Canine rotavirus monoclonal antibody and use thereof

Abstract

The application provides a pair of mouse monoclonal antibodies 4A5 and 2C12 with specific binding to canine rotavirus VP7 protein, and a colloidal gold detection kit prepared from the mouse monoclonal antibodies, which can detect various epidemic strains and standard strains of canine rotavirus with high sensitivity, has a high coincidence rate with an RT-PCR detection method, and can detect epidemic strains and target strains in different regions, has the advantages of rapidness, simplicity, accuracy, and can meet the urgent needs of the present clinical stage.

Description

Technical Field

[0001] This invention relates to canine rotavirus monoclonal antibodies, hybridoma cells that secrete the monoclonal antibodies, and a double-antibody sandwich detection kit containing monoclonal antibody pairs and its applications, belonging to the field of biotechnology. Background Technology

[0002] Canine ratavirus (CRV) belongs to the family Reoviridae, genus Rotavirus, and its nucleic acid type is double-stranded RNA. This disease primarily affects puppies and is an acute, contagious disease characterized by diarrhea. It is most prevalent in autumn and winter, especially during the cold season from late winter to early spring. CRV is mainly found in the epithelial cells of the small intestinal mucosa, and can also be found in the reticular cells of the mesenteric lymph node cortex and paracortex. Transmission primarily occurs through the digestive tract, with infected dogs becoming infected through fecal contamination of utensils and the surrounding environment. The main sources of infection are infected dogs and asymptomatic carriers. CRV usually causes severe infection in puppies, while adult dogs often present with subclinical infection. Puppies often experience severe diarrhea with watery to mucous stools, but their appetite and body temperature remain relatively stable. Initially, puppies may exhibit lethargy, decreased appetite, reluctance to move, rough coat, and fecal contamination of the skin around the anus. Typically, vomiting precedes diarrhea, with stools being mucous or watery, and varying in color (yellow, white, gray, etc.). Severe cases may involve bloody stools, severe dehydration, hypothermia, and rapid heartbeat, often ending in death. Currently, there is no effective vaccine or specific treatment for canine rotavirus. Antibiotics and sulfonamides are ineffective against this disease; clinical treatment primarily involves maintaining electrolyte balance and correcting acid-base imbalances, with limited effectiveness. Furthermore, the clinical manifestations and epidemiological patterns of diarrhea caused by different diseases are very similar, making clinical differential diagnosis extremely difficult.

[0003] CRV pathogen detection methods include virus isolation, electron microscopy, immunofluorescence assay, and RT-PCR. Currently, commercially available diagnostic reagents include RT-PCR, quantitative real-time PCR, and colloidal gold test strips. The first two fall under the category of nucleic acid diagnostics, offering high sensitivity, but are cumbersome to operate, require expensive equipment, and demand high skill levels from both personnel and equipment. They are suitable for mature laboratory testing but not for rapid on-site detection of this virus. While immunogold detection technology is relatively mature, numerous factors influence the results, including the molecular weight and concentration of antigen and antibody, the amount of gold particles used, the characteristics of the sample, and interfering substances present in the sample, significantly affecting the sensitivity and specificity of the detection. Therefore, there is an urgent need to establish a highly sensitive, rapid, accurate, simple, and effective CRV diagnostic method. Furthermore, co-infection with circulating strains from different periods may occur clinically; therefore, a highly sensitive CRV diagnostic method capable of detecting circulating strains from different periods is required. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a pair of monoclonal antibodies that can specifically bind to canine rotavirus, as well as a colloidal gold test strip containing the monoclonal antibody pair and its applications.

[0005] This invention relates to a monoclonal antibody that specifically binds to the VP7 protein of canine rotavirus.

[0006] This invention relates to monoclonal antibodies that specifically bind to circulating strains of canine rotavirus at different times.

[0007] The present invention also relates to a kit for detecting canine rotavirus with high sensitivity, good specificity and good repeatability.

[0008] This invention also relates to a canine rotavirus detection kit with a long shelf life under normal temperature conditions.

[0009] The present invention also relates to a kit capable of highly sensitively detecting circulating strains of canine rotavirus at different times.

[0010] The present invention also relates to a kit capable of highly sensitive detection of prevalent canine rotavirus strains in different regions.

[0011] The present invention also relates to a kit for detecting the fluctuation pattern of canine rotavirus antigen that is consistent with the detection method of RT-PCR.

[0012] The present invention also relates to single-chain antibodies that exhibit good reactivity with canine rotavirus.

[0013] The present invention also relates to single-chain antibodies that exhibit good reactivity with circulating strains of canine rotavirus at different times. Detailed Implementation

[0014] definition

[0015] The term "canine ratavirus" (CRV) belongs to the family Reoviridae and the genus Rotavirus. Its nucleic acid type is double-stranded RNA. CRV is an acute gastrointestinal infectious disease that primarily affects dogs, especially puppies, with a high morbidity rate. Common symptoms include rapid onset, yellow-green watery stools mixed with mucus, dehydration, fever, rough and disheveled coat, and fecal contamination around the anus. Severe cases present with lethargy, loss of appetite, watery diarrhea, small amounts of blood in the stool, and may even lead to death.

[0016] The term "canine rotavirus structural proteins" refers to the structural proteins VP4, VP6, and VP7, which are associated with CRV antigenicity; namely, serogroup antigen VP6, neutralizing antigen VP7, and hemagglutinin antigen VP4. VP4 and VP7 are the main recognition factors for rotavirus, both being independent viral neutralization targets that mediate serum specificity. Based on the antigenicity of VP4 and VP7, group A rotaviruses can be divided into 14 G types (G1–G14) and 21 P types (P1–P24). Currently, the vast majority of canine rotaviruses belong to group A. VP7 is encoded by gene 9 or genes 7 and 8 (depending on the strain), has a molecular weight of 37 Ku, and in most rotavirus strains, VP7 is 1062 bp long, accounting for approximately 30% of the total viral protein. It is the main glycoprotein and neutralizing antigen of the viral envelope, forming the smooth portion of the viral envelope. VP7 is an N-linked oligomannose glycoprotein that determines the G serotype of the virus. VP7 is also a Ca2+ protein. 2+ These binding proteins are involved in viral morphology and stability. VP7 and VP4 are arranged in a regular and orderly manner within the rotavirus capsid, and their interactions influence the expression of each other's antigenicity and biological characteristics.

[0017] The term "monoclonal antibody" refers to an antibody derived from a substantially homologous group of antibodies, meaning that the individual antibodies comprising this group are identical, except for the possible presence of a small number of spontaneous mutations. Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies. Preferably, the monoclonal antibody includes monovalent or single-chain antibodies, double-chain antibodies, chimeric antibodies, humanized antibodies, and derivatives, functional equivalents, and homologs of the above antibodies, as well as antibody fragments and any polypeptide containing an antigen-binding domain. An antibody encompasses any specific binding factor having a desired specific binding domain; therefore, this term covers antibody fragments, derivatives, humanized antibodies, and functional equivalents and homologs of antibodies, as well as any polypeptide containing an antigen-binding domain, whether natural or synthetic. Examples of antibodies are immunoglobulin subtypes (such as IgG, IgE, IgM, IgD, and IgA) and their subtypes; they can also be fragments containing an antigen-binding domain such as Fab, scFv, Fv, dAb, Fd; and double-chain antibodies (diabodies). Chimeric molecules or equivalents containing antigen-binding domains fused to another polypeptide are also included. Cloning and expression of chimeric antibodies are described in EP.A.0120694 and EP.A.0125023. Antibodies can be modified in many ways, and DNA recombination techniques can be used to generate other antibodies or chimeric molecules that retain the original antibody specificity. Such techniques may include introducing DNA encoding the variable region or complementarity-determining region (CDRs) of an immunoglobulin into the constant region or constant region plus frame region of a different immunoglobulin, see EP.A.184187, GB2188638A, or EP.A.239400. Genetic mutations or other alterations can also be made to hybridoma cells or other antibody-producing cells, which may or may not alter the binding specificity of the produced antibody. The "monoclonal antibody" used in this invention can also be prepared using hybridoma methods, because the DNA sequence encoding the murine antibody of this invention can be obtained using conventional methods well known to those skilled in the art, such as artificially synthesizing nucleotide sequences according to the amino acid sequence disclosed in this invention or amplifying them by PCR. Therefore, recombinant DNA methods can also be used, and the sequence can be ligated into a suitable expression vector using various methods well known in the art. Finally, under conditions suitable for antibody expression of this invention, the transformed host cells are cultured, and then purified using conventional isolation and purification methods well known to those skilled in the art to obtain the monoclonal antibody of this invention. The antibody comprises a polypeptide chain geometry linked together by disulfide bridges; two polypeptide backbones, called the light chain and the heavy chain, constitute all major structural classes (isotypes) of the antibody. Both the heavy chain and the light chain can be further divided into subregions called variable regions and constant regions. The heavy chain includes a single variable region and three distinct constant regions, while the light chain includes a single variable region (different from the variable region of the heavy chain) and a single constant region (different from the constant region of the heavy chain). The variable regions of the heavy and light chains are responsible for the binding specificity of the antibody.

[0018] The term "heavy chain variable region" refers to a polypeptide of 110 to 125 amino acids in length, whose amino acid sequence corresponds to the heavy chain amino acid sequence of the monoclonal antibody of the present invention, starting from the N-terminal amino acid of the heavy chain. Similarly, the term "light chain variable region" refers to a polypeptide of 95 to 115 amino acids in length, whose amino acid sequence corresponds to the light chain amino acid sequence of the monoclonal antibody of the present invention, starting from the N-terminal amino acid of the light chain. Those skilled in the art will readily recognize that, based on the amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody specifically disclosed in this invention, one or more amino acids can be added, deleted, or substituted using conventional genetic engineering and protein engineering methods to obtain conserved variants that still maintain specific binding to canine rotavirus. The monoclonal antibody of this invention also includes its active fragment or conserved variant.

[0019] The term "conservative variant" refers to a variant that essentially retains the characteristics of its parent polypeptide, such as basic immunobiological, structural, regulatory, or biochemical properties. Generally, the amino acid sequence of a conservative variant of a polypeptide differs from that of the parent polypeptide, but the difference is limited to ensure that the sequence of the parent polypeptide and the conservative variant is generally very similar and identical in many regions. Differences in the amino acid sequence between the conservative variant and the parent polypeptide can be, for example, substitutions, additions, and deletions of one or more amino acid residues and any combination thereof. The substituted or inserted amino acid residues may or may not be encoded by the genetic code. Conservative variants of polypeptides can arise naturally or can be unnaturally occurring variants. Unnaturally occurring conservative variants of polypeptides can be produced through mutagenesis or direct synthesis. Invention Details

[0021] This invention relates to a variable region sequence of a monoclonal antibody 4A5 that specifically binds to canine rotavirus, wherein the heavy chain variable region of the monoclonal antibody 4A5 is encoded by SEQ.ID No.1 or its degenerate sequence; and the light chain variable region of the monoclonal antibody 4A5 is encoded by SEQ.ID No.2 or its degenerate sequence.

[0022] The variable region sequence of the monoclonal antibody 4A5 of this invention can specifically bind to canine rotavirus, and the antigenic epitope it binds to is the antigenic epitope on the VP7 protein.

[0023] This invention relates to an antibody or antibody fragment that specifically binds to canine rotavirus, wherein the heavy chain variable region of the antibody or antibody fragment is encoded by SEQ.ID No. 1 or its degenerate sequence, and the light chain variable region of the antibody or antibody fragment is encoded by SEQ.ID No. 2 or its degenerate sequence; the antibody is a monoclonal antibody or a genetically engineered antibody; wherein the genetically engineered antibody includes single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies; the antibody or antibody fragment specifically binds to the canine rotavirus VP7 protein. This invention also relates to monoclonal antibody 4A5, wherein the heavy chain variable region of monoclonal antibody 4A5 is encoded by SEQ.ID No. 1 or its degenerate sequence, and its light chain variable region is shown in SEQ.ID No. 2 or its degenerate sequence. Monoclonal antibody 4A5 is a canine rotavirus monoclonal antibody, further being a monoclonal antibody against the canine rotavirus VP7 protein; it has an IFA titer ≥1:3200 against CRV and exhibits good reactivity with CRV.

[0024] The present invention also relates to a hybridoma cell line 4A5, which secretes the monoclonal antibody 4A5.

[0025] This invention relates to a variable region sequence of a monoclonal antibody 2C12 that specifically binds to canine rotavirus, wherein the heavy chain variable region of the monoclonal antibody 2C12 is encoded by SEQ.ID No.3 or its degenerate sequence; and the light chain variable region of the monoclonal antibody 2C12 is encoded by SEQ.ID No.4 or its degenerate sequence.

[0026] The variable region sequence of the monoclonal antibody 2C12 of this invention can specifically bind to canine rotavirus, and the antigenic epitope it binds to is the antigenic epitope on the VP7 protein.

[0027] This invention relates to an antibody or antibody fragment that specifically binds to canine rotavirus, wherein the heavy chain variable region of the antibody or antibody fragment is encoded by SEQ.ID No. 3 or its degenerate sequence, and the light chain variable region of the antibody or antibody fragment is encoded by SEQ.ID No. 4 or its degenerate sequence; the antibody is a monoclonal antibody or a genetically engineered antibody; wherein the genetically engineered antibody includes single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies; the antibody or the antibody fragment specifically binds to the canine rotavirus VP7 protein. This invention also relates to monoclonal antibody 2C12, wherein the heavy chain variable region of the monoclonal antibody 2C12 is encoded by SEQ.ID No. 3 or its degenerate sequence, and its light chain variable region is encoded by SEQ.ID No. 4 or its degenerate sequence.

[0028] The monoclonal antibody 2C12 of this invention can specifically bind to canine rotavirus, and the antigenic epitope it binds to is located on the VP7 protein, and it binds to a different antigenic epitope than the monoclonal antibody 4A5.

[0029] The monoclonal antibody 2C12 is a canine rotavirus monoclonal antibody, and further, a monoclonal antibody against the canine rotavirus VP7 protein; its IFA titer against CRV is ≥1:3200, indicating good reactivity with CRV.

[0030] The present invention also relates to a hybridoma cell line 2C12, which secretes the monoclonal antibody 2C12.

[0031] Hybridoma cell lines 4A5 and 2C12 can effectively secrete monoclonal antibodies 4A5 and 2C12, respectively, and the purity of the secreted monoclonal antibodies 4A5 and 2C12 is high.

[0032] The present invention also relates to the application of the antibody or antibody fragment or monoclonal antibody, wherein the application is epitope identification research and canine rotavirus antigen reactivity research.

[0033] The present invention also relates to a kit comprising an effective amount of the monoclonal antibody 4A5, an effective amount of the gold-labeled monoclonal antibody 2C12, and a detection reagent for detecting canine rotavirus antigen-antibody reaction; or the kit comprising an effective amount of the monoclonal antibody 2C12, an effective amount of the gold-labeled monoclonal antibody 4A5, and a detection reagent for detecting canine rotavirus antigen-antibody reaction.

[0034] In one embodiment of the present invention, the kit includes a colloidal gold test strip. The colloidal gold test strip includes: a base plate having a first end and a second end, and a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad sequentially arranged along the direction from the first end to the second end. The nitrocellulose membrane contacts the gold-labeled pad or the sample pad and the gold-labeled pad, allowing the conjugate of canine rotavirus antigen and the monoclonal antibody 2C12 to migrate thereto towards the second end of the base plate. The gold-labeled pad contains colloidal gold. The labeled monoclonal antibody 2C12 is attached to a nitrocellulose membrane comprising a detection line and a control line. The detection line is immobilized with the monoclonal antibody 4A5, and the control line is immobilized with goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody. The immobilized content of monoclonal antibody 4A5 is 1.0–3.0 mg / ml, the concentration of monoclonal antibody 2C12 when labeled with colloidal gold is 8–32 μg / ml, and the content of goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody is 1.0–4.0 mg / ml.

[0035] The monoclonal antibody 4A5 is present in fixed concentrations of 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml, 2.1 mg / ml, 2.2 mg / ml, 2.3 mg / ml, 2.4 mg / ml, 2.5 mg / ml, 2.6 mg / ml, 2.7 mg / ml, 2.8 mg / ml, 2.9 mg / ml, or 3.0 mg / ml.

[0036] The concentration of the monoclonal antibody 2C12 when labeled with colloidal gold is 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, 24 μg / ml, 25 μg / ml, 26 μg / ml, 27 μg / ml, 28 μg / ml, 29 μg / ml, 30 μg / ml, 31 μg / ml, or 32 μg / ml.

[0037] The content of the goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody can be 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml, 2.1 mg / ml, 2.2 mg / ml, 2.3 mg / ml, 2.4 mg / ml, 2.5 mg / ml, 2.6 mg / ml, 2.7 mg / ml, 2.8 mg / ml, 2.9 mg / ml, 3.0 mg / ml, 3.1 mg / ml, 3.2 mg / ml, 3.3 mg / ml, 3.4 mg / ml, 3.5 mg / ml, 3.6 mg / ml, 3.7 mg / ml, 3.8 mg / ml, 3.9 mg / ml, or 4.0 mg / ml.

[0038] The gold-labeled monoclonal antibody 2C12 was sprayed at concentrations of 1.0 μl / cm, 1.1 μl / cm, 1.2 μl / cm, 1.3 μl / cm, 1.4 μl / cm, 1.5 μl / cm, 1.6 μl / cm, 1.7 μl / cm, 1.8 μl / cm, 1.9 μl / cm, 2.0 μl / cm, 2.1 μl / cm, 2.2 μl / cm, 2.3 μl / cm, 2.4 μl / cm, 2.5 μl / cm, 2.6 μl / cm, 2.7 μl / cm, 2.8 μl / cm, 2.9 μl / cm, or 3.0 μl / cm.

[0039] The double-antibody sandwich method detection kit of the present invention can effectively detect canine rotavirus, and can detect strains circulating at different times and in different regions with high sensitivity, and can accurately detect various types of target tissues.

[0040] This invention ensures higher detection sensitivity by selecting the fixed content of monoclonal antibody 4A5, the concentration of monoclonal antibody 2C12 colloidal gold labeling, and the content of goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody.

[0041] In a preferred embodiment of the present invention, the fixed concentration of the monoclonal antibody 4A5 is 2.0–3.0 mg / ml, the concentration of the monoclonal antibody 2C12 when colloidal gold-labeled is 20–28 μg / ml, the content of the goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody is 2.0–4.0 mg / ml, and the spray volume of the gold-labeled monoclonal antibody 2C12 is 1.0 μl / cm–3.0 μl / cm; the kit also includes a sample processing solution, which is a phosphate buffer containing 1% V / V Triton X-100.

[0042] In a preferred embodiment of the present invention, the monoclonal antibody 4A5 has a fixed content of 2.0 mg / ml, the monoclonal antibody 2C12 has a colloidal gold labeling concentration of 24 μg / ml, the goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody has a content of 2.0 mg / ml, and the gold-labeled monoclonal antibody 2C12 is sprayed at a rate of 2.0 μl / cm to 3.0 μl / cm.

[0043] In a more preferred embodiment of the present invention, the spray volume of the gold-labeled monoclonal antibody 2C12 is 2.0 μl / cm to 3.0 μl / cm.

[0044] In one embodiment of the present invention, the sample pad, gold label pad, nitrocellulose membrane and absorbent pad arranged sequentially along the direction from the first end to the second end in the kit are in contact with each other when they are adjacent, while the non-adjacent components are not in contact with each other.

[0045] In one embodiment of the present invention, the test samples of the kit are canine rotavirus in canine anal swabs, feces, virus cultures and other liquid samples.

[0046] The present invention also relates to a method for preparing the reagent kit, wherein the preparation method includes:

[0047] Step 1) Label the monoclonal antibody 2C12 with colloidal gold to form a gold-labeled antibody, and prepare a gold-labeled pad;

[0048] Step 2) Fix the monoclonal antibody 4A5, goat anti-mouse secondary antibody or goat anti-mouse polyclonal antibody to one end of the nitrocellulose membrane as the detection line and the quality control line, respectively;

[0049] Step 3) Prepare the sample processing solution and dispense it;

[0050] Step 4) Adhere the gold label pad prepared in Step 1), the nitrocellulose membrane prepared in Step 2), and the absorbent paper to the base plate in sequence, and cut them; assemble them together with the sample processing solution prepared in Step 3) to form a kit.

[0051] In one embodiment of the present invention, the monoclonal antibody 2C12 labeled in step 1) is 8-32 μg / ml, the monoclonal antibody 4A5 labeled in step 2) is 1-3 mg / ml, and the goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody is 2.0-3.0 mg / ml.

[0052] In a preferred embodiment of the present invention, the monoclonal antibody 2C12 labeled in step 1) is 24 μg / ml, and the monoclonal antibody 4A5 labeled in step 2) is 2.0 mg / ml.

[0053] In one embodiment of the present invention, the sample processing solution in step 3) is a phosphate buffer containing 1% V / V Triton X-100.

[0054] The present invention also relates to a detection method for the aforementioned kit, wherein the detection method comprises: inserting the collected sample into a sample processing tube to dissolve the sample in the sample processing solution as much as possible, adding the processed sample to the sample well of the colloidal gold test strip, and determining the result after 10 minutes.

[0055] The present invention also relates to the application of the kit for non-immunodiagnostic purposes, wherein the non-immunodiagnostic applications are epidemiological surveys and detection of isolated tissues.

[0056] The present invention also relates to a single-chain antibody, wherein the heavy chain variable region of the single-chain antibody is encoded by SEQ.ID No.1 or its degenerate sequence, or the heavy chain variable region of the single-chain antibody is encoded by SEQ.ID No.3 or its degenerate sequence, the light chain variable region of the single-chain antibody is encoded by SEQ.ID No.2 or its degenerate sequence, or the heavy chain variable region of the single-chain antibody is encoded by SEQ.ID No.4 or its degenerate sequence.

[0057] The single-chain antibodies all exhibited IFA titers ≥1:800 against canine rotavirus, indicating that they have good reactivity with canine rotavirus.

[0058] The beneficial effects of this invention are:

[0059] The monoclonal antibody prepared by this invention exhibits good reactivity with both current and previously prevalent strains.

[0060] The kit containing the monoclonal antibody pair of the present invention overcomes the problem of low detection sensitivity in the prior art, avoids the occurrence of missed detection and false negatives, can detect epidemic strains in different periods and regions with high sensitivity, and can detect multiple types of targets with high sensitivity. It also has the advantages of being fast, simple and accurate, which is more conducive to its clinical application.

[0061] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0062] All chemical reagents used in the embodiments of this invention are of analytical grade and were purchased from Sinopharm Group.

[0063] The sample processing solution used in this invention is a phosphate buffer containing 1% V / V Triton X-100. The 1L volume of the phosphate buffer has the following formulation: 2.90g Na2HPO4·12H2O and 0.26g NaH2PO4·H2O, but is not limited to this formulation. Unless otherwise specified, the solution is diluted with phosphate buffer. However, this embodiment does not constitute a limitation of the invention under any circumstances.

[0064] Unless otherwise specified, the experimental methods described in this invention are all conventional methods; and the biological materials described are all commercially available unless otherwise specified.

[0065] Example 1: Isolation and Identification of Canine Rotavirus

[0066] A total of 96 anal swabs and fecal samples were collected from puppies exhibiting severe diarrhea, watery to mucous stools, loss of appetite, rough coat, and fecal contamination of the perianal skin, as well as those showing bloody stools, severe dehydration, lethargy, or even death. These samples were collected from veterinary hospitals in multiple provinces including Henan, Shandong, Anhui, Sichuan, Guangdong, Hubei, and Heilongjiang. Canine rotavirus (CRV) samples were tested using RT-PCR, with 45 samples being negative and 51 positive. CRV-positive samples were further tested for other common infectious diseases such as canine parvovirus, canine distemper virus, canine coronavirus, canine adenovirus, and canine parainfluenza virus. Fecal samples were also examined for parasite eggs. The results showed that 41 samples were mixed infections with canine rotavirus and other pathogens, while only 10 samples were infected with CRV alone. These 10 samples were diluted with PBS, filtered, and inoculated into MA104 cells. When 80% of the cells showed cytopathic effects, the virus was harvested by freeze-thawing at -20°C and stored at -70°C. Cells without cytopathic effects were passaged at a 1:3 ratio 96 hours after inoculation, and then blindly passaged for 3 generations. Cells without cytopathic effects were discarded. The harvested virus solution was then passaged and cultured using the method described above.

[0067] Structural protein gene sequencing and sequence alignment were performed on 10 viral fluid strains. Eight strains with homology of no less than 78% with the published CRV structural protein gene sequences were selected as representative strains for further research. They were named SL001, SL002, SL003, SL004, SL005, SL006, SL007, and SL008, respectively, all of which are currently circulating strains.

[0068] The viral load of the eight isolated CRV virus strains was determined. Each strain was then inoculated into a monolayer of MA104 cells, with healthy cells as a control. After culturing at 37°C and 5% CO2 for 48 hours, the cells were fixed with 80% acetone to prepare corresponding IFA antigen plates. 100 μl of CRV mouse positive serum diluted 1:400 was added to each virus inoculation well, with a negative control included. The cells were incubated at 37°C for 40–60 minutes. After washing with PBS, FITC-labeled goat anti-mouse IgG diluted 1:500 was added, and the cells were incubated at 37°C for 40–60 minutes. After washing with PBS, 50 μl of PBS was added, and the cells were observed under a fluorescence microscope. Results showed that the viral load of the eight isolates was less than 10... 5.49 ~10 6.20 TCID 50 The viral loads were between 10 / ml and all were able to react with CRV-positive serum, with strain SL006 having the highest viral load at 10 / ml. 6.20 TCID 50 The fluorescence was strongest at / ml, and the results are shown in Table 1.

[0069] Table 1. Virus content and IFA identification results of CRV strains

[0070]

[0071]

[0072] Example 2: Preparation, purification, identification, and testing of canine rotavirus monoclonal antibodies.

[0073] 2.1 Preparation and purification of canine rotavirus monoclonal antibodies

[0074] The CRV SL006 cell culture prepared in Example 1 was frozen and thawed, then centrifuged at 3000 rpm for 30 min, and the supernatant was the canine rotavirus solution. The harvested virus solution was concentrated 10-fold using PEG20000 to serve as an immunogen, and 4-6 week old female Balb / C mice were immunized. For the first immunization, Freund's complete adjuvant was used for emulsification; for the second and third immunizations, Freund's incomplete adjuvant was used. The immunization dose was 200 μl per mouse. IFA antigen plates were prepared using the SL006 strain, and the serum titer of mice after three immunizations was detected. The highest IFA titer in mouse serum was defined as the highest dilution corresponding to the wells where yellow-green fluorescence was observed. The highest serum titer after immunization was 1:12800.

[0075] Cell fusion was performed using mice with the highest IFA titers. Three days prior to fusion, concentrated virus solution was injected intraperitoneally into the mice for shock immunization. SP2 / 0 cells were mixed with centrifuged immune spleen cells at a ratio of 1:10, and polyethylene glycol (PEG) 1500 was used as a fusion agent for cell fusion. The fused cells were dropped into 96-well cell plates coated with feeder cells and cultured in a 37°C, 5% CO2 incubator. After 10 days, the cell supernatant was collected, and hybridoma cells were screened using the IFA method. After 3-4 rounds of screening and subcloning, 30 hybridoma cell lines that stably secreted monoclonal antibodies against CRV virus were obtained. Antigen plates prepared using the 8 CRV strains prepared in Example 1 and the standard strain Cu-1 purchased from ATCC were used to identify the reactivity of the supernatant of the 30 positive hybridoma cells with different strains. Results: The supernatant of 4 hybridoma cell lines reacted with the other 7 isolates and the standard strain Cu-1, namely 1H3, 5F3, 2C12, and 4A5. Ascites fluid was prepared from the four hybridoma cell lines in female mice. The ascites titer was detected by IFA method. The IFA titers of the supernatant of hybridoma cell lines 1H3, 5F3, 2C12 and 4A5 were not lower than 1:8. The titers of the monoclonal antibodies after purification of the ascites fluid were not lower than 1:1600. The evaluation results of the monoclonal antibodies after purification of the supernatant and ascites fluid of the four hybridoma cell lines are shown in Table 2.

[0076] Table 2. Monoclonal antibody titers after purification from hybridoma cell supernatant and ascites.

[0077]

[0078] The ascites fluids of the four strains were purified using Protein G affinity chromatography to obtain purified monoclonal antibodies. The evaluation results are shown in Table 2. SDS-PAGE gel electrophoresis was then used for identification, and the results showed that the purity of all four monoclonal antibodies was not less than 85%. The protein content was quantified using a BCA protein quantification kit, and the results showed that the protein contents of the four monoclonal antibodies were 4.6 mg / ml, 5.4 mg / ml, 5.9 mg / ml, and 4.8 mg / ml, respectively.

[0079] 2.2 Study on monoclonal antibody pairing assay for test strips

[0080] 2.2.1 Preliminary Study on Antibody Pairing

[0081] Colloidal gold was labeled with purified monoclonal antibodies 1H3, 5F3, 2C12, and 4A5, respectively, and paired with the detection monoclonal antibodies 1H3, 5F3, 2C12, and 4A5, respectively. The prepared test strips were used to detect CRV virus solutions diluted 1:2, 1:8, 1:32, and 1:128. 6.61 TCID 50 / ml), and negative controls were performed using canine negative fecal and anal swabs. The results showed that the test strip had the highest detection sensitivity when the gold-labeled monoclonal antibody was 2C12 and the detection monoclonal antibody was 4A5. A 1:32 dilution of CRV virus solution could still detect a positive result. Negative fecal and anal swab tests were all negative. The sensitivity of other antibody-paired modes was lower than 1:32 (10 5.10 TCID 50 ( / ml). Therefore, the optimal pairing method was determined to be: 2C12 as the gold standard monoclonal antibody and 4A5 as the detection monoclonal antibody.

[0082] Table 3. Pairing results of monoclonal antibodies

[0083]

[0084] 2.3 Identification of the characteristics of canine rotavirus monoclonal antibodies 4A5 and 2C12

[0085] 2.3.1 Identification of Monoclonal Antibody Types and Subclasses

[0086] The subtypes of the two monoclonal antibodies were identified using a monoclonal antibody subtype identification kit. The results showed that the heavy chain subtypes of the two monoclonal antibodies, 4A5 and 2C12, were IgG2a and IgG2b, respectively, and the light chain subtype was kappa for both.

[0087] 2.3.2 Monoclonal antibody specificity identification

[0088] The specificity of canine rotavirus monoclonal antibodies 4A5 and 2C12 was evaluated using common canine viruses. The assay was performed using in vitro fluorescence (IFA). A valid assay was achieved when healthy cell control wells showed no fluorescence and positive control wells showed yellow-green fluorescence. Wells inoculated with the virus showed yellow-green fluorescence and were considered positive; wells inoculated with the virus showed no yellow-green fluorescence and were considered negative. Results: Monoclonal antibodies 2C12 and 4A5 did not react with canine parvovirus (CPV), canine distemper virus (CDV), canine parainfluenza virus (CPIV), canine adenovirus type 1 (CAV-1), or canine adenovirus type 2 (CAV-2), but reacted only with canine rotavirus. This indicates that monoclonal antibodies 4A5 and 2C12 are specific monoclonal antibodies against canine rotavirus.

[0089] 2.4 Determination of the variable region sequences of monoclonal antibodies 4A5 and 2C12

[0090] Based on the sequence characteristics of murine monoclonal antibodies, primer sequences for the heavy chain variable region were designed:

[0091] P1: 5'-ACTAGTTGACGTGGTCTCTAGGGTCACTTTAGTTTCCT-3'

[0092] P2: 5'-CGGAAGCTTCCAGCGRCCARKCCATATACIGRTGG-3'

[0093] Design primer sequences for the light chain variable region:

[0094] P3: 5'-GCCATCTCATGRAGWCATTKWCYCAAGTCTTT-3'

[0095] P4: 5'-CGGACGCTTACTGCCTGGTAAGAAGATGGA-3'

[0096] Two hybridoma cell lines, 4A5 and 2C12, were collected. RNA was extracted and reverse transcribed to serve as a template. The variable region sequences of these cells were amplified using the primers described above. The amplified products were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. Results: The heavy chain and light chain variable regions of monoclonal antibody 4A5 are shown in SEQ.ID No. 1 and SEQ.ID No. 2, respectively; the heavy chain and light chain variable regions of monoclonal antibody 2C12 are shown in SEQ.ID No. 3 and SEQ.ID No. 4, respectively.

[0097] 2.5 Identification of recognition proteins of monoclonal antibodies 4A5 and 2C12

[0098] 2.5.1 Identification of viral proteins by monoclonal antibodies

[0099] Using canine rotavirus nucleotides as templates, primers were designed using RT-PCR to amplify the gene sequences of VP4, VP6, and VP7 proteins. The amplified target genes were ligated into the pCAGGS plasmid, transformed into competent DH5α cells, and plasmids were extracted from positive colonies after inoculation with culture medium. After confirming the plasmids were correctly identified by enzyme digestion, sequencing was performed. The results showed that the sequences ligated into the vector were correct, indicating successful construction of recombinant plasmids expressing different proteins. The recombinant pCAGGS-CRV-VP4, pCAGGS-CRV-VP6, and pCAGGS-CRV-VP7 plasmids were transfected into Vero cells, and IFA antigen plates were prepared. IFA detection was performed using monoclonal antibodies as primary antibodies. The results showed that monoclonal antibodies 2C12 and 4A5 both exhibited yellow-green fluorescence upon reaction with the recombinant VP7 protein, indicating positive detection. No yellow-green fluorescence was observed upon reaction with other recombinant proteins, indicating that monoclonal antibodies 2C12 and 4A5 both recognize the VP7 protein of canine rotavirus.

[0100] 2.5.2 Identification of similarities and differences in monoclonal antibody recognition of antigenic epitopes

[0101] The detection was performed using a monoclonal antibody additive assay: Canine rotavirus was coated onto a 96-well ELISA plate, blocked, and then reacted with a first monoclonal antibody at a saturated concentration. After washing and drying, another monoclonal antibody at a saturated concentration was added. After the two monoclonal antibodies had reacted, HRP-labeled goat anti-mouse IgG was added and reacted with the plate. After washing and color development, the A value was measured. The additive index (AI) of the pairwise superposition of monoclonal antibodies was calculated using the formula: AI = [(A1.2 - A1) / A2] × 100%, where A1 and A2 are the A values ​​of monoclonal antibodies 1 and 2, and A1.2 is the A value of monoclonal antibody 1 superimposed on monoclonal antibody 2. If AI is greater than 50%, it indicates that the two monoclonal antibodies recognize different antigenic epitopes; if AI is less than 50%, it indicates that the two monoclonal antibodies bind to the same or similar antigenic epitopes. Results: The amplification indices (AI) of monoclonal antibodies 2C12 and 4A5 were 75% and 82%, respectively, both greater than 50% (see Table 4). This indicates that monoclonal antibodies 2C12 and 4A5 bind to different antigenic epitopes of the canine rotavirus VP7 protein, further confirming their applicability in establishing a double-antibody sandwich method for antigen detection.

[0102] Table 4. Appreciation Index (AI) of Pairwise Combinations of Monoclonal Antibodies

[0103]

[0104] Example 3: Preparation and application of test strips

[0105] 3.1 Preparation and Detection of Colloidal Gold Test Strips

[0106] 3.1.1 Preparation and Detection of Test Strips

[0107] Heat a 0.01 w / v HAuC14 aqueous solution to boiling. While stirring, add a 1 w / v sodium citrate solution. At this point, the color of the chloroauric acid aqueous solution changes from pale yellow to black and then to wine red. After the solution color stabilizes, continue heating and stirring for 15 minutes. Stop heating, allow to cool naturally to room temperature, and then restore to the original volume with purified water. Store in a sealed container at 2–8℃. Before labeling, adjust the pH of the colloidal gold solution to 8.0 with 0.1 mol / L K₂CO₃ and stir at a constant speed for 30 minutes. Then add the monoclonal antibody 2C12 (final concentration 10–30 μg / ml) to the colloidal gold solution and stir at a constant speed for 30 minutes. Block by adding 10 w / v BSA dropwise and stirring at a constant speed for 30 minutes. After incubation at 2–8℃ for 2 hours, centrifuge at 2000 rpm for 20 minutes at 4℃, collect the supernatant, and then centrifuge at 12000 rpm for 30 minutes at 4℃. Collect the precipitate, resuspend it in 1 / 10 volume of gold-labeled buffer to obtain the gold-labeled monoclonal antibody. Coat the sample with the gold-labeled monoclonal antibody 2C12 using spraying or soaking to prepare a gold-labeled pad. Spray monoclonal antibody 4A5 (coating concentration of 0.5–3.0 mg / ml) and goat anti-mouse secondary antibody (coating concentration of 1–4 mg / ml) onto a nitrocellulose membrane to serve as the detection line (T) and control line (C), respectively. Attach the sample pad, gold-labeled pad, nitrocellulose membrane, and absorbent pad to a base plate to obtain the canine rotavirus colloidal gold test strip. The sample processing tube contains a sample processing solution prepared with phosphate buffer.

[0108] During testing, place the sample to be tested in the sample processing tube, ensuring the sample is dissolved as much as possible in the sample processing solution. Break off the cap of the sample processing tube containing the sample, and add 4 drops (approximately 100 μl) of the mixed sample to the center of the sample well on the test strip. After 10 minutes, observe and record the results in the test area of ​​the test strip, and make a judgment according to the judgment criteria. Result judgment criteria: If the control line develops color, the test is valid; if the test line develops color, the result is positive; if no color develops, the result is negative. If the control line does not develop color, the test is invalid, and regardless of whether the test line develops color, the result is considered invalid and must be repeated.

[0109] 3.1.2 Optimization of the working concentration of monoclonal antibodies in the test strip

[0110] (1) Selection of detection line coating concentration

[0111] Monoclonal antibody 4A5 was diluted to 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, and 3.0 mg / ml with diluent, coated onto nitrocellulose membranes, and then test strips were assembled. Canine rotavirus at different dilutions was detected, and the color development of the test and control bands was observed. Results showed that when monoclonal antibody 4A5 was diluted to 0.5 mg / ml, the test line was blurry; when diluted to 1.0 mg / ml, the test line was also faint; the clarity was better at 2.0 mg / ml to 3.0 mg / ml than at 1.0 mg / ml (see Table 5). Considering cost, a dilution of 2.0 mg / ml was chosen for coating.

[0112] Table 5 Optimization of Detection Line Coating Concentration

[0113]

[0114] Note: "+" indicates positive, and "-" indicates negative.

[0115] (2) Selection of control line coating concentration

[0116] Commercially available goat anti-mouse IgG was diluted to 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, and 4.0 mg / ml, and used as the coating solution to coat the control line area of ​​a nitrocellulose membrane. Test strips were then assembled to detect canine rotavirus at different dilutions, and the color bands of the test and control lines were observed. Results showed that the control line was slightly fainter when goat anti-mouse IgG was diluted to 1.0 mg / ml; the clarity was better at 2.0 mg / ml to 4.0 mg / ml than at 1.0 mg / ml, but the color was darker at 3.0 mg / ml to 4.0 mg / ml (see Table 6). Considering cost, a dilution of goat anti-mouse IgG to 2.0 mg / ml was chosen for coating.

[0117] Table 6. Optimization of control line coating concentration.

[0118]

[0119] Note: "+" indicates positive, and "-" indicates negative.

[0120] (3) Optimization of gold-labeled monoclonal antibody concentration

[0121] Gold-labeled monoclonal antibodies were labeled according to the concentrations listed in Table 7. Fixed monoclonal antibody 4A5 and goat anti-mouse secondary antibody were both coated at 2.0 mg / ml to prepare test strips. The results were evaluated using SL006 virus solution, negative anal swabs, and negative stool samples (all identified by RT-PCR). The results are shown in Table 7: When the gold-labeled monoclonal antibody was labeled at a concentration of 4 μg / ml or 36 μg / ml, the sensitivity for detecting CRV virus solution was 10. 6.45 Or 106.18 TCID 50 The concentration of gold-labeled monoclonal antibody at 8–32 μg / ml showed false positives in clinical samples; however, when the labeling concentration was 8–32 μg / ml, the sensitivity for detecting CRV virus fluid was 10. 4.65 ~10 6.10 TCID 50 / ml, correctly detecting clinical samples. However, when the gold-labeled monoclonal antibody concentration is 20–28 μg / ml, the sensitivity for detecting CRV virus solution is 10. 4.65 ~10 5.39 TCID 50 The concentration of gold-labeled monoclonal antibody at 24 μg / ml provides better detection sensitivity; the optimal sensitivity for detecting CRV virus solution is 10. 4.65 TCID 50 / ml.

[0122] Table 7 Optimization of different gold-labeled monoclonal antibody concentrations

[0123]

[0124] (4) Optimization of the gold-labeled pad manufacturing process

[0125] The gold-labeled monoclonal antibody 2C12 was sprayed onto a glass fiber membrane at spray rates of 1.0 μl / cm, 2.0 μl / cm, and 3.0 μl / cm, respectively. The coated glass fiber membranes were then dried in a drying room (temperature 20–25℃, relative humidity below 20%) for 2–3 hours. Test strips were then assembled, and different dilutions of canine rotavirus were tested. The color bands and background of the test and control lines were observed. Results showed that at a spray rate of 1.0 μl / cm, the test of a 1:32 dilution of CRV virus was negative; at spray rates of 2.0 μl / cm–3.0 μl / cm, all tests of a 1:32 dilution of CRV virus were positive (see Table 8). Considering cost, a spray rate of 2.0 μl / cm for the gold-labeled monoclonal antibody 2C12 was chosen for drying and assembling the test strips.

[0126] Table 8 Optimization of the gold-labeled pad manufacturing process

[0127]

[0128] Note: "+" indicates positive, and "-" indicates negative.

[0129] Monoclonal antibody 4A5 and goat anti-mouse IgG were diluted to 2.0 mg / ml and 2.0 mg / ml, respectively, and used as coating solutions for the detection line and control line. These solutions were then streaked onto the detection line and control line of the nitrocellulose membrane, respectively, as the preparation process for the nitrocellulose membrane. The gold-labeled monoclonal antibody 2C12 was prepared at a concentration of 24 μg / ml and uniformly sprayed onto the glass cellulose membrane using a spraying device at a spray rate of 2 μl / cm, as the preparation process for the gold-labeled pad, which was used for subsequent evaluation.

[0130] Example 4: Application of the test strip

[0131] 4.1 Sensitivity Testing

[0132] The sensitivity of the colloidal gold test strip prepared in Example 3 was evaluated using eight CRV virus strains isolated in Example 1 and the standard strain Cu-1 purchased from ATCC, and compared with two commercially available test strips. Commercially available test strip 1 was purchased from Kuailing Biotechnology, and commercially available test strip 2 was purchased from Anjie Pharmaceutical Co., Ltd. of South Korea. The test results are shown in Table 9: the commercially available test strips showed false negatives and low sensitivity in detecting multiple clinical CRV strains, while the test strip in this example showed positive results in all cases and a sensitivity (10-10) of low sensitivity. 4.69 ~10 5.18 TCID 50 The sensitivity ( / ml) is significantly higher. Furthermore, the sensitivity of the test strip of this invention in detecting the standard strain, namely the previously prevalent strain Cu-1 and the currently prevalent strain, is not significantly different, and the detection sensitivity of each strain is significantly higher than that of commercially available test strips in the prior art.

[0133] Table 9. Sensitivity of test strips for detecting different CRV strains

[0134]

[0135] 4.2 Specificity Detection

[0136] The colloidal gold test strip prepared in Example 2 was used to test canine parvovirus, canine parainfluenza virus, canine adenovirus type 1, canine adenovirus type 2, and canine distemper virus fluids, and the results were all negative. The colloidal gold test strip for canine rotavirus was used to test anal swabs and fecal samples infected with canine parvovirus, canine adenovirus, canine distemper virus, and canine coronavirus alone or in combination, as well as anal swabs and fecal samples from healthy dogs, and the results were all negative, indicating that the test strip has good specificity.

[0137] 4.3 Repeatability and Reliability Testing

[0138] Three batches of self-made test strips were used to test the repeatability of the same 15 samples at three different locations in China. The results were consistent, indicating that the self-made test strips had good repeatability and reliability.

[0139] 4.4 Shelf life

[0140] Sensitivity, specificity, and repeatability tests were performed on the test strips after being stored at room temperature for 3, 6, 12, 18, 24, and 27 months, respectively. The concordance rate of the test results was 100%, indicating that the test strips can be stored at room temperature for 24 months or even 27 months. In addition, the test strips were placed at 37°C for 6 days and 9 days, respectively, and the concordance rate of the test results was 100%, indicating that the test strips can be stored at 37°C for 6 to 9 days under extreme or practical conditions.

[0141] 4.5 Clinical Application

[0142] A total of 180 clinical samples were collected from clinical trial sites in seven provinces: Hebei, Henan, Heilongjiang, Shandong, Sichuan, Jiangsu, and Guangdong. These included 40 anal swabs and 50 fecal samples from dogs exhibiting clinical symptoms of canine rotavirus infection, and 40 anal swabs and 50 fecal samples from dogs without clinical symptoms. The samples were tested using the test strip of this invention, the CRV RT-PCR method, commercial test strip 1 (Kuailing Biotechnology), and commercial test strip 2 (Anjie, Korea). The results are shown in Table 10. All three test strips showed negative results for RT-PCR-negative samples, indicating good specificity. However, the positive concordance rates for RT-PCR-positive samples using the two commercial test strips were 68% and 72%, respectively, both lower than the 82% positive concordance rate of the test strip of this invention, indicating false negatives and missed detections. The test strip of this invention has superior clinical applicability.

[0143] Table 10 Comparison of Clinical Test Results

[0144]

[0145] 4.6 Detection of antigen fluctuation patterns

[0146] Three healthy, susceptible puppies aged 2-3 months (numbered G1, G2, and G3) were infected with canine rotavirus strain SL006 (10 μL / kg). 6.6 TCID 50 Three dogs were challenged with rotavirus via oral administration of 2 ml / dog and subcutaneous administration of 2 ml / dog. Anal swabs and fecal samples from the challenged dogs were tested using the test strip of this invention and the established canine rotavirus RT-PCR method. The results are shown in Table 11: the test strip detected positive samples on day 2, and fecal samples tested positive on days 3-8, turning negative on day 10; RT-PCR tests of fecal samples from challenged dogs showed positive results on days 2-9, turning negative on day 11. This indicates that the antigen fluctuation pattern detected by the test strip of this invention is consistent with that detected by the RT-PCR method and can be used for real-time clinical detection.

[0147] Table 11 Results of antigen fluctuation pattern detection

[0148]

[0149] In summary, the test strip prepared by this invention overcomes the problems of low sensitivity and poor broad-spectrum detection of canine rotavirus in existing technologies, largely avoiding missed detections and false negatives. Furthermore, it conforms to the detection pattern of RT-PCR antigen fluctuations, providing technical support for animal health and reducing the risk of asymptomatic dogs as a source of infection. It has the advantages of being rapid, simple, accurate, and broad-spectrum, facilitating clinical application in non-diagnostic canine rotavirus detection, particularly in epidemiological surveys, health checkups, and screening studies.

[0150] Example 5: Preparation and Application of Genetically Engineered Antibodies

[0151] The heavy chain variable region and light chain variable region gene sequences of the monoclonal antibodies 4A5 and 2C12 in Example 2 were amplified, and the heavy chain variable region genes and light chain variable region genes were linked by linking peptides to construct recombinant plasmids 4A5-ScFv, 2C12-ScFv, 4A5 heavy + 2C12 light -ScFv, and 2C12 heavy + 4A5 light -ScFv, respectively. The ScFv gene was inserted into the pCDNA-3.1 vector to construct the pCDNA-4A5-ScFv, pCDNA-2C12-ScFv, pCDNA-4A5 heavy + 2C12 light -ScFv, and pCDNA-2C12 heavy + 4A5 light -ScFv eukaryotic expression systems, respectively, which were then transfected into MDCK cells for expression.

[0152] Following the IFA method described in Example 2, the expressed single-chain antibodies 4A5, 2C12, 4A5 heavy + 2C12 light, and 2C12 heavy + 4A5 light (numbered sequentially as single-chain antibody 1, single-chain antibody 2, single-chain antibody 3, and single-chain antibody 4) were subjected to IFA titer testing. The results are shown in Table 12: the IFA titers of single-chain antibodies 1 to 4 against different CRV strains were all ≥1:800, indicating that single-chain antibodies 1-4 have good reactivity with different CRV strains.

[0153] Table 12 Results of IFA titer assay for genetically engineered antibodies

[0154]

[0155] The results above show that SEQ.ID No.1, SEQ.ID No.2, SEQ.ID No.3 and SEQ.ID No.4 can be used for the preparation of genetically engineered antibodies against canine rotavirus and for the evaluation of reactivity to different CRV strains.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. SEQUENCE LISTING <110> Luoyang Putai Biotechnology Co., Ltd. <120> Canine rotavirus monoclonal antibody pairs and their applications <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 366 <212> DNA <213> Artificial sequence <400> 1 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactattaca tgtattgggt tcgccagact 120 ccggaaaaga ggctggagtg ggtcgctacc attagtgatg ttagttacac ctactatcca 180 gacagtgtga aggggcgatt caccacctcc agagacaatg ccaacaacaa cctgtatctg 240 gaaatgagca gtctgaagtc tgaggacaca gccatgtatt attgtgtaag agcggtcgta 300 ttctatgata attattttca ctactttgac tactggggcc aaggcacccc tctcacagtc 360 tcctca 366 <210> 2 <211> 333 <212> DNA <213> Artificial sequence <400> 2 gacattgtgc tgacacagtc tcctgcttc ttagctgtat ctctggggca gagggccacc 60 atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120 caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga ttctgcttac 300 acgttcggag gggggaccaa gctggaaata aaa 333 <210> 3 <211> 348 <212> DNA <213> artificial sequence <400> 3 caggtccagc tgcagcagtc tggagatgat ctggtaaagc ctggggcctc agtgaagctg 60 tcctgcaagg cctctggcta caccttcacc agctactgga ttaattggat aaaacagagg 120 cctggacagg gccttgagtg gataggacgt tttgctcctg gaagtggtag ttcttactac 180 aatgaaatgt tcaagggcaa ggcaacactg actgtagaca catcctccag tacagcctac 240 attcagctca gcagcctggc atctgaggac tctgctgtct atttctgtgc aagaggactt 300 gggtactttg actattgggg ccaaggcacc actctcacag tctcctca 348 <210> 4 <211> 322 <212> DNA <213> artificial sequence <400> 4 gacgttgtga tgaccccatc tcacaaattc atgtccacat cattaggaga cagggtcagc 60 atcacctgca aggccggtca ggatgtgggt actggtgtag cctggtctca acagaaacca 120 gggcaatttc ctaaattact gatttactgg gcatccaccc gacacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccattagcaa tgtgcagtct 240 gaagacttgg cagattattt ctgtcaacaa tatagctcct atcctctgac gttcggtgga 300 ggcaccaggc tggaaatcaa ac 322

Claims

1. An antibody or antibody fragment that specifically binds to canine rotavirus, wherein, The heavy chain variable region of the antibody or antibody fragment is encoded by SEQ ID No. 1 or its degenerate sequence, and the light chain variable region of the antibody or antibody fragment is encoded by SEQ ID No. 2 or its degenerate sequence; the antibody or antibody fragment specifically binds to the canine rotavirus VP7 protein.

2. The antibody or antibody fragment according to claim 1, wherein, The antibody is a monoclonal antibody.

3. The antibody or antibody fragment according to claim 1, wherein, The antibody is a genetically engineered antibody; wherein, the genetically engineered antibody includes single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies.

4. Monoclonal antibody 4A5, of which, The heavy chain variable region of the monoclonal antibody 4A5 is encoded by SEQ ID No. 1 or its degenerate sequence, and its light chain variable region is encoded by SEQ ID No. 2 or its degenerate sequence.

5. An antibody or antibody fragment that specifically binds to canine rotavirus, wherein, The heavy chain variable region of the antibody or antibody fragment is encoded by SEQ ID No. 3 or its degenerate sequence, and the light chain variable region of the antibody or antibody fragment is encoded by SEQ ID No. 4 or its degenerate sequence; the antibody or antibody fragment specifically binds to the canine rotavirus VP7 protein.

6. The antibody or antibody fragment according to claim 5, wherein, The antibody is a monoclonal antibody.

7. The antibody or antibody fragment according to claim 5, wherein, The antibody is a genetically engineered antibody; wherein, the genetically engineered antibody includes single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies.

8. Monoclonal antibody 2C12, wherein, The heavy chain variable region of the monoclonal antibody 2C12 is encoded by SEQ ID No. 3 or its degenerate sequence, and its light chain variable region is encoded by SEQ ID No. 4 or its degenerate sequence.

9. The use of the antibody or antibody fragment according to any one of claims 1 to 3 and 5 to 7, or the monoclonal antibody 4A5 according to claim 4, or the monoclonal antibody 2C12 according to claim 8, in the preparation of epitope identification research reagents or canine rotavirus antigen reactivity research reagents for non-diagnostic purposes.

10. A reagent kit, wherein, The kit comprises an effective amount of the monoclonal antibody 4A5 as described in claim 4, an effective amount of the gold-labeled monoclonal antibody 2C12 as described in claim 8, and a detection reagent for detecting canine rotavirus antigen-antibody reaction; or the kit comprises an effective amount of the monoclonal antibody 2C12 as described in claim 8, an effective amount of the gold-labeled monoclonal antibody 4A5 as described in claim 4, and a detection reagent for detecting canine rotavirus antigen-antibody reaction.

11. The kit according to claim 10, wherein, The kit includes a colloidal gold test strip, comprising: a base plate having a first end and a second end, and sequentially arranged along the direction from the first end to the second end: a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane contacts the gold-labeled pad or the sample pad and the gold-labeled pad, allowing the conjugate of canine rotavirus antigen and the monoclonal antibody 2C12 to migrate towards the second end of the base plate. The gold-labeled pad contains the colloidal gold-labeled monoclonal antibody 2C12. The nitrocellulose membrane includes a detection line and a control line. The detection line is immobilized with the monoclonal antibody 4A5, and the control line is immobilized with goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody. The immobilized concentration of monoclonal antibody 4A5 is 1.0–3.0 mg / ml, the concentration of the colloidal gold-labeled monoclonal antibody 2C12 is 8–32 μg / ml, and the concentration of the goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody is 1.0–4.0 mg / ml.

12. The kit according to claim 11, wherein, The monoclonal antibody 4A5 has a fixed concentration of 2.0–3.0 mg / ml, the monoclonal antibody 2C12 has a colloidal gold labeling concentration of 20–28 μg / ml, the goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody has a concentration of 2.0–4.0 mg / ml, and the gold-labeled monoclonal antibody 2C12 has a spray volume of 1.0 μl / cm–3.0 μl / cm. The kit also includes a sample processing solution, which is a phosphate buffer containing 1% V / V Triton X-100.

13. The kit according to claim 12, wherein, The monoclonal antibody 4A5 has a fixed concentration of 2.0 mg / ml, the monoclonal antibody 2C12 has a colloidal gold labeling concentration of 24 μg / ml, the goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody has a content of 2.0 mg / ml, and the gold-labeled monoclonal antibody 2C12 is sprayed at a rate of 2.0 μl / cm to 3.0 μl / cm.

14. The kit according to claim 13, wherein, The gold-labeled monoclonal antibody 2C12 was sprayed at a rate of 2.0 μl / cm.

15. The kit according to claim 11, wherein, In the kit, the sample pad, gold label pad, nitrocellulose membrane, and absorbent pad, arranged sequentially from the first end to the second end, are in contact with each other when adjacent components are in contact, while non-adjacent components are not in contact with each other.

16. A single-chain antibody, wherein, When the heavy chain variable region of the single-chain antibody is encoded by SEQ ID No. 1 or its degenerate sequence, the light chain variable region of the single-chain antibody is encoded by SEQ ID No. 2 or SEQ ID No. 4 or their degenerate sequence; when the heavy chain variable region of the single-chain antibody is encoded by SEQ ID No. 3 or its degenerate sequence, the light chain variable region of the single-chain antibody is encoded by SEQ ID No. 2 or SEQ ID No. 4 or their degenerate sequence.

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