenic and non-pathogenic serotype 4 avian adenovirus. The detection limit of this ELISA is 1×10 4 The HP-FAdV-4 double antibody sandwich ELISA kit developed by the present invention provides technical support and product assurance for distinguishing highly pathogenic and non-pathogenic serotype 4 avian adenovirus.

[0036] The present invention discloses a double-antibody sandwich ELISA kit, prepared using ELISA technology, capable of distinguishing highly pathogenic and nonpathogenic serotype 4 avian adenoviruses, and its application in testing clinical samples. The kit for detecting highly pathogenic serotype 4 avian adenovirus prepared by the present invention can clinically distinguish highly pathogenic serotype 4 avian adenoviruses from nonpathogenic serotype 4 avian adenoviruses, providing technical support and product assurance for the precise prevention and control of the spread of highly pathogenic serotype 4 avian adenoviruses in China. Therefore, the present invention has considerable market application value.

[0037] The present invention uses the monoclonal antibody 2C5 and the monoclonal antibody 6F11 prepared in this experiment against the highly pathogenic FAdV-4 Hexon protein to establish a double-antibody sandwich ELISA method for detecting highly pathogenic FAdV-4 in China, providing a useful tool for the effective differential diagnosis of HP-FAdV-4 epidemics in the Chinese poultry industry.

[0038] The single monoclonal antibody 2C5 in the present invention can be used as a capture antibody in a double-antibody sandwich ELISA, but cannot be used as an enzyme-labeled antibody. Monoclonal antibody 6F11 can be used as an enzyme-labeled antibody, but lacks the antigen-capturing ability in a double-antibody sandwich ELISA test. Therefore, single monoclonal antibodies 2C5 or 6F11 can only be used to distinguish HP-FAdV-4 from FAdV-4 through IFA and WB methods. However, IFA and WB require expensive instruments and equipment and are not conducive to the rapid clinical diagnosis of HP-FAdV-4. Therefore, in the present invention, 2C5 is used to capture antigens, and HRP-labeled monoclonal antibody 6F11 is used to detect the antigens captured by monoclonal antibody 2C5. The successfully established double-antibody ELISA kit can efficiently and quickly distinguish HP-FAdV-4 and is more suitable for large-scale clinical use.

[0039] The double-antibody sandwich ELISA of the present invention requires a capture antibody (coating antibody) and a detection antibody (enzyme-labeled antibody). Among them, the monoclonal antibody 2C5 can specifically recognize the highly pathogenic FAdV-4 and can be detected by IFA and WB. Monoclonal antibody 2C5 has good antibody capture ability in the double-antibody sandwich ELISA and can be used as a coating antibody, but after coupling with HRP, its ability to detect antigens is poor and cannot be used as a detection antibody in the double-antibody sandwich ELISA test. For this purpose, another monoclonal cell line 6F11 was further proposed. The monoclonal antibody 6F11 obtained can specifically recognize all FAdV-4 strains and has no cross-reaction with other serotypes of FAdV. It can be used to detect highly pathogenic FAdV-4 using IFA and WB. Monoclonal antibody 6F11 lacks antibody capture ability in the double-antibody sandwich ELISA, but has good antigen detection ability after coupling with HRP and can be used as a detection antibody in the double-antibody sandwich ELISA. Although monoclonal antibody 2C5 and monoclonal antibody 6F11 can play a role in the detection of FAdV by relying on IFA and WB tests respectively, and can effectively detect highly pathogenic FAdV-4 and FAdV-4, the consumables of WB test and IFA test are expensive and require specialized personnel and equipment. The specific double antibody sandwich ELISA of the present invention is a highly specific and sensitive immunoassay method, which is widely used in the quantitative detection of proteins (such as cytokines, hormones, viral antigens, etc.). Its core advantages include: 1. High specificity (High Specificity), using two antibodies (capture antibody + detection antibody) to bind to different epitopes of the target protein respectively to reduce cross-reactions. It is suitable for complex samples (such as serum, cell lysate) to reduce background interference. 2. High sensitivity (High Sensitivity), the signal amplification mechanism (such as enzyme-substrate reaction, biotin-streptavidin system) can detect low-abundance proteins (pg / mL level), which is more sensitive than direct ELISA or indirect ELISA and suitable for trace sample analysis.

[0040] The present invention utilizes the excellent antigen capture ability of monoclonal antibody 2C5 and the excellent antigen detection ability of monoclonal antibody 6F11 after enzyme labeling. For the first time, monoclonal antibody 2C5 is used as a coating antibody, and monoclonal antibody 6F11 is enzyme-labeled with HRP as a detection antibody. The combination of the two antibodies creates a double-antibody sandwich ELISA kit that can specifically detect the highly pathogenic FAdV-4. This is convenient and fast, and can be used for batch testing of multiple clinical samples. Single 2C5 or 6F11 cannot be used to create a double-antibody sandwich ELISA kit, and FAdV-4 identification can only be performed by Western blotting or IFA tests, which are time-consuming and labor-intensive, making large-scale batch detection of FAdV-4 impossible. Furthermore, the kit has certain sample requirements, making it unsuitable for large-scale clinical promotion.

[0041] The present invention utilizes a combination of monoclonal antibodies 2C5 and 6F11 to create a double-antibody sandwich ELISA kit. Single antibodies 2C5 or 6F11 are unable to complete a double-antibody sandwich ELISA test and can only identify FAdV-4 via IFA and WB, making them unsuitable for large-scale clinical application. Monoclonal antibodies 2C5 and 6F11 are purified, and antibody 6F11 is labeled with HRP. A series of experiments optimize and determine various test conditions for a double-antibody sandwich ELISA. The resulting double-antibody sandwich ELISA kit exhibits high sensitivity and specificity, is convenient, and can be quickly and clinically applied to the detection of a variety of samples. In the present invention, the OD450 value reaches a maximum of 1.152 when only 2C5 and 6F11 are combined. When used alone or in other combinations, the OD value is significantly lower than 1.152.

[0042] In the present invention, clinical samples naturally infected with HP-FAdV-4 and not infected with HP-FAdV-4 were selected for sample testing. The results showed that the double-antibody sandwich ELISA kit of the present invention can effectively detect liver samples and kidney samples of chickens naturally infected with HP-FAdV-4, and has practical value. The samples used were also verified by the single antibody 2C5 IFA test, and the detection results were 100% consistent with the monoclonal antibody 2C5.

[0043] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0044] (1) Strong specificity: The kit of the present invention has strong reactivity with HP-FAdV-4 recombinant Hexon protein and HP-FAdV-4 in clinical samples, but has no cross-reaction with LP-FAdV-4 and other serotypes of FAdV and common avian viruses.

[0045] (2) Good reproducibility: The kit of the present invention has good intra-batch and inter-batch reproducibility.

[0046] (3) Wide applicability: It can detect a variety of clinical samples including fecal samples and tissue samples.

[0047] (4) Rapid: The kit of the present invention is easy and rapid to operate.

[0048] Therefore, the double-antibody sandwich ELISA kit established in the present invention is convenient, fast, has good specificity and repeatability, and can be used for rapid detection of HP-FAdV-4 in the laboratory or clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 To verify the purification effect of monoclonal antibodies 2C5 and 6F11, M: protein marker; lane 1: purified monoclonal antibody 2C5; lane 2: purified monoclonal antibody 6F11.

[0050] Figure 2 This is the specificity test result of the double antibody sandwich ELISA kit.

[0051] Figure 3 These are the sensitivity test results of the double-antibody sandwich ELISA kit. A: Sensitivity results for detecting HP-FAdV-4; B: Sensitivity results for detecting His-Hexon recombinant protein.

[0052] Figure 4 This is the test result of the double antibody sandwich ELISA kit on clinical samples. DETAILED DESCRIPTION

[0053] The present invention will be further described below with reference to the accompanying drawings and examples.

[0054] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. Experimental methods without specific conditions specified in the examples are generally performed under conventional conditions or the conditions recommended by the manufacturer.

[0055] Mouse myeloma cells (SP2 / 0), chicken hepatoma cells (LMH), FAdV-1 ( VR-432 TM ),FAdV-4B1-7(non-pathogenic FAdV-4,LP-FAdV-4)( VR-829 TM ),FAdV-6( VR-831 TM ),FAdV-7( VR-832 TM ),FAdV-9( VR-833 TM ) and FAdV-10( VR-834 TM ) was purchased from the American Type Culture Collection (ATCC).

[0056] FAdV-8a (JSSQ15), FAdV-11 (380), HP-FAdV-4 (highly pathogenic FAdV-4 strain SD2015, Outbreaks of serotype 4 fowl adenovirus with novel genotype, China, Emerging Microbes and Infections (2016) 5, e50; doi: 10.1038 / emi.2016.50; published online 25 May 2016), FAdV-8a (JSSQ15) and FAdV-11 (380), Marek's disease virus (MDV), infectious bronchitis virus (IBV), avian reticuloendotheliosis virus (REV), chicken infectious anemia virus (CAV), H9N2 avian influenza virus (AIV) and subgroup J avian leukosis virus (ALV) were preserved and provided by Yangzhou University.

[0057] Monoclonal antibody 2C5 targeting highly pathogenic serotype 4 Hexon, and its hybridoma cell 2C5 and recombinant truncated protein of FAdV-4 Hexon (104-291 aa), were prepared according to CN202410802456.3 and publicly deposited in the aforementioned patent. They were provided by Yangzhou University. Monoclonal antibody 6F11 targeting serotype 4 Hexon was prepared and stored by the present invention. 96-well ELISA plates were purchased from Corning. TMB single-component colorimetric solution was purchased from Solebo Technology Co., Ltd.

[0058] Formula of main reagents:

[0059] Carbonate coating solution: Weigh 0.159 g of Na2CO3 and 0.293 g of NaHCO3 into a 100 mL glass bottle, add 100 mL of deionized water to fully dissolve, and store at 4°C.

[0060] 5% skim milk: Weigh 2.5 g of skim milk powder into a 50 mL centrifuge tube, add PBST solution to 50 mL, and vortex to mix.

[0061] 2M H2SO4 stop solution: Pipette 10.8 mL of concentrated sulfuric acid and slowly add it into 89.2 mL of deionized water. Mix slowly and set aside.

[0062] Preparation of the supernatant of cells infected with FAdV-4, i.e., the SD2015 virus solution: LMH cells were plated into 96-well plates. After approximately 9 days of growth, when the cell colony density reached approximately 60% to 80% of the well bottom area, the LMH cells were infected with FAdV4 (SD2015) at an MOI of 0.01. The cytopathic effect was observed daily. When the cytopathic effect reached approximately 70%, the cells and virus solution were harvested and centrifuged at 12,000 rpm for 10 minutes. The supernatant was aspirated to obtain the SD2015 virus solution.

[0063] Example 1

[0064] Preparation of hybridoma cell line 6F11

[0065] 1. Preparation of monoclonal antibodies

[0066] Purified prokaryotically expressed recombinant truncated FAdV-4 Hexon (104-291aa) protein was mixed with equal amounts of Freund's adjuvant and emulsified. Five-week-old female BALB / c mice were subcutaneously injected at multiple sites at 50 μg per mouse. Booster immunizations were performed 21 and 35 days after immunization using recombinant Hexon protein emulsified in incomplete Freund's adjuvant. Blood was collected 49 days after the initial immunization for antibody titer measurement. BALB / c mice with the highest antibody titers were selected and given a pulse immunization with 50 μg of recombinant Hexon protein intraperitoneally 3 days prior to cell fusion.

[0067] Aseptically remove the spleen from the immunized mouse and place it in a sterile container. Grind the spleen with an instrument to prepare a splenocyte suspension. The splenocytes were then fused with SP2 / 0 myeloma cells at a ratio of 5:1 using a PEG fusion agent. The cells were resuspended in HAT medium, aliquoted into 96-well cell culture plates, and cultured in a 37°C, 5% CO2 cell culture incubator. The cell culture supernatant was collected 12 days after fusion for later use. During this time, LMH cells were plated into 96-well plates. Approximately 9 days after growth, when the cell colony density reached approximately 60%–80% of the well bottom area, the LMH cells were infected with FAdV4 (SD2015) and monitored daily for cytopathic effects. When the cytopathic effect reached approximately 70%, the cells were fixed with pre-cooled acetone-ethanol (3:2 acetone-ethanol) at room temperature for 5–10 minutes. The fixative was discarded, the plates were washed once with PBS, and the plates were then ventilated in a fume hood for 3 hours. After the residual acetone and ethanol in the cell plate evaporated, the hybridoma supernatant was added to the cell plate as the primary antibody at 50 μl / well. The cell plate was then incubated in a 37°C water bath for 45 minutes. The primary antibody was discarded and the plate was washed three times with PBS. FITC-labeled goat anti-mouse immunoglobulin was diluted 1:150 and added to the cell plate (protected from light) at 50 μl / well. The cell plate was incubated in a 37°C water bath for 45 minutes. The secondary antibody was discarded and the plate was washed three times with PBS (protected from light). PBS was added and the results were analyzed using an inverted fluorescence microscope. A cell line that tested positive for IFA was obtained. The positive hybridoma cells screened were subcloned to establish a hybridoma cell line that stably secretes anti-FAdV-4, designated hybridoma cell line 6F11. The hybridoma cell strain 6F11 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on March 21, 2024, with the accession number CGMCC NO.45830, and was classified as mouse hybridoma cell.

[0068] 2. Preparation of monoclonal antibodies against hybridomas

[0069] The established monoclonal antibody 6F11 was cultured in a 25 cm2 T25 cell culture flask. After adding 6 mL of HT medium containing 10% serum, the cells were cultured in vitro for 2 days. The hybridoma cells formed a dense monolayer and the culture medium turned yellow. The culture supernatant was collected and centrifuged at 4°C, 12000 rpm for 10 minutes to remove cells and debris, thereby obtaining the 6F11 hybridoma monoclonal antibody.

[0070] 3. Preparation of Ascites

[0071] After the hybridoma cells were screened and cultured, 1×10 7 / 200μl was injected into BLAB / C mice with 200μl. After 7 days, when the abdominal circumference of the mice increased, the ascites was collected with a syringe needle, centrifuged at 4000r / min for 10min, and the supernatant was filtered through 0.45μm to obtain the ascites mAb product.

[0072] 4. Identification of monoclonal antibody antigen subclasses

[0073] The subclass of monoclonal antibody 6F11 was identified according to the instructions of the monoclonal antibody subclass identification kit (Beijing Biolong Biotechnology Co., Ltd., catalog number: KLB4015524). The result showed that the heavy chain subclass of monoclonal antibody 6F11 was IgG1.

[0074] 5. Determination of the titer of monoclonal antibodies in hybridoma cell supernatant and ascites

[0075] The specific steps for ELISA titer determination of supernatant and ascites are as follows: Hexon truncated protein is coated on the ELISA plate at 1 μg / well and incubated at 37°C for 1 hour and 4°C overnight. The next day, the liquid in the wells was discarded, and the wells were washed three times, each time for 2 minutes; 200 μl of 5% skim milk blocking solution was added to each well and incubated at 37°C for 1 hour; 6F11 hybridoma supernatant and ascites diluted 1:101-109 with PBS were added respectively and incubated at 37°C for 1 hour; after washing three times, they were patted dry; HRP-labeled goat anti-mouse IgG (1:10000 dilution) was added and incubated at 37°C for 1 hour; after washing three times, they were patted dry; 100 μl / well of color development solution was added and color was developed at room temperature in the dark for 15 minutes; the reaction was terminated, 50 μl of 2 mol / L sulfuric acid was added to each well, and the mixture was shaken and mixed. The OD450nm value was measured by a microplate reader. The titer of the monoclonal antibody secreted by the 6F11 hybridoma cells was 1:3200, and the titer of the prepared ascites antibody was 1:106. Furthermore, using the IFA assay, LMH cells infected with FAdV-4 (SD2015 strain) were fixed, and the ascites and supernatant were each diluted 10 times, and the titer was determined by indirect immunofluorescence. The IFA titer of the 6F11 hybridoma cell supernatant was 1:800, and the titer of the ascites was 1:25,000.

[0076] 6. Stability experiment of monoclonal antibodies secreted by hybridoma cell lines

[0077] After 12 months of cryopreservation, the hybridoma cells were removed from liquid nitrogen and revived. Indirect immunofluorescence assays were then performed to assess the secretion of monoclonal antibodies (MAbs). The results showed that the titer of the supernatant secreted by the hybridoma cell line 6F11 was consistent with that before cryopreservation, indicating stable antibody secretion. Testing of MAbs secreted by hybridomas after 30 passages also demonstrated the stability of MAb secretion by the hybridoma cell line.

[0078] Example 2

[0079] The establishment process of the double antibody sandwich ELISA antigen detection method

[0080] 1. Preparation and Purification of Monoclonal Antibodies

[0081] The preserved hybridoma cell lines 2C5 and 6F11 were expanded and cultured at 1×10 7 200 μl was injected into 5-week-old female BLAB / C mice. After the abdominal circumference of the mice increased on day 7, the ascites was collected with a syringe needle and centrifuged at 4000 rpm for 10 min. The supernatant was filtered through 0.45 μm to obtain the crude ascites mAb product. The monoclonal antibody was purified using a GE Protein pre-packed column and dialyzed against PBS to obtain the purified monoclonal antibody. Figure 1 As shown in the figure, SDS-PAGE gel electrophoresis analysis of the purified monoclonal antibodies 2C5 and 6F11 revealed a clear single protein band at 50kDa and 25kDa, corresponding to the heavy and light chains of the antibodies, respectively, with no other contaminants on the gel. This result strongly demonstrates that the monoclonal antibody purification operation was successful, yielding the target monoclonal antibodies of high purity.

[0082] 2. Preparation of enzyme-labeled monoclonal antibodies

[0083] The enzyme-labeled anti-FAdV-4 monoclonal antibodies were labeled with horseradish peroxidase using Thermo Fisher Scientific's EZ-Link Plus Activated Peroxidase Kit (EZ-Link Plus Activated Peroxidase Kit) according to the kit's instructions. After dialysis with PBS, the HRP-labeled monoclonal antibodies 2C5-HRP and 6F11-HRP were obtained.

[0084] 3. Selection of double antibody sandwich pairing antibodies

[0085] The purified monoclonal antibodies 2C5 and 6F11 were coated as capture antigens (coating antibodies) and cross-matched with enzyme-labeled antibodies HRP-2C5 and HRP-6F11. The group with the highest OD value was selected as the optimal antibody sandwich combination.

[0086] The coating method can be performed as follows: dilute the coating antibody to 5 μg / mL in bicarbonate buffer and add 100 μl per well to the ELISA plate. The plate is incubated at 4°C for 12 hours, washed twice with PBST, and then patted dry. Block the plate with 5% skim milk (250 μl / well). Block the plate at 37°C for 2 hours and then wash three times with PBST. Add supernatant from FAdV-4-infected cells to positive wells, and supernatant from LMH cells to corresponding negative wells. Incubate the plate at 37°C for 1 hour and wash the plate three times for 2 minutes each. Then, dilute the two enzyme-labeled antibodies 1:800, incubate the plate at 37°C for 1 hour, and wash the plate three times for 2 minutes each. Add the substrate colorimetric solution and incubate the plate at 37°C in the dark for 15 minutes. The colorimetric reaction is terminated by adding 50 μL of 2 M concentrated H₂SO₄, and the OD450 nm is measured using a microplate reader. The results are shown in Table 1. Based on these results, monoclonal antibody 2C5 was selected as the capture antibody (coating antibody) and HRP-6F11 was selected as the detection antibody. In subsequent condition screening, unless otherwise specified, follow the procedure in step 3, varying the conditions. Once an optimal condition is determined, explore the next optimal condition by following step 3 and the previous optimal condition, until the optimal condition is obtained.

[0087] Table 1 Optimal combination of capture antibody and detection antibody

[0088]

[0089] Furthermore, the experiment showed that only when 2C5 and 6F11 were combined, the OD450 value was the highest, reaching 1.152. When used alone or in other combinations, the OD value was far lower than 1.152, demonstrating the synergistic effect of the combination.

[0090] 4. Determination of the coating concentration of monoclonal antibodies and the working concentration of enzyme-labeled antibodies

[0091] Monoclonal antibody 2C5 was selected as the capture antigen, and enzyme-labeled monoclonal antibody HRP-6F11 was used as the detection antibody. The optimal antibody coating concentration and the optimal working concentration of the enzyme-labeled secondary antibody were determined using a checkerboard titration method. Following the method in step 3 above, the following steps were performed: The coating antibody 2C5 and the enzyme-labeled antibody HRP-6F11 were diluted separately in carbonate coating solution. The wells with the highest P / N ratio and an OD450nm value of approximately 1.0 were selected. The corresponding concentrations of the coating antibody 2C5 and enzyme-labeled antibody HRP-6F11 were considered optimal. The results are shown in Tables 2 and 3. The optimal coating concentrations for the coating antibody 2C5 and the enzyme-labeled antibody HRP-6F11 were determined to be 8 μg / mL, and 0.6 μg / mL, respectively.

[0092] Table 2 OD450 value detection results of chessboard titration method

[0093]

[0094] Table 3 P / N values of chessboard titration method

[0095]

[0096] 5. Optimization of optimal response time

[0097] Following the reaction conditions established above, following the method in step 3, FAdV-4 antigen (SD2015 strain supernatant) was added and incubated at room temperature for 30 and 60 minutes, respectively. The cells were then washed three times and patted dry. The 6F11-HRP enzyme-labeled antibody was then added and incubated at room temperature for 15, 30, 45, 60, and 75 minutes, respectively. The cells were washed three times and patted dry. The substrate was added and color was developed at room temperature for 15 minutes. The reaction was then terminated by adding 50 μl of stop solution to each well. The OD450 value was read and the P / N ratio was calculated. The results are shown in Table 4. Based on the results and for convenience, the final incubation time for both the antigen and secondary antibody was 60 minutes.

[0098] Table 4 Optimization of optimal reaction time between antigen and secondary antibody

[0099]

[0100] 6. Optimal conditions and operating procedures for the double antibody sandwich ELISA kit

[0101] (1) Dilute mouse anti-HP-FAdV-4 Hexon protein monoclonal antibody (2C5) with antibody coating solution, add 100 μL / well to the ELISA plate, and coat at 4°C for 12 h.

[0102] (2) Wash the plate twice with washing solution for 2 min each time, add blocking solution, 250 μL per well, and block in a 37°C water bath for 2 h;

[0103] (3) Wash the ELISA plate three times with washing solution for 2 minutes each time. Add the antigen sample to be tested to the ELISA plate, set up parallel samples, and incubate at 37°C for 1 hour. At the same time, set up positive and negative controls.

[0104] (4) Wash the plate three times with washing buffer for 2 min each time, add HRP-labeled monoclonal antibody (6F11) diluted in PBS, and incubate at 37°C for 1 h;

[0105] (6) Wash the plate three times with washing solution for 2 minutes each time, add TMB substrate color development solution, and develop in a dark room for 15 minutes;

[0106] (7) Add stop solution and read the value.

[0107] 7. Determination of positive and negative critical values

[0108] Using the established sandwich ELISA method, 32 FAdV-free negative samples were tested. These samples included LMH cell culture supernatant samples, healthy non-immune chicken feces, and liver and kidney tissue samples. The experimental conditions were as follows: the average OD value of the positive control (PC) was greater than 1.0; the average OD value of the negative control (NC) was less than 0.086; the positive-negative critical value = the average OD450 value of the negative sample + 3SD (standard deviation). The final results are shown in Table 5. The average OD450nm value of the negative sample was 0.0674 and the standard deviation was 0.0062. When OD ≥ 0.086, it was judged as positive, and when OD < 0.086, it was judged as negative.

[0109] Table 5 Test results of 32 negative samples

[0110]

[0111]

[0112] 8. Specificity

[0113] Different serotypes of avian adenovirus (FAdV-1, HP-FAdV-4, LP-FAdV-4, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10, FAdV-11, MDV, AIV, ALV, REV, IBV, CAV) were detected according to the method in step 6 to evaluate the specificity of the sandwich ELISA method. Figure 2 As shown in the results, the OD450 values of LP-FAdV-4 and other serotypes of FAdV and common avian viruses were between 0.059 and 0.075, which were lower than the cut-off value of ELISA, while the OD450 value of HP-FAdV-4 reached 2.187, demonstrating the high specificity of this kit in detecting HP-FAdV-4.

[0114] 9. Sensitivity

[0115] HP-FAdV-4 and His-Hexon fusion proteins at different dilutions were tested according to the method in step 6 to explore the sensitivity of sandwich ELISA in detecting FAdV-4. Figure 3 As shown in A, the detection limit (Log10) of FAdV-4 strain SD15 is 10 4 TCID50 / ml. Figure 3As shown in B, the detection limit of the purified His-Hexon fusion protein in the ELISA was 3.125 ng / ml. Notably, the OD450 values of both FAdV-4 and the His-Hexon fusion protein showed an effective dose-dependency, further highlighting the specificity of the ELISA method established in this study for FAdV-4.

[0116] 10. Repeatability test

[0117] According to the optimal working conditions of the sandwich ELISA determined above, 12 different HP-FAdV-4 cell supernatants were used to perform intra-batch and inter-batch repeatability tests. The results are shown in Table 6. The inter-batch and intra-batch coefficients of variation of the HP-FAdV-4 double antibody sandwich ELISA method established in this experiment were less than 10%, indicating that this method has good repeatability during detection.

[0118] Table 6 Intra-plate repeatability test results

[0119] 1 2 3 4 5 6 1 hole 1.423 1.397 1.214 1.351 1.276 1.286 2 holes 1.415 1.369 1.278 1.368 1.342 1.204 3 holes 1.444 1.377 1.216 1.445 1.357 1.185 4 holes 1.465 1.425 1.252 1.383 1.304 1.182 Mean 1.437 1.392 1.240 1.387 1.320 1.214 SD 0.022 0.025 0.031 0.041 0.037 0.049 CV% 1.6％ 1.8％ 2.5％ 3％ 2.8％ 4.0％

[0120] Table 7 Inter-plate repeatability test results

[0121]

[0122]

[0123] 10. Testing of clinical samples

[0124] To explore whether the double antibody sandwich ELISA kit can be used to detect FAdV-4 clinical samples, 28 liver samples and kidney samples from chickens naturally infected with FAdV-4 were collected as positive samples, and 10 liver samples from SPF chickens not infected with FAdV-4 were collected as negative samples. All 56 positive samples were verified by PCR. The kit and ELISA method constructed by the present invention were used for detection. Figure 4 As shown, the ELISA test results of 56 diseased chicken tissue samples were all positive, with the OD450 values of positive samples ranging from 0.258 to 1.540, and an average of 0.773. The results show that the sandwich ELISA method established in the present invention can be used to detect HP-FAdV-4 antigen in clinical specimens.

[0125] In summary, the dual-antibody ELISA method established based on monoclonal antibodies 2C5 and 4H7 has high sensitivity, strong specificity, and good reproducibility, providing a useful tool for the effective differential diagnosis of HP-FAdV-4 prevalent in the Chinese poultry industry.

Claims

1. An application of a combination of anti-serum type 4 avian adenovirus monoclonal antibody 6F11 and 2C5 in the preparation of a kit for detecting highly pathogenic serotype 4 avian adenovirus, wherein the anti-serum type 4 avian adenovirus monoclonal antibody 6F11 is secreted by hybridoma cell 6F11, the deposit number of the hybridoma cell line 6F11 is CGMCC NO.45830, and the deposit date is March 21, 2024; the anti-highly pathogenic serotype 4 avian adenovirus monoclonal antibody 2C5 is secreted by hybridoma cell line 2C5, the deposit number of the hybridoma cell line 2C5 is CGMCC NO.45829, and the deposit date is March 21, 2024.

2. A double antibody sandwich ELISA detection kit for highly pathogenic serotype 4 avian adenovirus, characterized in that: The kit includes anti-serum type 4 avian adenovirus monoclonal antibody 6F11 and anti-highly pathogenic serum type 4 avian adenovirus monoclonal antibody 2C5. The anti-serum type 4 avian adenovirus monoclonal antibody 6F11 is secreted by hybridoma cell 6F11, the preservation number of the hybridoma cell line 6F11 is CGMCC NO.45830, and the preservation date is March 21, 2024. The anti-highly pathogenic serum type 4 avian adenovirus monoclonal antibody 2C5 is secreted by hybridoma cell line 2C5, the preservation number of the hybridoma cell line 2C5 is CGMCC NO.45829, and the preservation date is March 21, 2024.

3. The double antibody sandwich ELISA detection kit for highly pathogenic serotype 4 avian adenovirus according to claim 2, characterized in that: The monoclonal antibody 6F11 can specifically recognize the Hexon protein of FAdV-4.

4. The double antibody sandwich ELISA detection kit for highly pathogenic serotype 4 avian adenovirus according to claim 2, characterized in that: The monoclonal antibody 2C5 only reacts with highly pathogenic FAdV (HP-FAdV-4), but has no antigen-antibody reaction with non-pathogenic FAdV (LP-FAdV-4) and other common avian viruses.

5. The double antibody sandwich ELISA detection kit for highly pathogenic serotype 4 avian adenovirus according to claim 2, characterized in that: The kit comprises a pre-coated ELISA plate and an enzyme-labeled antibody. The pre-coated ELISA plate is coated with the anti-highly pathogenic serum type 4 avian adenovirus monoclonal antibody 2C5, and the enzyme-labeled antibody is the anti-serum type 4 avian adenovirus monoclonal antibody 6F11 labeled with horseradish peroxidase.

6. The double antibody sandwich ELISA detection kit for highly pathogenic serotype 4 avian adenovirus according to claim 5, characterized in that: The coating amount of the monoclonal antibody 2C5 is 8-10 μg / mL, and the concentration of the enzyme-labeled antibody 6F11 is 0.6-1 μg / mL.

7. The double antibody sandwich ELISA detection kit for highly pathogenic serotype 4 avian adenovirus according to claim 6, characterized in that: The kit also includes a positive standard substance, a negative standard substance, an antibody coating solution, a sample diluent, a color developer, a stop solution and a washing solution.

8. Use of the double antibody sandwich ELISA detection kit for highly pathogenic serotype 4 avian adenovirus according to claim 2 in preparing reagent materials for detecting highly pathogenic serotype 4 avian adenovirus in clinical samples.

9. The use according to claim 8, characterized in that The highly pathogenic serotype 4 avian adenovirus in the clinical sample preferably includes representative strains of the highly pathogenic serotype 4 avian adenovirus strains SD2015, AHFY15, JH13, and JSCZ15.

Images