Test strip for detecting canine parvovirus and hybridoma cell, monoclonal antibody and application

Abstract

This invention relates to the field of biodetection technology, and more particularly to a fluorescent test strip for detecting canine parvovirus, along with hybridoma cells, monoclonal antibodies, and their applications. The test strip of this invention has fluorescent microspheres coated with canine parvovirus monoclonal antibody and fluorescently labeled rabbit IgG within the conjugate pad; a detection line and a control line are disposed on the surface of a nitrocellulose membrane, with the detection line coated with canine parvovirus monoclonal antibody and the control line coated with rabbit anti-IgG; wherein the canine parvovirus monoclonal antibody is secreted using hybridoma cells with accession number C202355. The test strip of this invention can be used to simultaneously detect feline parvovirus, canine parvovirus, and various genotypes of canine parvovirus, exhibiting high sensitivity and specificity, and possessing the technical advantages of accuracy, speed, and simplicity.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a test strip for detecting canine parvovirus, hybridoma cells, monoclonal antibodies, and their applications. Background Technology

[0002] Canine parvovirus (CPV) is an acute, highly contagious viral disease caused by canine parvovirus infection in puppies. CPV is a small, non-enveloped, single-stranded negative-sense DNA virus with an icosahedral symmetry structure and a genome length of approximately 5 kb. It primarily encodes two non-structural proteins (NS1 and NS2) and two structural proteins (VP1, VP2, and VP3). VP2 is the main capsid protein, accounting for 90% of the total nucleocapsid, and is also the main structural component and immunogenic protein, promoting the production of neutralizing antibodies. The disease was first reported in 1970, with my country first noting its first case in 1982, and has since spread globally. The incubation period is approximately 3–7 days. Clinically, it manifests as severe gastroenteritis, vomiting, diarrhea, and fever. Puppies and purebred dogs are most susceptible, and the mortality rate is relatively high, thus severely impacting the dog breeding and pet industries in China and worldwide. Therefore, early prevention and detection are of paramount importance for controlling this disease.

[0003] Common methods for detecting canine parvovirus include hemagglutination assay (HA) and hemagglutination inhibition assay (HI), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and immunochromatographic assay. Each of these methods has its advantages and disadvantages. Compared to immunochromatographic assay, while the other methods are relatively sensitive, they are more complex to operate, take longer, and require specific instruments and skilled personnel, making them unsuitable for rapid on-site diagnosis.

[0004] Immunochromatography is a novel diagnostic technique developed in recent years based on the specific reaction of antigens and antibodies. The main biomarkers are colloidal gold, quantum dots, and time-resolved fluorescent microspheres. Currently, colloidal gold immunochromatography is the primary method for detecting CPV. However, this method suffers from drawbacks such as poor stability and low sensitivity, easily leading to false positives and false negatives, which is detrimental to the early detection and prevention of the disease.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a fluorescent test strip for detecting canine parvovirus, along with hybridoma cells, monoclonal antibodies, and applications. The test strip of this invention can simultaneously detect feline parvovirus, canine parvovirus, and various genotypes of canine parvovirus.

[0007] This invention provides a fluorescent test strip for detecting canine parvovirus, comprising a PVC base plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially fixed; the conjugate pad is coated with fluorescent microspheres labeled with canine parvovirus monoclonal antibody and fluorescently labeled rabbit IgG; a detection line and a control line are disposed on the surface of the nitrocellulose membrane, the detection line being coated with canine parvovirus monoclonal antibody and the control line being coated with rabbit anti-IgG; the canine parvovirus monoclonal antibody is obtained by secretion using hybridoma cells with accession number C202355.

[0008] Optionally, within the detection range: the coating concentration of canine parvovirus monoclonal antibody is 0.4–1.0 mg / mL, preferably 0.6 mg / mL, and the coating amount is 0.5–2 μL / cm, preferably 1 μL / cm; within the control range: the coating concentration of rabbit anti-IgG is 0.4–1.0 mg / mL, preferably 0.5 mg / mL, and the coating amount is 0.5–2 μL / cm, preferably 1 μL / cm.

[0009] Optionally, the coating amount of the fluorescent microsphere-labeled canine parvovirus monoclonal antibody in the binding pad is 4–10 μg, preferably 6 μg; preferably, the mass ratio of the fluorescently labeled canine parvovirus monoclonal antibody to rabbit anti-IgG is 1:1.

[0010] Optionally, the mass ratio of fluorescent microspheres to canine parvovirus monoclonal antibody is 1:20-40, preferably 1:30; the mass ratio of fluorescent microspheres to rabbit IgG is 1:40-80, preferably 1:50.

[0011] The present invention also proposes a method for preparing the fluorescent test strip, comprising at least the following steps:

[0012] S1. Prepare fluorescent microsphere-labeled canine parvovirus monoclonal antibody and fluorescent microsphere-labeled rabbit IgG, respectively;

[0013] S2. Seal the conjugate pad and the sample pad separately, and spray fluorescent microspheres labeled with canine parvovirus monoclonal antibody and fluorescent microspheres labeled with rabbit IgG onto the conjugate pad;

[0014] S3. Detection lines and control lines are sprayed onto the surface of the nitrocellulose membrane, respectively; the detection lines are coated with canine parvovirus monoclonal antibody; the control lines are coated with rabbit anti-IgG.

[0015] S4. Assemble the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad to obtain the test strip.

[0016] Optionally, S1 includes: after activating the fluorescent microspheres, adding canine parvovirus monoclonal antibody and rabbit IgG for incubation, and then adding BSA solution for blocking; preferably, activation is performed using EDC and NHS solutions; preferably, the incubation time is 1 to 3 hours and the incubation temperature is 20 to 25°C.

[0017] Optionally, S2 includes: sealing the sample pad with a sample pad sealing solution, the sample pad sealing solution being a boric acid solution containing 0.75% Tween-20, 1% PEG20000, and 3% BSA; and sealing the conjugate pad with a conjugate pad sealing solution, the conjugate pad sealing solution being a boric acid solution containing 3% trehalose, 2% BSA, 0.75% Tween-20, and 0.5% Triton X-100; preferably, the sealing time is 1 to 3 hours, and the temperature is 36 to 38°C.

[0018] This invention also proposes a hybridoma cell line that secretes viral monoclonal antibodies, with accession number CCTCC NO:C202355.

[0019] This invention also proposes a canine parvovirus monoclonal antibody, which is obtained by secretion from hybridoma cells with accession number CCTCC NO:C202355.

[0020] The present invention also proposes the application of the canine parvovirus monoclonal antibody in the preparation of formulations, kits or vaccines for the detection of canine parvovirus.

[0021] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0022] The test strip of this invention can be used to simultaneously detect feline parvovirus and canine parvovirus, and has high sensitivity and specificity, as well as the technical advantages of being accurate, rapid and convenient.

[0023] The canine parvovirus monoclonal antibody of the present invention has the technical advantages of high specificity and strong binding ability. Attached Figure Description

[0024] Figure 1 The results of SDS-PAGE experiments on recombinant VP2 protein;

[0025] Figure 2 The results of SDS-PAGE were obtained to collect the eluent from the purified antibody.

[0026] Figure 3 The results of Western blot experiments using 6A8 mAb as a primary antibody against CPV;

[0027] Figure 4The results are from an indirect immunofluorescence assay (IFA).

[0028] Figure 5 This is a graph showing the results of the blood coagulation reaction;

[0029] Figure 6 Experimental results comparing the 6A8 mAb coating amount of T-line;

[0030] Figure 7 To compare experimental results of combining 6A8 mAb labeled with fluorescent microspheres in the pad;

[0031] Figure 8 The results are experimental findings comparing reaction times;

[0032] Figure 9 The experimental results are for the detection limit;

[0033] Figure 10 and Figure 11 These are specific experimental results;

[0034] Figure 12 and Figure 13 The results are from experiments on sensitivity.

[0035] Figure 14 The results show the specificity of each genotype of canine parvovirus.

[0036] Figure 15 The results are experimental findings on stability.

[0037] Preservation Information

[0038] The hybridoma cell line CPV VP2 6A8 of this invention was deposited on March 8, 2023, at the China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO:C202355. Detailed Implementation

[0039] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0040] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0041] This invention provides an example of obtaining a VP2 protein gene sequence from GenBank:MK332007.1. Experimental animals were immunized with this recombinant protein to screen for a hybridoma cell line with optimal performance. BALB / c mice were immunized four times using the VP2 recombinant protein as an immunogen. The first dose consisted of 50 μg of protein-emulsified complete Freund's adjuvant. The second and third doses consisted of 50 μg of protein-emulsified incomplete Freund's adjuvant. A final booster immunization was performed by intraperitoneal injection of 100 μg of protein. Cell fusion was performed three days after the booster immunization. Using the VP2 recombinant protein as an antigen, cell culture supernatant was screened from the hybridoma cell culture supernatant using a specific enzyme-linked immunosorbent assay (ELISA). Hybridoma clones producing VP2-specific antibodies were subcloned into single-cell clones, and purified monoclonal antibodies were prepared from ascites fluid. High-titer hybridoma cell lines were screened based on monoclonal antibody titer. Identification was performed using indirect ELISA, and specificity was detected using indirect immunofluorescence assay (IFA) and Western blotting.

[0042] This invention provides a test strip for detecting canine parvovirus prepared using this monoclonal antibody. The innovative use of time-resolved fluorescence immunochromatographic assay (TRFIA) leverages the long lifetime and ultrasensitivity of lanthanide chelates, allowing for time-resolved fluorescence measurement and effectively eliminating interference from non-specific fluorescence. Common elements such as europium are used as markers. Therefore, compared to colloidal gold test strips, this invention significantly improves both sensitivity and specificity.

[0043] Based on the characteristics of TRFIA and the obtained recombinant VP2 protein, this invention proposes a time-resolved fluorescence immunochromatographic test strip for rapid detection of CPV. This allows for rapid and sensitive detection of CPV, thereby controlling the spread and prevalence of the disease and providing an accurate, rapid, and convenient detection method for the prevention and control of canine parvovirus disease.

[0044] The test strip for detecting canine parvovirus in this embodiment of the invention includes a PVC base plate. A sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially fixed on the PVC base plate. The conjugate pad is coated with fluorescent microspheres labeled with canine parvovirus monoclonal antibody and fluorescently labeled rabbit IgG. A test line and a control line are set on the surface of the nitrocellulose membrane. The test line (T line) is coated with canine parvovirus monoclonal antibody, and the control line (C line) is coated with rabbit anti-IgG. A positive result is indicated when both the test line (T line) and the control line (C line) turn red; a negative result is indicated when the test line does not develop color but the control line (C line) does; and a negative result is considered a test strip failure. When the sample to be tested is chromatographyd forward through the capillary action to the conjugate pad, if the sample contains CPV antigen, the fluorescently labeled antibody on the conjugate pad will undergo a specific antigen-antibody reaction with its corresponding antigen, forming an antigen-antibody complex. The two complexes then continue chromatography forward, forming a red band at the test line T. Meanwhile, the fluorescently labeled rabbit IgG antibody also reaches the control line C through capillary action and binds to the goat anti-rabbit IgG antibody to form a red band.

[0045] As an improvement to this invention, the coating amount of fluorescently labeled canine parvovirus monoclonal antibody in the detection line is 0.5–2 μL / cm, preferably 1 μL / cm; the coating amount of rabbit anti-IgG in the control line is 0.5–2 μL / cm, preferably 1 μL / cm. If the coating amount is too large, it will result in a non-specific reaction; if the coating amount is too small, insufficient color development may occur.

[0046] As an improvement of this invention, the coating amount of fluorescently labeled canine parvovirus monoclonal antibody in the binding pad is 0.5-2 μL / cm, preferably 1 μL / cm; if the coating amount is too large, it will have the defect of non-specific reaction; if the coating amount is too small, it may have the defect of insufficient color development.

[0047] As an improvement of this invention, the mass ratio of fluorescently labeled canine parvovirus monoclonal antibody to rabbit anti-IgG is 1:1.

[0048] As an improvement to this embodiment of the invention, the fluorescent microspheres were labeled with Eu-time-resolved fluorescent nanospheres purchased from Nanjing Microtest Biotechnology Co., Ltd.

[0049] As an improvement of this invention, in the fluorescently labeled canine parvovirus monoclonal antibody, the mass ratio of fluorescent microspheres to canine parvovirus monoclonal antibody is 1:20-40, preferably 1:30.

[0050] As an improvement of this invention, the mass ratio of fluorescent microspheres to rabbit IgG is 1:40-80, preferably 1:50. This invention also relates to a method for preparing the test strip, comprising at least the following steps:

[0051] S1. Prepare fluorescent microsphere-labeled canine parvovirus monoclonal antibody and fluorescent microsphere-labeled rabbit IgG, respectively;

[0052] S2. Seal the conjugate pad and the sample pad separately, and spray fluorescent microspheres labeled with canine parvovirus monoclonal antibody and fluorescent microspheres labeled with rabbit IgG onto the conjugate pad;

[0053] S3. Detection lines and control lines are sprayed onto the surface of the nitrocellulose membrane, respectively; the detection lines are coated with canine parvovirus monoclonal antibody; the control lines are coated with rabbit anti-IgG.

[0054] S4. Assemble the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad to obtain the test strip.

[0055] As an improvement of this embodiment of the invention, S1 includes: after activating the fluorescent microspheres, adding canine parvovirus monoclonal antibody and rabbit IgG for incubation, and then adding BSA solution for blocking; wherein, the activation is performed using EDC and NHS solutions, the incubation time is 1 to 3 hours, and the incubation temperature is 20 to 25°C.

[0056] As an improvement to an embodiment of the present invention, S2 includes:

[0057] The sample pad is sealed using a sample pad sealing solution, which can be a boric acid solution containing 0.75% Tween-20, 1% PEG20000, and 3% BSA. The conjugate pad is sealed using a conjugate pad sealing solution, which can be a boric acid solution containing 3% trehalose, 2% BSA, 0.75% Tween-20, and 0.5% Triton X-100. Preferably, the sealing time is 1–3 hours, and the temperature is 36–38°C.

[0058] This invention also relates to a hybridoma cell line that secretes viral monoclonal antibodies, with accession number CCTCCNO:C202355.

[0059] This invention also relates to a canine parvovirus monoclonal antibody, obtained by secretion from hybridoma cells with accession number CCTCC NO: C202355. The canine parvovirus monoclonal antibody obtained through screening in this invention has the technical advantages of high titer and strong antigen-binding ability.

[0060] The embodiments of the present invention also relate to the application of the above-mentioned canine parvovirus monoclonal antibody in the preparation of formulations, kits or vaccines for detecting canine parvovirus.

[0061] Example

[0062] Materials and Methods

[0063] 1.1 Protein and Serum

[0064] Feline kidney cell line (F81), SP2 / 0 cells, 60 clinical samples, canine parvovirus (CPV), canine coronavirus (CCoV), canine adenovirus (CAV), and canine distemper virus (CDV) were all provided by the Institute of Special Agricultural Products, Chinese Academy of Agricultural Sciences. BABLC mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute; rabbit IgG antibodies and goat anti-rabbit IgG antibodies were purchased from Thermo Fisher Scientific.

[0065] 1.2 Main Reagents, Equipment and Kits

[0066] Chloroauric acid was purchased from Sigma-Aldrich; trisodium citrate was purchased from Sinopharm Group; sample pads, absorption pads, conjugated pads, nitrocellulose membranes (NC), and polyvinyl chloride (PVC) were purchased from Shanghai Jieen Biotechnology Co., Ltd.; potassium carbonate was purchased from Sinopharm Chemical Reagent Co., Ltd.; 1% carboxyl-modified time-resolved fluorescent microspheres and a fluorescence quantitative rapid detector were purchased from Nanjing Microtest Co., Ltd.

[0067] [1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride] and bovine serum albumin were purchased from Sigma-Aldrich.

[0068] The ultrapure water system was purchased from Milli-QILLIPORE, France; the Biodot AD3200 diagnostic spray system was purchased from Biodot Inc.; the Biodot CM4000 flatbed automatic cutting machine was purchased from Biodot, USA; and the JY200 electronic scale was purchased from Shanghai Precision Scientific Instruments Co., Ltd.

[0069] Both the BioDot AD3200 gold standard diagnostic positioning system and the BioDot CM4000 flat-top automatic peeler were purchased from BioDot.

[0070] Example 1

[0071] This example illustrates the process of obtaining the antigen protein:

[0072] 1. Identification and expression of VP2 protein

[0073] 1.1 Obtain the VP2 protein gene sequence:

[0074] 1.2 The VP2 protein gene sequence was tandemly linked with a SUMO tag and a His tag. The VP2 gene sequence (GenBank: MK332007.1 SEQ ID NO:1) and the SUMO tag were then tandemly linked with the His tag and sent to Sangon Biotech Co., Ltd. (Shanghai, China) for synthesis. The synthesized VP2-SUMO-His gene fragment was cloned into the E. coli expression vector pET-28a, and the recombinant plasmid was named pET-28a-VP2.

[0075] SEQ ID NO:1

[0076]

[0077] 1.3 The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain recombinant Escherichia coli, named E. coli-CPV-VP2.

[0078] 1.4 The target protein was expressed by recombinant engineered bacteria induced by isopropyl β-d-1-thiogalactopyranoside.

[0079] The purification of VP2 recombinant protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed by Western blotting.

[0080] The experimental results obtained from SDS-PAGE are as follows: Figure 1 As shown in the figure. According to SDS-PAGE analysis, the VP2-SUMO-His recombinant protein (hereinafter referred to as VP2 recombinant protein) has good reactivity and specificity, and its size is approximately 76 kDa.

[0081] Example 2

[0082] This example illustrates the preparation and identification process of monoclonal antibodies:

[0083] 1. Production of VP2 monoclonal antibody

[0084] The VP2 recombinant protein prepared in Example 1 was used as an immunogen to immunize BABL / c mice four times. The first immunization was performed using Freund's complete adjuvant emulsified with 50 μg of VP2 recombinant protein. The second and third immunizations were performed using Freund's incomplete adjuvant emulsified with 50 μg of VP2 recombinant protein. A final booster immunization was performed by intraperitoneal injection of 100 μg of VP2 recombinant protein into BABL / c mice. Fusion occurred three days after the booster immunization.

[0085] Using the recombinant VP2 protein prepared in Example 1 as an antigen, cell culture supernatant from wells containing hybridoma colonies was screened by ELISA. Hybridoma clones producing VP2-specific antibodies were subcloned into single-cell clones (monoclonus), and purified mAbs were prepared from ascites fluid.

[0086] The selected hybridoma cell line was deposited under the accession number CCTCC NO:C202355. The monoclonal antibody secreted by this hybridoma cell line was named 6A8 mAb.

[0087] 2. Identification of VP2 monoclonal antibodies

[0088] The results of SDS-PAGE of the purified antibody eluent are as follows: Figure 2 As shown, by Figure 2It can be seen that the relative molecular weights of the heavy and light chains of the purified VP2 antibody are approximately 55 and 25 kDa, respectively, indicating that the purified antibody bands are correct and have high purity.

[0089] Western blot analysis was performed using 6A8 mAb as the primary antibody and CPV, and the results are as follows: Figure 3 As shown, by Figure 3 It can be seen that 6A8 mAb only reacts with VP2 protein.

[0090] 3. Indirect immunofluorescence assay (IFA)

[0091] The reactivity of VP2 recombinant protein with 6A8 mAb was analyzed. VP2-SUMO-His recombinant protein was separated by SDS-PAGE and then transferred to an NC membrane. The membrane was blocked in TBST with 5% (w / v) skim milk at room temperature for 1.5 h, followed by incubation with 6A8 mAb (1:2000 dilution) at room temperature for 1 h. After washing three times with TBST (containing 0.1% Tween-20), the membrane was incubated with HRP-conjugated goat anti-mouse IgG (1:20000; Abcam, Cambridge, MA, USA) at room temperature for 1 h. Protein expression was then detected using an electrochemiluminescence (ECL) kit (Engreen Biosystems, Beijing, China) and exposed to a developer (Azure c300, USA). After washing three times with PTBST, protein expression was detected using an electrochemiluminescence (ECL) kit (Engreen Biosystems, Beijing, China) and a developer (Bio-Rad, USA). Indirect immunofluorescence (IFA) results are shown below. Figure 4 As shown.

[0092] Depend on Figure 4 It can be seen that 6A8 mAb showed specific fluorescent staining, while no fluorescence was found in the blank cell group.

[0093] Example 3

[0094] This embodiment illustrates the method for preparing the test strip.

[0095] 1. Preparation of fluorescent microspheres labeled with 6A8 mAb (prepared in Example 2) and rabbit IgG

[0096] Take two 800 μL portions of 0.5 M borate buffer (pH 8.5) and add them to 2 mL centrifuge tubes respectively. Add 200 μL of 1% fluorescent microspheres and mix quickly. Then add 20 μL of 10 mg / mL EDC and 50 μL of 10 mg / mL NHS. Mix by pipetting and activate at room temperature for 15 min, then centrifuge at 14000 rpm for 10 min. Resuspend in 1000 μL of borate buffer. Repeat the above steps twice. If there is obvious precipitate that cannot be mixed, sonicate at 100 W for 3 s, 3 s for 1 min until the fluorescent microspheres are completely dispersed. Add 60 μg of 6A8 mAb (prepared in Example 2) and 100 μg of rabbit IgG to the solution respectively. Incubate at room temperature for 2 hours. Add 100 μL of 10% BSA solution for blocking. Incubate at room temperature for 2 hours, then centrifuge at 14000 rpm. After centrifugation for 10 min, discard the supernatant and resuspend the microspheres by adding 1000 μL of fluorescent microsphere labeling protectant (0.1% BSA in 0.05 M borate buffer, pH 8.2). Repeat the above steps twice. If there is obvious precipitate that cannot be mixed, sonicate at 100 W for 3 s for 3 s for 1 min and store at 4 °C for later use.

[0097] 5.2 Assembly of Fluorescent Test Strips

[0098] The test strip consists of a nitrocellulose membrane (NC membrane), a conjugate pad, a sample pad, an absorbent pad, and PVC. The sample pad is sealed at 37°C for 2 hours with a sample pad blocking solution (a boric acid solution containing 0.75% Tween-20, 1% PEG20000, and 3% BSA). After removal, it is dried in an oven and sealed in an aluminum foil bag for later use. The conjugate pad is sealed at 37°C for 2 hours with a conjugate pad blocking solution (a boric acid solution containing 3% trehalose, 2% BSA, 0.75% Tween-20, and 0.5% Triton X-100). After removal, it is dried in an oven. Using a three-dimensional spray coating instrument, fluorescently labeled 6A8 mAb and rabbit IgG are uniformly mixed at a 1:1 ratio and sprayed onto the sealed conjugate pad at a rate of 1 μL / cm. The coating amount of fluorescently labeled 6A8 mAb on the conjugate pad of each test strip is 6 μg, and the coating amount of fluorescently labeled rabbit IgG is 6 μg. Dry at 37℃ for 3 hours, then seal in aluminum foil bags for later use. Coat the NC membrane with rabbit anti-IgG (0.5 mg / mL) at the control line C, and with 6A8 mAb (19 μg / mL) at the detection line T, using a coating amount of 1 μL / cm for both. Dry at 37℃ for 1 hour. Finally, attach the NC membrane, conjugate pad, sample pad, and absorbent pad sequentially to a PVC base plate, and cut into 4 mm wide test strips using a cutter. Seal in aluminum foil bags for later use.

[0099] Example 4

[0100] This example illustrates how to use the test strip:

[0101] The sample to be tested was diluted with PBS solution at a ratio of 1:5. 100 μL of the diluted sample solution was dropped into the sample slot of the test strip prepared in Example 3. The results were observed after reacting at room temperature for 10 min.

[0102] If both the test line (T) and the control line (C) turn red, the result is positive; if the test line does not show color but the control line (C) does, the result is negative; if the control line (C) does not show color, the test strip is considered invalid.

[0103] Experimental Example 1

[0104] This experimental example illustrates the optimal labeling amount of monoclonal antibody 6A8 mAb within the T line when preparing fluorescent test strips:

[0105] The examples were prepared according to the method of Example 3, except that the concentrations of 6A8 mAb at the T line were 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, respectively.

[0106] Positive sample (T+): TCID50 = 10 3.9 The cytotoxicity was achieved by diluting the sample 10-fold with sample dilution buffer;

[0107] Negative samples (T-): with sample dilution buffer;

[0108] Positive and negative samples were added separately and detected using a fluorescence analyzer. The T+ and T- values ​​of the positive control and healthy dog ​​serum were measured using a reading instrument. The T+ / T- ratio was denoted as P, and the coating concentration corresponding to the highest P value was taken as the optimal coating concentration. The experimental results are as follows: Figure 6 As shown.

[0109] Depend on Figure 6 As shown, the P value is the largest when the concentration of monoclonal antibody 6A8 mAb at the T line is 0.6 mg / mL. Therefore, 0.6 mg / mL is determined to be the optimal coating concentration.

[0110] Experiment Example 2

[0111] This experimental example illustrates the optimal coating amount of monoclonal antibody 6A8mAb labeled with fluorescent microspheres in the pad when preparing fluorescent test strips.

[0112] The examples were prepared according to the method of Example 3, except that the amounts of the monoclonal antibody 6A8 mAb labeled with fluorescent microspheres in the binding pad were 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg, respectively.

[0113] Positive sample (T+): TCID50 = 10 3.9 The cytotoxicity was achieved by diluting the sample 10-fold with sample dilution buffer;

[0114] Negative samples (T-): with sample dilution buffer;

[0115] Positive and negative samples were added separately and detected using a fluorescence analyzer. The T+ / T- ratio was denoted as N, and the labeling amount corresponding to the largest N value was set as the optimal labeling amount. The experimental results are as follows: Figure 7 As shown.

[0116] Depend on Figure 7 It can be seen that the N value reaches its highest value when the fluorescent microspheres are labeled with 6 μg of 6A8 mAb.

[0117] Experimental Example 3

[0118] This experimental example illustrates the determination of reaction time.

[0119] The detection was performed according to the method in Example 4, with the following difference:

[0120] Positive sample (T+): TCID50 = 10 3.9 The cytotoxicity was achieved by diluting the sample 10-fold with sample dilution buffer;

[0121] Negative samples (T-): with sample dilution buffer;

[0122] Positive and negative samples were added separately, and fluorescence analysis was performed using a fluorescence analyzer at intervals of 2, 4, 6, 8, 10, 12, 14, and 16 minutes. Fluorescence values ​​were recorded promptly, and the T-value was measured. A trend graph of the T-value over time was also plotted. The experimental results are as follows: Figure 8 As shown.

[0123] Depend on Figure 8 It can be seen that the fluorescence value at the T-line increases with time. When the time exceeds 10 minutes, the fluorescence value decreases briefly and then tends to plateau. Therefore, 10 minutes is chosen as the optimal reaction time.

[0124] Experiment Example 4

[0125] The specificity, sensitivity, and stability of the test strips prepared in Example 3 were verified.

[0126] 1. Limit of detection:

[0127] Fifty CPV-negative samples were tested according to the conditions in Example 4. T-line fluorescence values ​​were read, and the results were statistically analyzed to calculate the average value. The value was 1195, and the standard deviation (SD) was 566. Therefore, a fluorescence value of 2893 or higher is considered positive, and a value below 2893 is considered negative. The results are as follows: Figure 9 As shown.

[0128] 4.3 Specificity

[0129] The test strips of this invention were used to detect CPV, CCoV, CDV, and CAV positive samples. The test results showed that the test strips were only positive for CPV positive samples, and there was no non-specific cross-reaction with the other samples. The results are as follows. Figure 10 and Figure 11 As shown.

[0130] 4.4 Sensitivity

[0131] CPV positive (TCID) 50 =10 3.9 When the control (0.1 mL) was diluted 3200 times, the test strip of this invention was used for detection, and the experimental results are as follows: Figure 12 and Figure 13 As shown.

[0132] like Figure 12 and Figure 13 As can be seen, the test result of the fluorescent test strip was still positive, therefore the sensitivity of the fluorescent test strip was 1:3200.

[0133] 4.5 Specificity experiments against different genotypes of canine parvovirus:

[0134] The test strips of this invention were used to detect positive samples for FPV, MEV, CPV-2c, CPV-2, and CPV-2a. The test results showed that the test strips were positive for various genotypes of multiple canine parvoviruses. Figure 14 As shown.

[0135] 4.6 Stability

[0136] The test strips were stored at room temperature (18–25°C) and 4°C for 7 and 5 months, respectively. The experimental results are as follows: Figure 15 As shown.

[0137] Depend on Figure 15 It is known that the test strips of the present invention can be stably stored for 7 and 5 months at room temperature (18-25℃) and 4℃, respectively.

[0138] 4.6 Compliance Rate Test

[0139] Using the fluorescent test strips of this invention, 60 clinical samples were simultaneously tested by qPCR. The results showed that qPCR detected 38 positive samples and 22 negative samples; the fluorescent test strips detected 37 positive samples and 23 negative samples. The experimental results are shown in Table 1. All samples tested positive for FPV, CPV-2, CPV-2a, CPV-2b, CPV-2c, New CPV-2a, and CPV-2b.

[0140] Table 1

[0141]

[0142] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fluorescent test strip for detecting canine parvovirus, characterized in that, It includes a PVC base plate, on which a sample pad, a bonding pad, a nitrocellulose membrane, and an absorbent pad are fixed in sequence. The conjugation pad is coated with fluorescent microspheres labeled with canine parvovirus monoclonal antibody and fluorescently labeled rabbit IgG. The surface of the nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with canine parvovirus monoclonal antibody, and the control line is coated with goat anti-rabbit IgG antibody. The canine parvovirus monoclonal antibody was obtained by secretion from hybridoma cells with accession number CCTCC NO:C202355.

2. The fluorescent test strip according to claim 1, characterized in that, Within the detection line, the coating concentration of the canine parvovirus monoclonal antibody is 0.4–1.0 mg / mL, and the coating amount is 0.5–2 μL / cm. Within the quality control line, the coating concentration of the goat anti-rabbit IgG antibody is 0.4–1.0 mg / mL, and the coating amount is 0.5–2 μL / cm.

3. The fluorescent test strip according to claim 1, characterized in that, Within the detection line, the coating concentration of the canine parvovirus monoclonal antibody is 0.6 mg / mL, and the coating amount is 1 μL / cm. Within the quality control line, the coating concentration of the goat anti-rabbit IgG antibody is 0.5 mg / mL, and the coating amount is 1 μL / cm.

4. The fluorescent test strip according to claim 1, characterized in that, The amount of fluorescent microspheres in the binding pad labeled with canine parvovirus monoclonal antibody is 4–10 μg.

5. The fluorescent test strip according to claim 1, characterized in that, The amount of fluorescent microsphere-labeled canine parvovirus monoclonal antibody in the binding pad is 6 μg.

6. The fluorescent test strip according to claim 1, characterized in that, The mass ratio of fluorescently labeled canine parvovirus monoclonal antibody to rabbit anti-IgG was 1:

1.

7. The fluorescent test strip according to any one of claims 1 to 6, characterized in that, The mass ratio of fluorescent microspheres to the canine parvovirus monoclonal antibody is 1:20-40; 7. The mass ratio of fluorescent microspheres to rabbit IgG is 1:40-80.

8. The fluorescent test strip according to any one of claims 1 to 6, characterized in that, The mass ratio of fluorescent microspheres to the canine parvovirus monoclonal antibody was 1:30; the mass ratio of fluorescent microspheres to rabbit IgG was 1:

50.

9. The method for preparing the fluorescent test strip according to any one of claims 1 to 8, characterized in that, At least the following steps are included: S1. Prepare fluorescent microsphere-labeled canine parvovirus monoclonal antibody and fluorescent microsphere-labeled rabbit IgG, respectively; S2. Seal the conjugate pad and the sample pad respectively, and spray the fluorescent microsphere-labeled canine parvovirus monoclonal antibody and fluorescent microsphere-labeled rabbit IgG onto the conjugate pad; S3. Detection lines and control lines are respectively sprayed onto the surface of the nitrocellulose membrane; the detection lines are coated with canine parvovirus monoclonal antibodies; the control lines are coated with goat anti-rabbit IgG antibodies. S4. Assemble the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad to obtain the test strip.

10. The preparation method according to claim 9, characterized in that, S1 includes: After activating the fluorescent microspheres, the canine parvovirus monoclonal antibody and rabbit IgG were added for incubation, and then BSA solution was added for blocking.

11. The preparation method according to claim 10, characterized in that, The activation was performed using EDC and NHS solutions.

12. The preparation method according to claim 10, characterized in that, The incubation time is 1 to 3 hours, and the incubation temperature is 20 to 25°C.

13. The preparation method according to claim 9, characterized in that, S2 include: The sample pad was sealed using a sample pad sealing solution, which consisted of a boric acid solution containing 0.75% Tween-20, 1% PEG20000, and 3% BSA. The conjunctival pad was sealed using a conjunctival pad sealing solution, which consisted of a boric acid solution containing 3% trehalose, 2% BSA, 0.75% Tween-20, and 0.5% Triton X-100.

14. The preparation method according to claim 13, characterized in that, The sealing time is 1 to 3 hours, and the temperature is 36 to 38°C.

15. A hybridoma cell line that secretes viral monoclonal antibodies, with accession number CCTCC NO:C202355.

16. A monoclonal antibody against canine parvovirus, obtained by secretion from hybridoma cells with accession number CCTCC NO:C202355.

17. The use of the canine parvovirus monoclonal antibody as described in claim 16 in the preparation of formulations and kits for detecting canine parvovirus.

Images