A hybridoma cell strain, monoclonal antibody, test strip, kit and application thereof for feline leukemia virus

Abstract

The present application relates to a hybridoma cell strain, monoclonal antibody, test strip, kit and application thereof for feline leukemia virus, and belongs to the field of immunological technology. The present application uses feline leukemia virus P27 protein to immunize mice, and obtains specific hybridoma cell strains FeLV-06D8F10 strain and FeLV-02B6H12 strain from mouse ascites, purifies monoclonal antibodies, and applies them in detecting feline leukemia virus. The test strip described in the present application adopts the principle of colloidal gold immunochromatography, the kit detection method described in the present application is simple and easy to operate, can simultaneously detect feline leukemia virus antigens in different media of saliva and blood of cats, and realizes detection of feline leukemia virus antigens in different media. The saliva of pet cats in families is easier to collect, realizes self-detection of pet cats in families, and has a broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a hybridoma cell line, monoclonal antibody, test strip, reagent kit, and their applications against feline leukemia virus. Background Technology

[0002] Feline leukemia virus (FeLV) is a retrovirus. FeLV infection can cause diseases such as immunodeficiency, anemia, and lymphoma, and can also produce viremia, seriously endangering the life of infected cats.

[0003] It is reported that approximately 1% to 2% of stray cats in China are latent carriers of FeLV. These stray cats may act as the main reservoir hosts for FeLV, excreting it through saliva, urine, feces, and tears, which is a major route of virus spread. The infection rate is very low for domestic cats, but the infection rate is high for cats that roam freely indoors and outdoors, as biting is an effective route of transmission. Kittens are particularly susceptible to infection. FeLV can be transmitted horizontally (through secretions and excretions) and vertically (in utero). FeLV can enter a cat's body through oral-nasal inoculation, where the virus replicates in the oropharyngeal lymphatic tissue. If the body cannot effectively control the virus, it will invade the spleen, lymph nodes, intestinal epithelial cells, bladder, salivary glands, and bone marrow. At this point, the body's immune response can keep the virus latent in the bone marrow, preventing viremia and FeLV replication, but it may lead to further development of malignant tumors. If no immune response occurs, the virus may cause persistent viremia (40%), making the cat a carrier. Viremia typically appears 2–4 weeks after FeLV infection, and approximately 50% of cats with persistent viremia die within one year of FeLV infection. Therefore, early detection and diagnosis are crucial for the prevention and control of this disease. Summary of the Invention

[0004] The purpose of this invention is to provide a hybridoma cell line that produces monoclonal antibodies against feline leukemia virus, the monoclonal antibody, and its applications, so as to solve the problems existing in the prior art and provide novel antibodies for the diagnosis and prevention of feline leukemia virus.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a hybridoma cell line that produces monoclonal antibodies against feline leukemia virus, wherein the monoclonal antibody secreted by the hybridoma cell line is FeLV-06D8F10 or FeLV-02B6H12.

[0007] The amino acid sequence of the heavy chain variable region of the feline leukemia virus monoclonal antibody FeLV-02B6H12 is: QQLQQPGAAVKPGASVKSCKASGYTFPSFAMHWKQRPPGQGLEWIGIIASFPAGFTNYDKKSRATLTVDTSTSTAYMALTSDSAVYCIYSGAWAMDYWQGASVTVSS (SEQ ID NO.1), where CDR1 is SFAMH (SEQ ID NO.2), CDR2 is IIASFPAGFTNYDKKS (SEQ ID NO.3), and CDR3 is SGAWAMDY (SEQ ID NO.4).

[0008] The amino acid sequence of the light chain variable region is: DIVMQSPAAILAVSGQRKVTTCRAASSASSGKAYWFQKQKATSPKLIYKTSDSANLGVPAFSGSGSTSYSLTISREEATDAATYYCQQAGNSYLTTFGGTKLK (SEQ ID NO.5), where CDR1 is RAASSASSGKAY (SEQ ID NO.6), CDR2 is KTSDSANL (SEQ ID NO.7), and CDR3 is QQAGNSYLTT (SEQ ID NO.8);

[0009] The amino acid sequence of the heavy chain variable region of the feline leukemia virus monoclonal antibody FeLV-06D8F10 is: QVQLEEVAEVKKPAGSLKLCAGYSGFSTYAAMSWVRQQGKRLEWVATSSGGDFTYQYDSVKGRFTISALARNTLYLQYCVRSEDTAMFYCRPYYGSVGFYDVWGGPGTTTVSS (SEQ ID NO. 9), where CDR1 is AAMS (SEQ ID NO. 10), CDR2 is TSSGGDFTYQYDSVKG (SEQ ID NO. 11), and CDR3 is YYGSVGFYDV (SEQ ID NO. 12);

[0010] The amino acid sequence of the light chain variable region is: DIVLTQPELKKAETVGEKVAITCRAASGMYLHWYQQAPGQAPAKIYRTNTGASIYTESGVLDSFAGSGSEATISCMQDEVAATYYCQQGASSYAMDYFGAGGTSLALK (SEQ ID NO.13), where CDR1 is RAASGMYLH (SEQ ID NO.14), CDR2 is RTNTGASIYTES (SEQ ID NO.15), and CDR3 is QQGASSYAMDYF (SEQ ID NO.16).

[0011] In a preferred embodiment, the mark is an HRP mark or a FITC mark.

[0012] The present invention also provides the application of a monoclonal antibody against feline leukemia virus in the preparation of products for detecting feline leukemia virus.

[0013] Preferably, the method includes the step of generating the monoclonal antibody by secreting the hybridoma cell line.

[0014] In one embodiment, the product is a formulation, a kit, and / or a test strip.

[0015] The present invention also provides a test strip for detecting feline leukemia virus, the test strip containing the aforementioned monoclonal antibody.

[0016] In one embodiment, the test strip is used for detection by colloidal gold detection.

[0017] In a preferred embodiment, the test strip is prepared using FeLV-06D8F10 monoclonal antibody as the coating antibody and FeLV-02B6H12 monoclonal antibody as the labeling antibody.

[0018] Furthermore, this application provides a test strip for detecting feline leukemia virus (FLV). The test strip includes a base plate, on which, from left to right, a sample pad layer, a colloidal gold pad layer, a nitrocellulose membrane layer, and an absorbent layer are sequentially overlapped. The colloidal gold pad layer is coated with feline leukemia virus monoclonal antibody FeLV-02B6H12 labeled with colloidal gold particles. The nitrocellulose membrane layer is coated with feline leukemia virus monoclonal antibody FeLV-06D8F10 as a T detection line and with goat anti-mouse IgG as a control line. In this application, the feline leukemia virus monoclonal antibody is primarily targeted at the detection of feline leukemia virus.

[0019] The concentration of the feline leukemia virus monoclonal antibody FeLV-02B6H12 coated on the colloidal gold pad is 15 μg / mL to 25 μg / mL; the concentration of the feline leukemia virus monoclonal antibody FeLV-06D8F10 on the T detection line is 0.8 mg / mL to 1.2 mg / mL; and the concentration of the goat anti-mouse IgG on the quality control line is 0.6 mg / mL to 1.2 mg / mL.

[0020] The nitrocellulose membrane is adhered to the middle of the base plate. An absorbent layer is adhered to one side of the nitrocellulose membrane on the base plate, and a colloidal gold pad and a sample pad are adhered to the other side of the nitrocellulose membrane. One end of the sample pad presses down on one end of the colloidal gold pad by 1.0 mm to 2.0 mm, and the other end of the colloidal gold pad presses down on one end of the nitrocellulose membrane by 1.0 mm to 1.5 mm. One end of the absorbent layer presses down on the other end of the nitrocellulose membrane by 1.0 mm to 2.0 mm.

[0021] In this application, a method for preparing a test strip for detecting feline leukemia virus (FLV) is also provided for rapid and accurate detection. The method includes: S1, preparation of the colloidal gold pad 3: coating the colloidal gold pad with feline leukemia virus monoclonal antibody FeLV-02B6H12 labeled with colloidal gold particles; S2, preparation of the nitrocellulose membrane 4: coating the nitrocellulose membrane 4 with feline leukemia virus monoclonal antibody FeLV-06D8F10 as the T detection line 6 and with goat anti-mouse IgG as the control line 7; S3, preparation of the sample pad 2: firstly... The process involves preparing a treatment solution containing 0.5%–1.0% Tween-20, 0.5%–1.0% blocking agent, 0.2 mg / ml–0.5 mg / ml anti-feline erythrocyte serum, and PB buffer at pH 7.4. Next, the treatment solution is coated onto glass fibers to obtain a sample pad. S4, Test strip preparation: The sample pad layer 2 from step S3, the colloidal gold pad layer 3 from step S1, the nitrocellulose membrane layer 4 from step S2, and the absorbent layer 5 are sequentially overlapped from left to right on the base plate 1 to obtain a test strip for detecting feline leukemia virus. Further, commonly used blocking agents include BSA, sodium casein, and serum. In this application, the mass percentage of Tween-20 is further limited to 0.5%–1.0%.

[0022] In this application, as a preferred embodiment, the preparation of the colloidal gold pad 3 involves coating the colloidal gold pad with colloidal gold particles labeled with feline leukemia virus monoclonal antibody. The specific preparation method is as follows: the colloidal gold is prepared by the trisodium citrate reduction method. The feline leukemia virus monoclonal antibody is added to the colloidal gold at a ratio of 15 μg / mL to 25 μg / mL, and the mixture is allowed to stand for 10-15 min. The colloidal gold mixture is then centrifuged at 7000 rpm to 10000 rpm for 10 minutes. The supernatant is discarded, and the mixture is concentrated to obtain the colloidal gold particle-labeled feline leukemia virus monoclonal antibody, i.e., the colloidal gold complex. The colloidal gold complex is reconstituted and sprayed onto glass fiber to obtain the colloidal gold pad 3. As a further preferred embodiment, the concentration of the feline leukemia virus monoclonal antibody in the colloidal gold mixture is 2 times, i.e., the concentration of the colloidal gold complex prepared from the feline leukemia virus monoclonal antibody is preferably 40 μg / mL. In this application, as a preferred embodiment, the specific spraying process of the colloidal gold complex is as follows: glass fiber is placed in a stainless steel dish, 24 mL of colloidal gold complex is measured, and the concentration of feline leukemia virus monoclonal antibody labeled with colloidal gold particles in the colloidal gold complex is 40 μg / mL after concentration. The mixture is poured into the stainless steel dish to ensure that the colloidal gold complex is completely and uniformly absorbed. Then, it is placed in a 37°C electric heating drying oven for drying for 12 hours to obtain colloidal gold pad 3, which is stored for later use.

[0023] In this application, as a preferred embodiment, the preparation of the nitrocellulose membrane layer 4 is as follows: FeLV-06D8F10, a monoclonal antibody against feline leukemia virus (FLV), is coated onto the nitrocellulose membrane layer 4 as a T detection line 6, and goat anti-mouse IgG is coated onto it as a control line 7. The specific preparation method is as follows: The concentration of the feline leukemia virus monoclonal antibody FeLV-06D8F10 is diluted to 0.8 mg / mL to 1.2 mg / mL with 20 mmol / L PB buffer containing 1% to 2% sucrose and coated onto the nitrocellulose membrane. The membrane is then dried at 37°C for 16 hours to obtain the T detection line 6 coated with the feline leukemia virus monoclonal antibody on the nitrocellulose membrane layer 4; the concentration of the goat anti-mouse IgG is diluted to 0.6 mg / mL to 1.2 mg / mL with 20 mmol / L PB buffer containing 1% to 2% sucrose and coated onto the nitrocellulose membrane. The membrane is then dried at 37°C for 16 hours to obtain the control line 7 coated with the goat anti-mouse IgG on the nitrocellulose membrane layer 4.

[0024] In this application, as a preferred embodiment, the sample pad is prepared as follows: First, a treatment solution is prepared, containing 0.5%–1.0% Tween-20, 0.5%–1.0% blocking agent, 0.2 mg / ml–0.5 mg / ml anti-cat erythrocyte serum, and PB buffer at pH 7.4. Second, the treatment solution is coated onto glass fiber and dried to obtain the sample pad. The detailed process of preparing the treatment solution is as follows: 5.8 g of disodium hydrogen phosphate and 0.59 g of sodium dihydrogen phosphate are weighed and dissolved in 900 mL of ultrapure water, and 0.5%–1.0% of the blocking agent is added sequentially. 0% Tween-20, 0.5%–1.0% sodium caseinate, and 0.2 mg / ml–0.5 mg / ml anti-cat erythrocyte serum were thoroughly dissolved and mixed, and the pH was adjusted to 7.2–7.4. The solution was then diluted to 1 L with ultrapure water. Next, the treatment solution was coated onto glass fibers and dried. A further preferred method was to cut the glass fibers into 30 cm × 20 cm pads, coat the pads evenly with 30 ml of the treatment solution, and dry them in a 37°C electric heating oven for 16 hours to obtain sample pads for later use.

[0025] In this application, as a preferred embodiment, the test strip is prepared as follows: The sample pad 2 from step S3, the colloidal gold pad 3 from step S1, the nitrocellulose membrane 4 from step S2, and the absorbent layer 5 are sequentially overlapped from left to right on a base plate 1. The base plate 1 is a PVC board. The nitrocellulose membrane 4 is pasted in the middle of the base plate 1, and the absorbent layer 5 is pasted on one side of the base plate 1 where the nitrocellulose membrane is fixed. The colloidal gold pad 3 and the sample pad 2 are pasted on the other side of the base plate 1 where the nitrocellulose membrane is fixed. One end of the sample pad 2 presses down on one end of the colloidal gold pad 3 by 1.0mm to 2.0mm, and the other end of the colloidal gold pad 3 presses down on one end of the nitrocellulose membrane 4 by 1.0mm to 1.5mm. One end of the absorbent layer 5 presses down on the other end of the nitrocellulose membrane 4 by 1.0mm to 2.0mm. After flattening the product obtained in the above steps, it is cut into test strips 3.0mm wide, thus obtaining the test strip for detecting feline leukemia virus.

[0026] This application also provides a kit for detecting feline leukemia virus antigen, the kit comprising the test strip for detecting feline leukemia virus described above, and further comprising a blood pipette, a sampling swab, and a sample diluent for sampling; as a preferred embodiment, the sample diluent is a 20 mmol / L PBS buffer containing Tween-20 and a preservative.

[0027] Beneficial effects

[0028] This application provides a hybridoma cell line, monoclonal antibody, test strip, reagent kit, and their applications targeting feline leukemia virus (FLV). The invention utilizes FLV P27 protein to immunize mice, and specific hybridoma cell lines FeLV-06D8F10 and FeLV-02B6H12 are isolated from mouse ascites. Monoclonal antibodies are isolated and purified, and their application in detecting FLV is described. The monoclonal antibody used for detection exhibits high sensitivity and specificity, enabling accurate and rapid detection of FLV and possessing broad application prospects. It can be used in double-antibody sandwich ELISA assays for clinical samples, as well as in immunohistochemical assays for clinical diagnosis of pathological specimens. It also provides a convenient research tool for FLV research departments, with wide application value in the diagnosis and detection of FLV infection in definitive hosts, providing technical support for baseline surveys and evaluation of FLV control effectiveness. The test strips and reagent kits prepared based on this method are simple to operate and can be used in veterinary hospitals, homes, and other settings, effectively controlling the spread of the virus.

[0029] The test strip described in this application uses a colloidal gold pad labeled with monoclonal antibody FeLV-02B6H12 and a nitrocellulose membrane coated with monoclonal antibody FeLV-06D8F10 to improve reagent sensitivity and increase detection rate. The reagent kit and detection method described in this invention are simple to follow and highly operable, capable of simultaneously detecting feline leukemia virus in different media such as feline saliva and blood, achieving detection of feline leukemia virus antigen in different media. Saliva from pet cats is easier to collect in the home, enabling home testing of pet cats. Attached Figure Description

[0030] Figure 1 SDS-Page images for identifying feline leukemia virus P27 protein expression. PC1 is the BSA (1 μg) control, PC2 is the BSA (2 μg) control, M1 is the marker, NC: uninduced cell lysate, 1: cell lysate induced at 15℃ for 16 hours, 2: cell lysate induced at 37℃ for 4 hours, NC1: uninduced cell lysate supernatant, 3: cell lysate induced at 15℃ for 16 hours, 4: cell lysate induced at 37℃ for 4 hours, NC2: uninduced cell lysate precipitate, 5: cell lysate precipitate induced at 15℃ for 16 hours, 6: cell lysate precipitate induced at 37℃ for 4 hours.

[0031] Figure 2 Images of purified feline leukemia virus P27 protein, where M1 is the marker, BSA is the BSA (2μg) control, and R is the purified feline leukemia virus P27 protein.

[0032] Figure 3 This is a schematic diagram of the structure of a test strip for detecting feline leukemia virus. In the diagram: 1. Base plate; 2. Sample pad; 3. Colloidal gold pad; 4. Nitrocellulose membrane; 5. Absorbent layer; 6. T-line; 7. Control line.

[0033] Figure 4 This is a diagram illustrating when the test strip shows a negative result.

[0034] Figure 5 This is a diagram illustrating a positive result from the test strip.

[0035] Figure 6 This is a diagram illustrating when the test strip result is invalid.

[0036] Figure 7 This is a graph showing the specificity of the test strips in detecting different virus samples.

[0037] Figure 8 This is a graph showing the sensitivity results of the test strip. Detailed Implementation

[0038] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0039] Example 1: Expression and purification of feline leukemia virus p27 protein

[0040] The commercially available pET-30a(+) vector was used as the expression vector. The amino acid sequence of the feline leukemia virus P27 protein was referenced from GenBank, specifically NP_955574.1. Expression primers were designed based on its coding gene sequence. An NdeI restriction site was introduced into the upstream primer FeLVp27F, and a HindIII restriction site was introduced into the downstream primer FeLVp27R. Corresponding protective bases were added. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0041] The primer sequences are as follows:

[0042] FeLVp27-F: CATATGCCCTTGAGGGAGGGCC

[0043] FeLVp27-R:AAGCTTCAGAACTTTAGTCATCTCCTTG

[0044] The amplified PCR product was purified using a PCR cleaning kit and then double-digested with NdeI and HindIII. It was then ligated into the pET-30a(+) vector for enzyme digestion identification. Plasmids with the correct fragment were selected for further sequencing identification. The selected plasmid was named pET30a(+)-FeLVp27. The strain from which this plasmid was obtained by transforming *E. coli* BL21(DE3) competent cells was named pET30a(+)-FeLVp27-BL21(DE3).

[0045] The pET30a(+)-FeLVp27-BL21(DE3) strain and the BL21(DE3) strain containing the pET30a(+) empty vector were simultaneously inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and cultured overnight at 37°C with shaking. 5.0 mL of the overnight culture was inoculated into 500 mL of LB medium containing 25 μg / mL kanamycin and cultured at 37°C with shaking until the OD590nm value reached approximately 0.6. Amresco IPTG was then added to a final concentration of 1.0 mmol / L, and shaking was continued. After induction at 37°C for 6 h, the cells were harvested by centrifugation, the precipitate was resuspended in 10 mL of deionized water, and sonicated in an ice bath for 15 minutes at 200 W (5-second intervals). A small amount (approximately 200 μL) of the homogenized sonicated material was centrifuged, and the supernatant and precipitate were collected separately. The precipitate was resuspended in 200 μL of deionized water, and then two volumes of protein loading buffer were added to each. After boiling for 5 minutes, the mixture was centrifuged at 12,000 rpm and analyzed by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that feline leukemia virus P27 protein was efficiently expressed by E. coli BL21(DE3) under IPTG induction. The collected supernatant showed a molecular weight of 29 kDa, consistent with the expected size (see [link to original text]). Figure 1 ).

[0046] Recombinant feline leukemia virus P27 protein was purified using Ni-NTA resin affinity chromatography. The precipitate obtained after ultrasonic disruption was added to 2 mL of Buffer B solution per 100 mL of bacterial culture, mixed thoroughly, and incubated on a shaker at 220 rpm for 60 minutes. The mixture was then transferred to a 1.5 mL centrifuge tube and centrifuged repeatedly until no precipitate remained. The supernatant collected from centrifugation was transferred to a 25 mL serum bottle, and resin (Ni-NTA resin to supernatant) was added at a ratio of 1:4. The mixture was incubated at 37°C and 220 rpm for 60 minutes to allow the target protein to fully bind with the resin. The mixture was then added vertically to the column, allowing the liquid to flow out naturally. Once no liquid flowed out of the column, Buffer B was added. C. Wash twice with 1 mL of resin, then four times with 2 mL of resin, using 4 mL each time. Monitor the eluent with Coomassie Brilliant Blue staining solution continuously until the eluent no longer turns blue after being added to the staining solution. This indicates that most of the unbound proteins have been washed away. Then add Buffer E to elute the target protein. Collect the eluent and monitor it with Coomassie Brilliant Blue staining solution continuously until the eluent no longer turns blue after being added to the staining solution. This indicates that most of the target protein bound to the resin has been eluted. Perform 12% SDS-PAGE analysis on a small amount of the collected eluent. Figure 2 The results showed that high-purity feline leukemia virus P27 protein was obtained, ensuring the purity of the immunogen.

[0047] Example 2: Establishment of hybridoma cell lines

[0048] 2.1 Animal Immunization

[0049] After identification, the concentration of the immunogen was determined. For the first immunization, Freund's complete adjuvant and 100 μg / mouse protein were mixed in equal volumes and emulsified using a vortex mixer to form a water-in-oil emulsion. 6-8 week old BALB / c mice were used. After alcohol skin disinfection, the emulsified immunogen was injected subcutaneously at multiple points on the back, and the mice's condition was closely monitored. A second immunization was performed 14 days later, using Freund's incomplete adjuvant and 100 μg / mouse protein mixed in equal volumes, emulsified, and injected subcutaneously on the back. After three consecutive immunizations, blood was collected from the infraorbital sinus using standard methods, and the serum antibody titer was determined. When the titer reached 1:10000 or higher, fusion was initiated, and immunization was stopped. A pulse immunization was performed 3-4 days before fusion, with 100 μg / mouse (without adjuvant) of immunogen injected intraperitoneally. Spleen was harvested 3-4 days later for cell fusion.

[0050] 2.2 ELISA detection of polyclonal antibody titer

[0051] (1) Coating: Dilute the purified P27 protein to 1 μg / mL with coating buffer, add 100 μL to each well, incubate overnight at 4°C, discard the coating solution the next day, and wash three times with PBST.

[0052] (2) Blocking: After washing, block with 5% skim milk powder, 300 μL per well, incubate at 37°C for 2 h, discard the blocking solution, and wash three times with PBST.

[0053] (3) Primary antibody incubation: Dilute the immunized mouse serum from 1:200 to 1:409600 and add 100 μL to each well. At the same time, make negative control wells with the same dilution. Incubate at 37°C for 1 h and wash three times with PBST.

[0054] (4) Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody at a ratio of 1:2000 to each well, incubate at 37°C for 1 h, and wash three times with PBST.

[0055] (5) Color development: Add 100 μL of TMB substrate solution to each reaction well for color development, and incubate at room temperature in the dark for 10-15 min; add 50 μL of stop solution to each reaction well. Measure the OD450 of each reaction well using a microplate reader within 10 min.

[0056] 2.3SP / 20 myeloma cell resuscitation

[0057] SP / 20 myeloma cells were removed from the liquid nitrogen tank and thawed in a 37°C water bath until completely thawed. The cells were then placed in a 25cm² cell culture flask, and DMEM medium containing 10% fetal bovine serum was added. The flask was then incubated in a 37°C CO2 incubator. The cell status was observed, and the cells were passaged 24 hours before cell confluence. Cells with good growth and about 80% coverage of the bottom were selected, pipetted off with DMEM, collected in centrifuge tubes, washed once, and resuspended for later use.

[0058] 2.4 Preparation of immune spleen cells and feeder cells

[0059] One BALB / c mouse was euthanized by orbital bleeding, and the blood was collected and centrifuged to obtain serum, which was the positive serum. The mouse was disinfected by immersing it in 75% alcohol for 5 to 10 minutes. The disinfected mouse was then transferred to a laminar flow hood and fixed to a dissecting board, with the fore and hind limbs secured. The peritoneum was cut open to expose the spleen. The spleen was removed and washed twice in a petri dish to remove any adhering adipose tissue. It was then placed in a sterile homogenizer. 5 mL of DMEM basal medium was added to the homogenizer, and the cells were ground and extruded until the spleen turned white. The cell suspension was filtered into a sterile 50 mL centrifuge tube. 10 mL of culture medium was added to wash the homogenizer, and the cell suspension was filtered again into a 50 mL centrifuge tube. The liquid was centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, and the suspension was resuspended in DMEM basal medium for later use. Strict aseptic technique was maintained throughout the entire process. Feeder cells were obtained from the spleen of blank mice using the same method as for the preparation of immune spleen cells. After centrifugation, the cells were resuspended in 50 mL of HAT medium for later use.

[0060] 2.5 Cell Fusion

[0061] (1) Blow the cultured SP / 20 myeloma cells from the cell culture flask, wash twice with PBS, blow them down with DMEM, count them, and resuspend them. Adjust the cell concentration to 1 to 2 × 10⁷ cells / mL. Mix with resuspended immune spleen cells at a ratio of 1:5 to 1:10, and centrifuge at 1000 r / min for 10 min. Gently remove the supernatant, invert the centrifuge tube onto filter paper, and absorb any remaining culture medium.

[0062] (2) Rotate the centrifuge tube along the graduated surface to shake and break up the cell precipitate at the bottom of the tube, so that the cells are evenly spread at the bottom of the tube.

[0063] (3) Place the mixture of SP / 20 myeloma cells and immune spleen cells in an insulated beaker containing 37°C warm water. Add 1 mL of preheated 50% PEG dropwise to the mixed cells within 1 min while stirring with a pipette tip. Continue stirring for 30 s and let stand for 30 s.

[0064] (4) Then slowly add 10 mL of preheated DMEM medium at 37°C as the stop solution. The specific method is as follows: add 1 mL in the first minute, 2 mL in the second minute, 3 mL in the third minute, and 4 mL in the fourth minute. Each addition should be slow, with stirring while adding. Centrifuge at 1000 rpm for 10 min, and then discard the supernatant.

[0065] (5) Add 50 mL of HAT medium to a centrifuge tube and gently pipette the cells until they are resuspended and mixed. Then mix with the resuspended feeder cells and spread 200 μL per well in a 96-well plate. Incubate at 37°C in a 5% CO2 incubator.

[0066] 2.6 Screening of positive hybridoma cells

[0067] On the second day after the screening of hybridoma cells from the maternal clone, the cells were observed for contamination. By the fourth day, when obvious cell colonies were visible in the cell culture wells, a half-medium change was performed, i.e., 100 μL of medium was aspirated and replaced with 100 μL of HT medium. Observation continued until the cells reached 1 / 4 of their length, at which point the first full medium change was performed. A second full medium change was performed the night before antibody testing. Approximately 14 days after fusion, most cell culture wells showed vigorous cell growth. Microscopic examination of the cell culture wells revealed that the small, grape-like clusters of hybridoma cells became more prominent and continued to increase in size. The cell supernatant was detected using an indirect ELISA. The primary antibody was 100 μL of cell supernatant, and 100 μL of 1:1000 diluted positive and negative sera were added to the positive and negative control wells. The readings were taken at OD450. Twenty highly reactive maternal clones were selected (Elias results are shown in Table 1 below). Subcloning was then performed on these clones to obtain ten subclones with high ELISA titers (Elias results are shown in Table 2 below). Finally, two highly reactive hybridoma cells with a P / N ratio greater than 2.1 were selected.

[0068] Table 1. Screening results of positive maternal clone hybridoma cells

[0069]

[0070] Note: Coating antigen A is P27 protein, and coating antigen B is His tag protein.

[0071] Table 2. Screening results of positive subclonal hybridoma cells

[0072]

[0073] Note: Coating antigen A is a His-tagged protein, and coating antigen B is a P27 protein.

[0074] Example 3: Preparation and Identification of Feline Leukemia Virus Monoclonal Antibody

[0075] 3.1 Antibody Preparation

[0076] Female Balb / c mice aged 8–10 weeks were selected and injected intraperitoneally with 500 μl of sterile liquid paraffin per mouse. Seven days later, each mouse was injected with 500 μl of cell suspension, with a cell density of 2–4 × 10⁻⁶ cells / mL. 6 Cells / ml. Seven days after cell injection, significant abdominal distension was observed in the mice, and ascites fluid was collected using a 20-gauge needle.

[0077] The collected ascites fluid was centrifuged to remove impurities, then purified using a Protein G column. The column was equilibrated with 20 mM PBS (pH 7.2), eluted with Gly-HCl solution (pH 2.7), and neutralized with Tris-HCl (pH 9.0). The resulting monoclonal antibodies were then subjected to G25 column chromatography with the buffer replaced by PBS solution at pH 7.2. The antibody solution was concentrated using a 30 kDa ultrafiltration tube. After concentration, the FeLV-06D8F10 monoclonal antibody concentration was 2.12 mg / ml, and the FeLV-02B6H12 monoclonal antibody concentration was 2.00 mg / ml. The obtained monoclonal antibodies were aliquoted and stored at -20°C.

[0078] 3.2 Identification of Monoclonal Antibodies

[0079] 3.2.1 SDS-PAGE Identification

[0080] The antibodies were identified using the reduction method SDS-PAGE. The purity of FeLV-06D8F10 monoclonal antibody was 99%, and the purity of FeLV-02B6H12 monoclonal antibody was 99%.

[0081] After the above-mentioned antibody was cultured in a large-scale manner, it was sent to a gene company for sequencing, and the results identified it as follows:

[0082] The amino acid sequence of the heavy chain variable region of the feline leukemia virus monoclonal antibody FeLV-02B6H12 is: QQLQQPGAAVKPGASVKSCKASGYTFPSFAMHWKQRPPGQGLEWIGIIASFPAGFTNYDKKSRATLTVDTSTSTAYMALTSDSAVYCIYSGAWAMDYWQGASVTVSS (SEQ ID NO.1), where CDR1 is SFAMH (SEQ ID NO.2), CDR2 is IIASFPAGFTNYDKKS (SEQ ID NO.3), and CDR3 is SGAWAMDY (SEQ ID NO.4).

[0083] The amino acid sequence of the light chain variable region is: DIVMQSPAAILAVSGQRKVTTCRAASSASSGKAYWFQKQKATSPKLIYKTSDSANLGVPAFSGSGSTSYSLTISREEATDAATYYCQQAGNSYLTTFGGTKLK (SEQ ID NO.5), where CDR1 is RAASSASSGKAY (SEQ ID NO.6), CDR2 is KTSDSANL (SEQ ID NO.7), and CDR3 is QQAGNSYLTT (SEQ ID NO.8);

[0084] The amino acid sequence of the heavy chain variable region of the feline leukemia virus monoclonal antibody FeLV-06D8F10 is: QVQLEEVAEVKKPAGSLKLCAGYSGFSTYAAMSWVRQQGKRLEWVATSSGGDFTYQYDSVKGRFTISALARNTLYLQYCVRSEDTAMFYCRPYYGSVGFYDVWGGPGTTTVSS (SEQ ID NO. 9), where CDR1 is AAMS (SEQ ID NO. 10), CDR2 is TSSGGDFTYQYDSVKG (SEQ ID NO. 11), and CDR3 is YYGSVGFYDV (SEQ ID NO. 12);

[0085] The amino acid sequence of the light chain variable region is: DIVLTQPELKKAETVGEKVAITCRAASGMYLHWYQQAPGQAPAKIYRTNTGASIYTESGVLDSFAGSGSEATISCMQDEVAATYYCQQGASSYAMDYFGAGGTSLALK (SEQ ID NO.13), where CDR1 is RAASGMYLH (SEQ ID NO.14), CDR2 is RTNTGASIYTES (SEQ ID NO.15), and CDR3 is QQGASSYAMDYF (SEQ ID NO.16).

[0086] 3.2.3 ELISA Identification

[0087] (1) Coat the purified feline leukemia virus p27 protein onto the microplate at 1 μg / ml, 100 μl per well, and incubate at 2-8℃ for 12-15 hours.

[0088] (2) Add 300 μl of PBS (0.01 mol / L, pH 7.4, the same below) to each well, let stand for 3 minutes, discard the PBS, repeat the washing 3 times, and spin dry on the last time.

[0089] (3) Add PBS containing 1% BSA, 200 μl per well, and block at 37°C for 2 hours.

[0090] (4) Discard the blocking solution, add 300 μl of PBS to each well, let stand for 3 minutes, discard the PBS, and shake dry.

[0091] (5) Dilute the supernatant to be tested with PBS containing 1% BSA at ratios of 1:10, 1:100, 1:1000, 1:10000, and 1:100000.

[0092] (6) Discard the washing solution, add the diluted supernatant, positive control (positive serum of feline leukemia antibody from Suzhou Aiyi Animal Pharmaceutical Co., Ltd.) and negative control (clone-free culture supernatant), 100 μl per well, incubate at 37°C for 1 hour, add 300 μl of PBS to each well, let stand for 3 minutes, discard the PBS, repeat the washing 3 times, and finally spin dry.

[0093] (7) Add PBS containing 1% BSA to prepare goat anti-mouse enzyme-labeled secondary antibody diluted 1:10000, 100 μl per well, incubate at 37°C for 1 hour, discard the liquid in the well, add 300 μl PBS to each well, let stand for 3 minutes, discard the PBS, repeat the washing 5 times, and finally spin dry.

[0094] (8) Add 100 μl of TMB colorimetric solution to each reaction well and develop the color at 37°C in the dark for 10 minutes.

[0095] (9) Measure the OD650 value of each well using an ELISA reader. The test results are valid when the OD650nm value of the positive control is ≥0.6 and the OD650nm value of the negative control is <0.2. The OD650nm value of the test sample is ≥0.4 and is judged as positive, and the OD650nm value of the test sample is <0.4 and is judged as negative.

[0096] Example 4: Preparation of Diagnostic Test Strips

[0097] To quickly and accurately detect feline leukemia virus (FLV), a test strip for detecting FLV was prepared, such as... Figure 3 As shown, the test strip includes a base plate 1, on which a sample pad layer 2, a colloidal gold pad layer 3, a nitrocellulose membrane layer 4, and an absorbent layer 5 are sequentially overlapped from left to right. The colloidal gold pad layer 3 is coated with feline leukemia virus monoclonal antibody labeled with colloidal gold particles. The nitrocellulose membrane layer 4 is coated with feline leukemia virus monoclonal antibody as a T detection line 6 and with goat anti-mouse IgG as a quality control line 7.

[0098] in,

[0099] (1) Preparation of coating membrane

[0100] The FeLV-06D8F10 monoclonal antibody was diluted to 1.0 mg / ml with coating buffer (0.02 mol / L PB, 2% sucrose) and sprayed onto a nitrocellulose membrane at a rate of 1.0 μl / cm as the detection line. Commercially available goat anti-mouse IgG coating buffer was diluted to 1.0 mg / ml and sprayed onto a nitrocellulose membrane at a rate of 1.0 μl / cm as the quality control line. The prepared coated membranes were dried overnight at 37°C and stored in a sealed container.

[0101] (2) Preparation of gold-labeled pads

[0102] Adjust the pH of the colloidal gold solution to 8.0 with 0.1 mol / L K₂CO₃ solution. Add FeLV-02B6H12 monoclonal antibody to a final concentration of 20 μg / ml and let stand for 10 minutes. Add 10% BSA to the above solution to a final concentration of 0.2%. Let stand for 5 minutes, centrifuge at 10000 rpm for 10 minutes, discard the supernatant, and resuspend the precipitate in gold-labeled resuspending solution (0.02 mol / L borate, 10% sucrose, 1% BSA, pH 8.0) with 500 μl of the gold-labeled antibody precipitate obtained by centrifugation per 1 ml of colloidal gold solution. Mix well and spray onto a glass cellulose membrane at a spraying density of 40 μl / cm². 2 Dry at 37°C overnight and store in a sealed container.

[0103] (3) Test strip assembly

[0104] according to Figure 3 As shown, the components (absorbent paper, gold label pad, sample pad) are pasted onto the PVC base plate in sequence, cut into 3mm wide test strips, and assembled into the plastic casing to make the test strips.

[0105] The method for detecting feline leukemia virus antigen using the above-mentioned test strip includes the following steps:

[0106] For saliva samples, S1, place the saliva swab sample in the sample diluent and mix thoroughly; S2, add the test solution sample to the sample well of the reagent strip, let it stand for a period of time, and then determine the result. As a preferred embodiment, the detailed detection steps are as follows: S1, the sample is cat saliva. During sampling, the swab should be thoroughly moistened. Place the sampling swab in the sample diluent, and the detailed processing procedure is as follows: Place the sampling swab in the sample diluent, mix thoroughly, and then add 1 drop (40~50μL) to the sample well of the reagent strip. Let it stand for 8min~10min and then read the result. Results after a standing time greater than 15min are considered invalid.

[0107] For blood samples, S1, use a blood sample pipette to draw a certain amount of blood sample and add it to the sample well of the test strip; S2, add 1 drop (40~50μL) of sample diluent to the sample well of the test strip, let it stand for a period of time, and then determine the result. As another preferred embodiment, the detailed detection steps are as follows: S1, the sample is cat blood. During sampling, mix the blood sample thoroughly, use a pipette to draw blood to the mark, and add it to the sample well; S2, add 1 drop (40~50μL) of sample diluent to the sample well of the test strip, let it stand for 8min~10min, and then read the result. Results after a standing time of more than 15min are invalid.

[0108] The criteria for determining the results are as follows:

[0109] Negative, such as Figure 4 As shown: only one red band appears at the control line 7C, and no red band appears at the test line T of the feline leukemia virus antigen test strip, indicating that the feline leukemia virus antigen result is negative and the sample does not contain feline leukemia virus (FeLV).

[0110] Positive, such as Figure 5 As shown:

[0111] Two red bands appeared. One red band appeared at the T test line, and the other red band appeared at the control line 7C. The positive result indicates that the sample contains feline leukemia virus (FeLV).

[0112] Invalid, such as Figure 6 As shown: the absence of a red band at control line 7C indicates an operational error or reagent failure. If the result is invalid, the test must be repeated.

[0113] Specificity testing: To examine the specificity of the test strip, samples were taken from cats with feline panleukopenia, feline herpes simplex virus type 1 (FSV-1), and feline calicivirus. Samples from healthy cats served as negative controls, and samples from cats with feline leukemia served as positive controls. The tests were performed according to the methods described above, and the results are as follows: Figure 7 As shown, where, Figure 7 In the table, ① corresponds to the feline herpesvirus type 1 test result, ② corresponds to the feline panleukopenia virus test result, ③ corresponds to the feline calicivirus test result, ④ and ⑤ correspond to the test results of healthy cat samples, and ⑥ corresponds to the test result of feline leukemia virus. According to the test results, the feline panleukopenia virus, feline herpesvirus type 1, feline calicivirus, and healthy cat sample test results were negative, while the feline leukemia virus sample test result was positive.

[0114] Sensitivity detection of feline leukemia virus: Feline leukemia virus samples with concentrations of 101.0 TCID50 / mL, 102.0 TCID50 / mL, 102.5 TCID50 / mL, 103.0 TCID50 / mL, 103.5 TCID50 / mL, 104.0 TCID50 / mL, and 104.5 TCID50 / mL were tested according to the detection method described above. The test results are as follows: Figure 6 As shown, the limit of detection for the test strip is 102.5 TCID50 / mL.

[0115] Stability testing: The stability of different batches of feline leukemia virus (FLV) test strips was tested at different time points. The method involved testing with 102.5 TCID50 / mL FLV using the method described above. The test strips were stored at 37°C for 0, 1, 3, 7, 10, 14, 18, 20, 21, and 22 days. As shown in Table 3, the stability test results were the same across different batches, and all were positive.

[0116] Table 3. Results of stability test at 37℃ for different batches of feline leukemia virus test strips.

[0117]

[0118] In summary, this application provides a test strip for detecting feline leukemia virus (FLV) and a reagent kit prepared from the test strip. The test strip includes a base plate 1, on which, from left to right, a sample pad layer 2, a colloidal gold pad layer 3, a nitrocellulose membrane layer 4, and an absorbent layer 5 are sequentially overlapped. The colloidal gold pad layer 3 is coated with feline leukemia virus monoclonal antibody FeLV-02B6H12 labeled with colloidal gold particles. The nitrocellulose membrane layer 4 is coated with feline leukemia virus monoclonal antibody FeLV-06D8F10 as a T detection line 6 and with goat anti-mouse IgG as a control line 7. Detecting feline leukemia virus antigen using test strips and reagent kits is simple to operate, has good specificity, detection sensitivity, and stability, and can be used in veterinary hospitals, homes, and other places, effectively controlling the spread of the virus.

[0119] The test strip described in this application utilizes the colloidal gold immunochromatographic principle. The detection method of the reagent kit described in this invention is simple in procedure, highly operable, and can simultaneously detect feline leukemia virus antigen in different media, such as saliva and blood of felines, enabling the detection of feline leukemia virus antigen in different media. Pet cat saliva is easier to collect in the home, allowing for home testing of pet cats.

[0120] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against feline leukemia virus (FeLV), characterized in that, Place The monoclonal antibody is either feline leukemia virus monoclonal antibody FeLV-02B6H12 or feline leukemia virus monoclonal antibody FELV-06D8F10. The amino acid sequence of the heavy chain variable region of feline leukemia virus monoclonal antibody FeLV-02B6H12 is: QQLQQPGAAVKPGASVKSCKASGYTFPSFAMHWKQRPPGQGLEWIGIIASFPAGFTNYDKKSRATLTVDTSTSTAYMALTSDSAVYCIYSGAWAMDYWQGASVTVSS (SEQ ID NO.1), where CDR1 is SFAMH (SEQ ID NO.2), CDR2 is IIASFPAGFTNYDKKS (SEQ ID NO.3), and CDR3 is SGAWAMDY (SEQ ID NO.4). The amino acid sequence of the light chain variable region is: DIVMQSPAAILAVSGQRKVTTCRAASSASSGKAYWFQKQKATSPKLIYKTSDSANLGVPAFSGSGSTSYSLTISREEATDAATYYCQQAGNSYLTTFGGTKLK (SEQ ID NO.5), where CDR1 is RAASSASSGKAY (SEQ ID NO.6), CDR2 is KTSDSANL (SEQ ID NO.7), and CDR3 is QQAGNSYLTT (SEQ ID NO.8); The amino acid sequence of the heavy chain variable region of the feline leukemia virus monoclonal antibody FeLV-06D8F10 is: QVQLEEVAEVKKPAGSLKLCAGYSGFSTYAAMSWVRQQGKRLEWVATSSGGDFTYQYDSVKGRFTISALARNTLYLQYCVRSEDTAMFYCRPYYGSVGFYDVWGGPGTTTVSS (SEQ ID NO. 9), where CDR1 is AAMS (SEQ ID NO. 10), CDR2 is TSSGGDFTYQYDSVKG (SEQ ID NO. 11), and CDR3 is YYGSVGFYDV (SEQ ID NO. 12); The amino acid sequence of the light chain variable region is: DIVLTQPELKKAETVGEKVAITCRAASGMYLHWYQQAPGQAPAKIYRTNTGASIYTESGVLDSFAGSGSEATISCMQDEVAATYYCQQGASSYAMDYFGAGGTSLALK (SEQ ID NO.13), where CDR1 is RAASGMYLH (SEQ ID NO.14), CDR2 is RTNTGASIYTES (SEQ ID NO.15), and CDR3 is QQGASSYAMDYF (SEQ ID NO.16).

2. The monoclonal antibody as described in claim 1, characterized in that, The monoclonal antibody is further labeled with HRP or FITC.

3. The use of the monoclonal antibody against feline leukemia virus as described in claim 1 in the preparation of products for detecting feline leukemia virus.

4. The application as described in claim 3, characterized in that, The product is a formulation, reagent kit / or test strip.

5. A test strip for detecting feline leukemia virus, characterized in that, The test strip contains the monoclonal antibody as described in claim 1.

6. The test strip as described in claim 5, wherein the test strip comprises a base plate (1), and a sample pad layer (2), a colloidal gold pad layer (3), a nitrocellulose membrane layer (4), and an absorbent layer (5) are sequentially overlapped from left to right on the base plate (1), characterized in that, The colloidal gold pad (3) is coated with feline leukemia virus monoclonal antibody FeLV-02B6H12 labeled with colloidal gold particles; the nitrocellulose membrane (4) is coated with feline leukemia virus monoclonal antibody FeLV-06D8F10 as a T detection line (6) and coated with goat anti-mouse IgG as a quality control line (7).

7. The test strip for detecting feline leukemia virus as described in claim 6, characterized in that, The concentration of the feline leukemia virus monoclonal antibody FeLV-02B6H12 coated on the colloidal gold pad (3) is 15 μg / mL to 25 μg / mL; The concentration of the feline leukemia virus monoclonal antibody on the T test line (6) is 0.8 mg / mL to 1.2 mg / mL; The concentration of goat anti-mouse IgG mentioned above the quality control line (7) is 0.6 mg / mL to 1.2 mg / mL.

8. A method for preparing a test strip for detecting feline leukemia virus according to any one of claims 5-7, characterized in that, Includes the following steps: S1, Preparation of colloidal gold pad (3): FeLV-02B6H12, a feline leukemia virus monoclonal antibody labeled with colloidal gold particles, was coated on the colloidal gold pad; S2, Preparation of nitrocellulose membrane (4): FeLV-06D8F10 monoclonal antibody against feline leukemia virus was coated on the nitrocellulose membrane (4) as a T detection line (6), and goat anti-mouse IgG was coated on the membrane (4) as a quality control line (7). S3. Preparation of sample pad (2): First, prepare a treatment solution containing 0.5% to 1.0% Tween-20, 0.5% to 1.0% blocking agent, 0.2 mg / ml to 0.5 mg / ml cat erythrocyte blocking agent, and PB buffer at pH 7.4; second, coat the treatment solution onto glass fiber to obtain sample pad (2); S4. Preparation of test strip: On the base plate 1, from left to right, overlap the sample pad layer (2) in step S3, the colloidal gold pad layer (3) in step S1, the nitrocellulose membrane layer (4) in step S2, and the absorbent layer (5) to obtain the test strip for detecting feline leukemia virus.

9. A kit for detecting feline leukemia virus, characterized in that, The kit comprises the test strip for detecting feline leukemia virus as described in any one of claims 5-7, a blood pipette, a sampling swab, and a sample diluent.

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