Reagent for detecting avian leukemia virus and nanozyme immunochromatography test strip and application thereof

Abstract

The application provides a reagent for detecting avian leukemia virus, a nanozyme immunochromatography test strip and application thereof, and belongs to the technical field of immunology. In view of the problems of complicated operation, time consumption and strong equipment dependence of the existing avian leukemia virus detection method, the reagent comprises a capture antibody and a detection antibody; the heavy chain variable region of a monoclonal antibody A comprises an amino acid sequence as shown in (SEQ ID NO: 1), and the light chain variable region comprises an amino acid sequence as shown in (SEQ ID NO: 2); the heavy chain variable region of a monoclonal antibody B comprises an amino acid sequence as shown in (SEQ ID NO: 5), and the light chain variable region comprises an amino acid sequence as shown in (SEQ ID NO: 6); and the antigen binding fragment is a Fab fragment, a F(ab)2 fragment or a single-chain Fv fragment. The reagent is mainly used for detecting avian leukemia virus and is suitable for detecting samples such as avian cloaca swabs, fetal meconium, egg white or serum.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immunology. More particularly, the present application relates to a reagent for detecting avian leukosis virus (ALV) capsid protein (p27 protein) and a nanozyme immunochromatography test strip and application thereof. BACKGROUND

[0002] Avian leucosis (AL) is a chronic malignant tumor disease characterized by abnormal proliferation of lymphoid tissue caused by avian leukosis virus (ALV). As an important pathogen that threatens the global poultry industry, ALV has high vertical transmission efficiency, long incubation period, and continuous virus shedding after infection, and its harm runs through the entire cycle of poultry farming.

[0003] ALV belongs to the alpha retrovirus genus of the retrovirus family and is a single-stranded RNA virus. Currently, 11 subtypes of ALV have been found, of which 7 subtypes (A-E, J, and K) can infect chickens. It is worth noting that subtypes A, B, and J show strong pathogenicity, especially subtype J, which has become the most widely prevalent ALV subtype in the current Chinese poultry industry. The full-length genome of ALV is about 7.2 kb, which contains three main open reading frames (ORFs) in the 5' to 3' direction: the capsid protein gene (gag) that encodes the core structural protein of the virus; the polymerase gene (pol) that encodes key enzymes such as reverse transcriptase and integrase; and the envelope glycoprotein gene (env) that encodes the envelope protein determining the host tropism of the virus. Among them, the p27 protein encoded by the gag gene is the main component of the viral core capsid, which is highly conserved in different subgroups, contains multiple virus antigen sites that are easy to detect and has strong immunogenicity, making it a key marker in virology research and clinical diagnosis.

[0004] Currently, the basic strategy for preventing this disease in China is to detect and eradicate evolution in stages. Therefore, the diagnosis of this virus is particularly urgent. The detection methods of ALV mainly include pathogenology and serology detection, including virus isolation, indirect immunofluorescence, PCR, loop-mediated isothermal amplification (LAMP), ELISA, etc. Although these diagnostic methods have been greatly improved, these methods still have problems such as complex operation, time-consuming, or strong dependence on equipment, therefore, it is urgent to develop more efficient and portable detection tools. SUMMARY

[0005] An object of the present application is to solve at least the above problems and to provide at least the advantages described later.

[0006] Another objective of this invention is to provide a reagent and a nanozyme immunochromatographic test strip for detecting the capsid protein (p27 protein) of avian leukosis virus (ALV) and their applications.

[0007] To achieve these objectives and other advantages according to the present invention, a reagent for detecting avian leukosis virus is provided, characterized in that it comprises a capture antibody and a detection antibody; wherein,

[0008] When the capture antibody is monoclonal antibody A, the detection antibody is monoclonal antibody B; or when the capture antibody is monoclonal antibody B, the detection antibody is monoclonal antibody A.

[0009] The heavy chain variable region of the monoclonal antibody A contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:2.

[0010] The heavy chain variable region of the monoclonal antibody B contains an amino acid sequence as shown in SEQ ID NO:5, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6.

[0011] The antigen-binding fragment is a Fab fragment, an F(ab)2 fragment, or a single-chain Fv fragment.

[0012] Preferably, in the reagent for detecting avian leukosis virus, the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody A are ATPSQPTI, FILEILIL, and QEREVTTAWPGFLT, respectively; and the light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody A are KSVSTSGYSY, LVS, and QHIRELTR, respectively.

[0013] The heavy chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody B are VTHSLATP, LILTMVVL, and QDMMVTTWGLTT, respectively; the light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody B are QSLLNSGNQKNY, WAS, and QNDYDYPLT, respectively.

[0014] The present invention also provides a nanoenzyme immunochromatographic test strip, which includes a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad that are sequentially overlapped, wherein the nitrocellulose membrane is provided with a detection line and a control line;

[0015] The binding pad is coated with a nanozyme-labeled capture antibody, and the detection line is coated with a detection antibody;

[0016] The capture antibody and detection antibody are the capture antibody and detection antibody contained in the reagent for detecting avian leukosis virus mentioned above;

[0017] The quality control line is coated with goat anti-mouse IgG antibody.

[0018] Preferably, in the nanozyme immunochromatographic test strip, the concentration of the nanozyme-labeled capture antibody is 60 μg / mL; the concentration of the detection antibody coated on the detection line is 1 mg / mL; and the concentration of goat anti-mouse IgG coated on the control line (C) is 1 mg / mL.

[0019] The present invention also provides an application of the nanoenzyme immunochromatographic test strip described above in the detection of avian leukosis virus, wherein the test sample is avian cloacal swab, meconium, egg white or serum.

[0020] The present invention has at least the following beneficial effects:

[0021] 1. This invention first screened and obtained a monoclonal hybridoma cell line containing the avian leukosis virus p27 protein, then obtained a specific monoclonal antibody, and obtained the variable region gene sequence of the antibody using nested PCR amplification technology (which can be subsequently obtained using genetic engineering or protein engineering methods). Furthermore, based on the specific monoclonal antibody against the avian leukosis virus p27 protein, this invention developed a convenient, rapid, accurate, highly sensitive, and highly specific nanozyme immunochromatographic test strip for detecting avian leukosis virus.

[0022] 2. This invention develops a nanozyme antigen test strip for avian leukosis virus based on immunochromatographic technology, achieving "one-step" detection suitable for rapid on-site screening. This test strip combines the catalytic and signal amplification properties of nanomaterials, optimizing the shortcomings of traditional methods and improving the sensitivity of avian leukosis virus detection. The minimum detectable dose of the virus is 12.5 TCID. 50 / mL, the minimum detectable level for protein is 0.2 ng / mL.

[0023] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0024] Figure 1 The image shows the SDS-PAGE identification results of the purified avian leukosis virus p27 protein in Example 1 of this invention. M: protein marker; 1: purified recombinant pCold I-p27 protein.

[0025] Figure 2 This is a schematic diagram of the structure of the avian leukosis virus test strip in Embodiment 2 of the present invention.

[0026] Figure 3 This is a schematic diagram illustrating the determination of the detection results of the avian leukosis virus test strip in Embodiment 3 of the present invention.

[0027] Figure 4 This is a graph showing the virus sensitivity detection results of the avian leukosis virus nanozyme immunochromatographic test strip in Example 3 of the present invention.

[0028] Figure 5 This is a graph showing the sensitivity detection results of the avian leukosis virus nanozyme immunochromatographic test strip for p27 protein in Example 3 of the present invention.

[0029] Figure 6 This is a diagram showing the negative anal swab test results in Example 3 of the present invention.

[0030] Figure 7 This is a diagram showing the specific detection results of the avian leukosis virus test strip in Example 3 of the present invention. NC: sample dilution solution. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it based on the description.

[0032] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0033] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0034] In the description of this invention, the terms "lateral", "longitudinal", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0035] This invention specifically relates to a monoclonal antibody specific to the capsid protein (p27) of avian leukosis virus (ALV) and its application—a nanozyme immunochromatographic test strip containing this antibody. The amino acid sequences of the heavy chain variable regions of the monoclonal antibody or antigen-binding fragments one and two are shown in SEQ ID NO:1 and 5, respectively; the amino acid sequences of the light chain variable regions are shown in SEQ ID NO:2 and 6, respectively. Based on the double-antibody sandwich principle, this invention constructs an ALV nanozyme detection test strip. This test strip has significant technical advantages: ① High specificity, accurately identifying ALV, with no cross-reactivity with Newcastle disease virus, Marek's disease virus, infectious bursal disease virus, fowlpox virus, infectious laryngotracheitis virus, and avian influenza virus; ② High sensitivity, with a minimum detection limit of 12.5 TCID for viruses. 50 The detection limit for protein was 0.2 ng / mL, demonstrating good detection sensitivity.

[0036] In a first aspect, a monoclonal hybridoma cell line containing the avian leukosis virus (ALV) p27 protein was obtained through screening, thereby acquiring a specific monoclonal antibody. The variable region gene sequence of the antibody was obtained using nested PCR amplification technology (which can subsequently be obtained using genetic engineering or protein engineering methods). The monoclonal antibody is selected from monoclonal antibody A (monoclonal antibody or antigen-binding fragment one) or monoclonal antibody B (monoclonal antibody or antigen-binding fragment two). The heavy chain variable region of monoclonal antibody A contains the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:2. The heavy chain variable region of monoclonal antibody B contains the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:6.

[0037] The antigen-binding fragment is a Fab fragment, an F(ab)2 fragment, or a single-chain Fv fragment.

[0038] The amino acid sequences of the heavy chain variable regions CDR1 (SEQ ID NO:9), CDR2 (SEQ ID NO:10), and CDR3 (SEQ ID NO:11), and the light chain variable regions CDR1 (SEQ ID NO:12), CDR2, and CDR3 (SEQ ID NO:13) of the monoclonal antibody or antigen-binding fragment one (monoclonal antibody A) are shown in Table 1.

[0039] Table 1. Amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the heavy and light chains of monoclonal antibody A.

[0040]

[0041] The amino acid sequences of the heavy chain variable regions CDR1 (SEQ ID NO:14), CDR2 (SEQ ID NO:15), and CDR3 (SEQ ID NO:16), and the light chain variable regions CDR1 (SEQ ID NO:17), CDR2, and CDR3 (SEQ ID NO:18) of the monoclonal antibody or antigen-binding fragment two (monoclonal antibody B) are shown in Table 2.

[0042] Table 2. Amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the heavy and light chains of monoclonal antibody B.

[0043]

[0044] Secondly, biological materials related to the aforementioned monoclonal antibody or antigen-binding fragment thereof that specifically binds to the avian leukosis virus p27 protein are provided, wherein the biological material is any one of the following:

[0045] (a) A nucleic acid molecule containing a sequence of a heavy chain variable region and / or a light chain variable region encoding a monoclonal antibody or antigen-binding fragment that specifically binds to the avian leukosis virus p27 protein;

[0046] (b) an expression cassette containing the nucleic acid molecules in (a);

[0047] (c) A recombinant vector containing either the nucleic acid molecule in (a) or the expression cassette in (b);

[0048] (d) Recombinant eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c);

[0049] (e) A host cell containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

[0050] The nucleic acid sequence encoding the heavy chain variable region of monoclonal antibody A is shown in SEQ ID NO:3, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO:4; the nucleic acid sequence encoding the heavy chain variable region of monoclonal antibody B is shown in SEQ ID NO:7, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO:8.

[0051] The heavy chain constant region of the aforementioned ALV p27 protein monoclonal antibodies is all of the IgG1 type, and the light chain constant region is all of the Kappa type.

[0052] Thirdly, the present invention provides the application of the monoclonal antibody or antigen-binding fragment that specifically binds to the ALV p27 protein, the DNA encoding the monoclonal antibody or antigen-binding fragment, the vector, and the host cell in the preparation of avian leukosis virus detection reagents.

[0053] Fourthly, the present invention provides a reagent for detecting avian leukosis virus, comprising a capture antibody and a detection antibody; wherein,

[0054] The capture antibody and the detection antibody are independently selected from at least one of the above-mentioned monoclonal antibody A and monoclonal antibody B;

[0055] The capture antibody is selected from at least one of the monoclonal antibody or antigen-binding fragment one (monoclonal antibody A) that specifically binds to ALV p27 protein and monoclonal antibody or antigen-binding fragment two (monoclonal antibody B);

[0056] Alternatively, the detection antibody is selected from at least one of the monoclonal antibody or antigen-binding fragment one (monoclonal antibody A) that specifically binds to ALV p27 protein and monoclonal antibody or antigen-binding fragment two (monoclonal antibody B);

[0057] Alternatively, the capture antibody may be the specific ALV p27 protein-binding monoclonal antibody or antigen-binding fragment one (monoclonal antibody A), and the detection antibody may be the specific ALV p27 protein-binding monoclonal antibody or antigen-binding fragment two (monoclonal antibody B).

[0058] Alternatively, the capturing antibody may be the specific ALV p27 protein-binding monoclonal antibody or antigen-binding fragment two (monoclonal antibody B), and the detection antibody may be the specific ALV p27 protein-binding monoclonal antibody or antigen-binding fragment one (monoclonal antibody A).

[0059] Fifthly, the present invention provides a nanozyme immunochromatographic test strip for rapid detection of avian leukosis virus, the test strip containing the aforementioned reagents for detecting avian leukosis virus. Specifically, the test strip includes a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad being sequentially overlapped on the PVC base plate; the nitrocellulose membrane has a detection line T and a control line C, the detection line T being located near the conjugate pad, and the control line C being located near the absorbent pad; the conjugate pad is coated with the nanozyme-labeled capture antibody, the detection line T is coated with the detection antibody, and the control line C is coated with goat anti-mouse IgG.

[0060] Antibody capture and antibody detection specifically include the following:

[0061] The conjugate pad is coated with nanozyme-labeled monoclonal antibody A that specifically binds to ALV p27 protein (monoclonal antibody A), and the detection line T is coated with monoclonal antibody B that specifically binds to ALV p27 protein (monoclonal antibody B).

[0062] Alternatively, the conjugate pad may be coated with nanozyme-labeled monoclonal antibody B that specifically binds to ALV p27 protein, and the detection line T may be coated with monoclonal antibody A that specifically binds to ALV p27 protein.

[0063] Alternatively, the binding pad may be coated with a nanozyme-labeled monoclonal antibody A (monoclonal antibody A) that specifically binds to ALV p27 protein, and the detection line T may be coated with other existing monoclonal antibodies that specifically bind to ALV.

[0064] Alternatively, the binding pad may be coated with nanozyme-labeled monoclonal antibody B (monoclonal antibody B) that specifically binds to ALV p27 protein, and the detection line T may be coated with other existing monoclonal antibodies that specifically bind to ALV.

[0065] Alternatively, the conjugate pad may be coated with another existing monoclonal antibody that specifically binds to ALV and labeled with nanozyme, and the detection line T may be coated with the aforementioned monoclonal antibody A (monoclonal antibody A) that specifically binds to ALV p27 protein.

[0066] Alternatively, the binding pad may be coated with another existing monoclonal antibody that specifically binds to ALV and labeled with a nanozyme, and the detection line T may be coated with the aforementioned monoclonal antibody B that specifically binds to ALV p27 protein.

[0067] The nanozyme-labeled capture antibody was prepared by the following steps: the nanozyme was washed with deionized water; after centrifugation, the supernatant was discarded; the precipitate was resuspended in MES buffer, and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) were added simultaneously, mixed well, and activated in a shaker; after activation, the nanozyme was washed with MES buffer and then resuspended in MES buffer to obtain the activated nanozyme; ALV p27 protein monoclonal antibody A was incubated with the activated nanozyme solution overnight; Tris buffer was added to terminate the reaction; after magnetic separation, the nanozyme ALV p27 protein monoclonal antibody probe was obtained by resuspending in protective buffer.

[0068] The above-mentioned nanozyme ALV p27 protein monoclonal antibody probe was sprayed onto the binding pad;

[0069] The concentration of the nanozyme is 5 mg / mL;

[0070] The Tris buffer solution has a concentration of 0.05 mol / L and a pH of 7.2.

[0071] The protective buffer solution is 5% BSA-Tirs by mass concentration;

[0072] The composition of the ALV p27 protein monoclonal antibody A nanozyme-labeled solution is: each 1 mL of nanozyme-labeled solution contains 60 µg of ALV p27 protein monoclonal antibody A.

[0073] Preferably, the detection line T is coated with 1 mg / mL of ALV p27 protein monoclonal antibody B.

[0074] Preferably, the quality control line C is coated with 1 mg / mL of goat anti-mouse IgG.

[0075] The preparation method of nanozyme immunochromatographic test strips includes the following steps:

[0076] (1) Preparation of a binding pad coated with ALV p27 protein monoclonal antibody A labeled with nanozyme;

[0077] (2) Spray ALV p27 protein monoclonal antibody B (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) at intervals of 5-8 mm onto a nitrocellulose membrane, which will serve as the detection line T and the control line C, respectively.

[0078] (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are sequentially overlapped on the PVC base plate to obtain the nanoenzyme immunochromatographic test strip for detecting avian leukosis virus.

[0079] The beneficial effects of this invention are:

[0080] This invention screened and obtained monoclonal antibodies A and B against the p27 protein of avian leukosis virus, which can be used as capture antibodies or detection antibodies to detect avian leukosis virus.

[0081] The nanozyme immunochromatographic test strip for detecting avian leukosis virus provided by this invention can be used for the specific detection of avian leukosis virus. Simultaneously, the test strip can detect the virus in cultures up to a minimum limit of 12 TCID. 50 / mL, the minimum detectable amount of protein is 0.2ng / mL, and there is no cross-reactivity with Newcastle disease virus, Marek's disease virus, infectious bursal disease virus, fowlpox virus, infectious laryngotracheitis virus, and avian influenza virus.

[0082] The test strips prepared by this invention have the advantages of high specificity, high sensitivity, simple operation, short detection time, and intuitive result display, providing a real-time detection method for the on-site or laboratory detection of avian leukosis virus.

[0083] Example 1: Preparation of ALV p27 protein monoclonal antibody and amplification of antibody sequence

[0084] 1.1 Preparation of ALV p27 protein

[0085] Primers were designed based on the sequence of GenBank accession number KU375453. The PCR amplification product was inserted into the prokaryotic expression vector pCold I, and the recombinant plasmid was named pCold I-p27. This plasmid was transformed into *E. coli* BL21(DE3). The engineered bacteria expressing ALV p27 protein were inoculated into 100 mL of LB broth containing kanamycin. When the OD600 was approximately 0.8, IPTG was added to a final concentration of 1 mmol / L to induce expression. After 5 h, the precipitate was collected by centrifugation at 12000 rpm for 10 min. The precipitate was resuspended in an appropriate amount of PBS and sonicated for 10 min (3 s sonication, 3 s pause). After sonication, the precipitate was centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected and purified by nickel column chromatography. The purified product was the ALV p27 protein, which was identified by SDS-PAGE. The identification results are as follows: Figure 1 As shown. After obtaining the protein sample, the protein concentration was determined using a BCA kit (Beyotime), and then aliquoted and stored at -80℃ for later use.

[0086] 1.2 Preparation of ALV p27 protein monoclonal antibody and amplification of antibody sequence

[0087] Babl / c mice were immunized with purified ALV p27 recombinant protein at a dose of 30 μg / mouse. For the initial immunization, an equal volume of ALV p27 protein was emulsified with Freund's complete adjuvant and administered via subcutaneous injection at multiple sites on the back. Booster immunizations were given every two weeks for a total of four immunizations. For each booster immunization, an equal volume of ALV p27 recombinant protein was emulsified with Freund's incomplete adjuvant, and the immunization method was the same as the initial immunization. One week after the fourth immunization, blood was collected from the tails of the mice, and antibody titers were determined by ELISA. Spleen cells from the mouse with the highest antibody titer were collected and fused using standard methods to screen for hybridoma cell lines that specifically secrete monoclonal antibodies against ALV p27 protein. After expansion culture, the cells were injected into the peritoneal cavity of the mice, and the resulting ascites fluid was subjected to affinity chromatography using the PIERCE NAb™ Protein G Spin Purification Kit.

[0088] RNA was extracted from positive hybridoma cells and reverse transcribed into cDNA using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A). The antibody variable region gene was amplified using nested PCR. First, using the cDNA as a template, the antibody variable region gene was amplified using a first-round primer sequence of murine IgG and κ light chain antibodies. Then, using the first-round product as a template, the antibody variable region gene was amplified using a second-round primer sequence of murine IgG and κ light chain antibodies. The PCR reaction system was: 25 μL PrimeSTARMax Premix (2×), 0.2 μL each primer, 1 μL cDNA, and ddH2O to a final volume of 50 μL. The reaction program was: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 10 min. Primers for antibody variable region gene amplification were referenced in the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.). After amplification, 1% agarose gel electrophoresis was performed. The gene size of the heavy chain and κ light chain variable regions was approximately 300 bp. The target fragment was excised and recovered for sequencing. The sequencing results were compared with the antibody gene library (IMGT), and the sequencing results confirmed that the amplified sequences were the DNA sequences of the heavy chain and light chain variable regions of the monoclonal antibody. Specifically, the DNA sequences encoding the heavy chain variable regions of ALV p27 protein A and B monoclonal antibodies are shown in SEQ ID NO:3 and SEQ ID NO:7, respectively; the DNA sequences encoding the light chain variable regions of ALV p27 protein A and B monoclonal antibodies are shown in SEQ ID NO:4 and SEQ ID NO:8, respectively. The amino acid sequences of the heavy chain variable regions of ALV p27 protein A and B monoclonal antibodies are shown in SEQ ID NO:1 and SEQ ID NO:5, respectively; the amino acid sequences of the light chain variable regions of ALV p27 protein A and B monoclonal antibodies are shown in SEQ ID NO:2 and SEQ ID NO:6, respectively.

[0089] The amino acid sequences of the heavy and light chain variable regions CDR1, CDR2, and CDR3 of monoclonal antibody A are shown in Table 1. The amino acid sequences of the heavy and light chain variable regions CDR1, CDR2, and CDR3 of monoclonal antibody B are shown in Table 2.

[0090] Example 2: Preparation of Nanoenzyme Immunochromatographic Test Strips for Avian Leukosis Virus

[0091] 2.1 Preparation of nanozyme-ALV p27 protein monoclonal antibody probe

[0092] Take 500 μL of nanozyme solution (preferably prepared according to Publication 202411276039.6, a nanozyme and its preparation method and application), the nanozyme concentration is 5 mg / mL, wash three times with 1 mL of deionized water; after centrifugation, discard the supernatant; resuspend the precipitate with 1 mL of MES buffer solution, the pH of the 50 mM MES buffer solution is 6.0, and simultaneously add 10 μL of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 20 μL of NHS (N-hydroxysuccinimide), mix well, and place in a shaker for 30 min; the concentration of EDC and NHS is 20 mg / mL; the shaking speed is 70 rpm; after activation, wash once with 1 mL of MES buffer solution, and then resuspend with 500 μL of MES buffer solution to obtain activated nanozyme; take 50 μg of ALV p27 protein monoclonal antibody A was incubated overnight with activated nanozyme solution at 4°C; the reaction was terminated by incubation at room temperature for 30 min with 0.05 mol / L Tris buffer at pH 7.2; after magnetic separation, the nanozyme ALV monoclonal antibody probe was obtained by resuspending in 0.5 mL of 5% BSA Tris buffer.

[0093] 2.2 Preparation of Nanozyme Conjugate Pads

[0094] The prepared nanozyme-ALV p27 protein monoclonal antibody A probe was uniformly sprayed onto the conjugate pad using a spray film applicator, dried in a 37℃ oven for 2-3 hours, and then sealed for storage.

[0095] 2.3 Preparation of nitrocellulose membranes

[0096] ALV p27 protein monoclonal antibody B and goat anti-mouse IgG antibody were diluted to 1 mg / mL and sprayed onto a nitrocellulose membrane at a distance of 5-8 mm, serving as the detection line (T) and control line (C), respectively. After the lines were drawn, they were dried at 37℃-38℃ for 2-3 hours.

[0097] 2.4 Assembly of test strips

[0098] The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially connected on a PVC base plate to obtain a test strip. The test strip is then cut into strips according to the size of the test strip holder, and the cut test strips are then inserted into the test strip holder. A schematic diagram of the test strip structure is shown below. Figure 2 As shown.

[0099] Example 3: Application of Nanozyme Immunochromatographic Test Strips for Avian Leukosis Virus

[0100] 3.1 Instructions for using test strips

[0101] 3.1.1 Sample Preparation

[0102] (1) Samples include: poultry cloacal swabs, meconium, egg white or serum samples.

[0103] (2) Add the sample to a sample processing tube containing 1 mL of sample processing solution, shake back and forth to mix, so that the sample dissolves and is fully mixed with the sample processing solution, and then let stand for 1 min to allow any undissolved sample to precipitate. The sample processing solution is 0.01 mol / L pH 7.4 PBS solution.

[0104] 3.1.2 Sample Testing

[0105] Remove the sealed test strip and place it on a dry, stable surface. Use a pipette to draw an appropriate amount of the supernatant from step 3.1.1 and slowly add 6–7 drops (approximately 100–150 μL) into the sample well. Begin chromatography and time for 10 minutes, then observe the results. To amplify the detection signal, a peroxidase substrate such as DAB can be added after 10 minutes. The nanozyme activity will catalyze a chemical reaction in DAB to generate a large amount of brown precipitate.

[0106] 3.1.3. Judgment

[0107] (1) The appearance of two brown bands on the test strip (T: test line, C: control line) indicates a positive result (see [link to test strip]). Figure 3 A).

[0108] (2) If only one brown band appears on the test strip (C: control line), it is judged as negative (see Figure 3 B).

[0109] (3) If no brown band appears at the control line of the test strip, it is considered invalid (see Figure 3 C).

[0110] 3.2 Sensitivity of the test strip

[0111] With a titer of 10 5.5 TCID 50After diluting ALV virus at a ratio of 1:200 to 1 mL, and then performing a 2-fold serial dilution, the sensitivity of the avian leukosis virus nanoenzyme immunochromatographic test strip established in this invention was tested.

[0112] The results showed that when the avian leukosis virus nanozyme immunochromatographic test strip was used to detect 12.5 TCID... 50 The test result was positive when the viral solution was at a concentration of 6.25 TCID⁻¹ / mL; the result was positive when the viral concentration was detected using the avian leukosis virus nanozyme immunochromatographic test strip at 6.25 TCID⁻¹. 50 When the viral solution was / mL, the test result was negative ( Figure 4 The above results indicate that the test strip prepared in this embodiment has a sensitivity of 12.5 TCID for detecting avian leukosis virus. 50 / mL.

[0113] Purified p27 protein at a concentration of 0.1 mg / mL was serially diluted twofold to obtain concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10, and 0.05 ng / mL. When the p27 protein concentration was 0.2 ng / mL, the avian leukosis virus nanozyme immunochromatographic test strip established in this invention showed a positive result, while the protein sample at a concentration of 0.1 ng / mL showed a negative result. Figure 5 The above results indicate that the test strip prepared in this embodiment has a sensitivity of 0.2 ng / mL for detecting p27 protein.

[0114] 3.3 Specificity of the test strip

[0115] Ten negative anal swabs, numbered A1 to A10, were tested using test strips. The results showed a brownish band at the control line C, but no band was observed at the T line, indicating a negative test result. Figure 6 The test strips were used to detect different avian viruses, including Newcastle disease virus (NDV), Marek's disease virus (MDV), infectious bursal disease virus (IBDV), fowlpox virus (FPV), infectious laryngotracheitis virus (ILTV), and avian influenza virus (AIV). The results showed that only ALV was positive, while the others did not react, indicating that the method established in this invention can specifically identify ALV. Figure 7 As shown, the test strip developed in this invention has high specificity.

[0116] SEQ ID NO:1

[0117] PGASGGYPARLLATPSQPTIYTGCSRGLDRDLSGLDGFILEILILSTMRSSRARPHLLQTNPPAQPTCSSAAWPLRTLRSISVQEREVTTAWPGFLTGAKGLWSLSLQ

[0118] SEQ ID NO:2

[0119] GTISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKSN

[0120] SEQ ID NO:3

[0121] CCTGGGGCTTCAGGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAACCTACTATATACACTGGGTGCAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATTCTAATACTGAGTACAATGAGAAGTTCAAGGGCAAGGCCACACTTACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGGCCTCTGAGGACTCTGCGATCTATTTCTGTGCAAGAGAGGGAGGTAACTACGGCCTGGCCTGGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAG

[0122] SEQ ID NO:4

[0123] GGCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATCAAAC

[0124] SEQ ID NO:5

[0125] PGASGRYPARLLVTHSLATPSTGCNRAMERTLSGLDLLILTMVVLTTTRTSRARPHWLETCHPAQPTWNSSVWHLRTLQSITVQDMMV

[0126] SEQ ID NO:6

[0127] VTMSCKSSQSLLNSGNQKNYLTWYQQRPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYDYPLTFGT

[0128] SEQ ID NO:7

[0129] CCTGGAGCTTCAGGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATcAACTGGGTGCAACAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTGGTGCTAACTACAACCAGAACTTCAAGGGCAAGGCCACATTGGCTGGAGACATGTCATCCAGCACAGCCTACATGGAACTCCTCAGTCTGGCATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATATGATGGTTACTACGTGGGGTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG

[0130] SEQ ID NO:8

[0131] GTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAGACCAGGGCAGCCTCCTAAACTATTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATGATTATCCGCTCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAAC

[0132] SEQ ID NO:9

[0133] ATPSQPTI

[0134] SEQ ID NO:10

[0135] FILEILIL

[0136] SEQ ID NO:11

[0137] QEREVTTAWPGFLT

[0138] SEQ ID NO:12

[0139] KSVSTSGYSY

[0140] SEQ ID NO:13

[0141] QHIRELTR

[0142] SEQ ID NO:14

[0143] VTHSLATP

[0144] SEQ ID NO:15

[0145] LILTMVVL

[0146] SEQ ID NO:16

[0147] QDMMVTTWGLTT

[0148] SEQ ID NO:17

[0149] QSLLNSGNQKNY

[0150] SEQ ID NO:18

[0151] QNDYDYPLT

[0152] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0153] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A reagent for detecting avian leukosis virus, characterized in that, It includes capture antibodies and detection antibodies; among which, When the capture antibody is monoclonal antibody A, the detection antibody is monoclonal antibody B; or when the capture antibody is monoclonal antibody B, the detection antibody is monoclonal antibody A. The heavy chain variable region of the monoclonal antibody A contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

2. The heavy chain variable region of the monoclonal antibody B contains an amino acid sequence as shown in SEQ ID NO:5, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

6. The antigen-binding fragment is a Fab fragment, an F(ab)2 fragment, or a single-chain Fv fragment.

2. The reagent for detecting avian leukosis virus as described in claim 1, characterized in that, The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody A are ATPSQPTI, FILEILIL, and QEREVTTAWPGFLT, respectively; the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody A are KSVSTSGYSY, LVS, and QHIRELTR, respectively. The heavy chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody B are VTHSLATP, LILTMVVL, and QDMMVTTWGLTT, respectively; the light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody B are QSLLNSGNQKNY, WAS, and QNDYDYPLT, respectively.

3. A nanozyme immunochromatographic test strip, characterized in that, It includes a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped. The nitrocellulose membrane is provided with a detection line and a quality control line. The conjugate pad is coated with a nanozyme-labeled capture antibody, and the detection line is coated with a detection antibody. The capture antibody and the detection antibody are respectively the capture antibody and the detection antibody contained in the reagent for detecting avian leukosis virus according to claim 1; The quality control line is coated with goat anti-mouse IgG antibody.

4. The nanozyme immunochromatographic test strip as described in claim 3, characterized in that, The nanozyme-labeled capture antibody has a labeling concentration of 60 μg / mL; the detection line is coated with a detection antibody concentration of 1 mg / mL; and the control line is coated with goat anti-mouse IgG at a concentration of 1 mg / mL.

5. The application of the nanozyme immunochromatographic test strip according to any one of claims 3-4 in the detection of avian leukosis virus, characterized in that, The test samples are poultry cloacal swabs, meconium, egg white, or serum.

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