Monoclonal antibody of feline calicivirus, kit and application thereof

Abstract

The invention relates to a monoclonal antibody of feline calicivirus, a kit and application thereof, and belongs to the technical field of detection of feline calicivirus. The invention aims to provide a method for detecting feline calicivirus. The invention provides a monoclonal antibody of feline calicivirus, the amino acid sequence of a heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.2, and the amino acid sequence of a light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.3. And more means are provided for detecting the feline calicivirus.

Description

Technical Field

[0001] This invention belongs to the field of feline calicivirus detection technology, specifically relating to a monoclonal antibody, reagent kit, and application of feline calicivirus. Background Technology

[0002] Feline upper respiratory tract infection syndrome (FLIS) accounts for 6.7% of all reported feline diseases. One of the leading pathogens causing this syndrome is feline calicivirus (FCV), belonging to the genus Varicellavirus in the family Caliciviridae. Clinical manifestations of FCV infection include fever, conjunctivitis, rhinitis, oral ulcers, and chronic gingivostomatitis; less commonly, skin lesions, lameness, and pneumonia may also occur. Infection with highly virulent systemic strains (VS-FCV) can cause extensive cell lysis and systemic vasculitis, often leading to rapid death.

[0003] Currently available methods for detecting feline calicivirus (FCV) include virus neutralization assays, nucleic acid detection, and antigen- or antibody-based detection. While virus neutralization assays are considered the gold standard, their labor-intensive, time-consuming nature, and reliance on cell culture limit their application in large-scale sample screening. Polymerase chain reaction (PCR) and quantitative real-time PCR offer advantages such as high throughput, high sensitivity, and high specificity, but require expensive equipment and specialized technical knowledge. Isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), provide rapid, equipment-free alternatives for nucleic acid detection; however, they are not suitable for applications such as vaccine immunogenicity assessment. For antigen detection, immunochromatographic strips are widely used for point-of-care diagnosis in companion animals, but their sensitivity remains unsatisfactory. Among serological methods, enzyme-linked immunosorbent assay (ELISA) is commonly used to assess vaccine-induced immune responses. Indirect ELISA is cost-effective and simple to perform, but its specificity may be reduced due to serum heterogeneity. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting feline calicivirus.

[0005] The present invention provides a monoclonal antibody against feline calicivirus, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.3.

[0006] This invention provides a gene that encodes the aforementioned monoclonal antibody.

[0007] The present invention provides a recombinant vector containing the above-mentioned genes.

[0008] The present invention provides a recombinant host cell containing the above-described recombinant vector.

[0009] This invention provides the application of the above-mentioned monoclonal antibody in the preparation of a kit for detecting feline calicivirus, detecting antibodies against feline calicivirus, or detecting the efficacy of vaccines against feline calicivirus infection.

[0010] This invention provides the application of the above-mentioned gene in the preparation of a kit for detecting feline calicivirus, detecting antibodies against feline calicivirus, or detecting the efficacy of vaccines against feline calicivirus infection.

[0011] This invention provides the application of the above-mentioned recombinant vector in the preparation of a kit for detecting feline calicivirus, detecting antibodies against feline calicivirus, or detecting the efficacy of vaccines against feline calicivirus infection.

[0012] This invention provides the application of the above-mentioned host cells in the preparation of a kit for detecting feline calicivirus, detecting antibodies against feline calicivirus, or detecting the efficacy of vaccines against feline calicivirus infection.

[0013] To further specify, the kit is a blocking ELISA kit.

[0014] The present invention provides a kit for detecting feline calicivirus, the kit comprising the above-mentioned monoclonal antibody.

[0015] Beneficial Effects: This study successfully prepared nine monoclonal antibodies (MAbs) capable of recognizing linear and conformational epitopes of the FCV VP1 protein. Among them, MAb 8F9 (subtype IgG2a / κ) exhibited the strongest blocking activity and was selected to establish a blocking ELISA for detecting feline calicivirus antibodies. The developed detection method is simple to operate, yields stable and reproducible results, and is highly suitable for large-scale FCV antibody screening, post-vaccination immune response assessment, and antibody monitoring in SPF cat populations. In summary, this method provides reliable technical support for the control of FCV-related diseases and the health management of cat populations. In this study, the E. coli prokaryotic expression system was used to achieve high-yield and high-purity expression of the FCV VP1 protein, thereby minimizing non-specific interference associated with crude whole virus antigen. After three immunizations, indirect ELISA showed a mouse serum titer exceeding 1:51,200 (Figure 2), and indirect immunofluorescence assay confirmed a titer greater than 1:1,600, indicating that the recombinant VP1 protein has strong immunogenicity. Nine hybridoma cell lines secreting blocking monoclonal antibodies (MAbs) were generated from mice immunized with VP1 using cell fusion technology. Ascites was induced in BALB / c mice via intraperitoneal inoculation, and the MAbs were purified to high purity using rProtein G Beads affinity chromatography. These MAbs, acting as competitive antibodies, bind to the purified VP1 protein as a coating antigen, specifically recognizing different epitopes within VP1 and effectively blocking the non-specific binding of unrelated antibodies in the test serum. This strategy significantly improves the specificity and accuracy of the resulting immunoassay method. Attached Figure Description

[0016] Figure 1. Recombinant pMAL C5x Construction of the VP1 vector and purification and identification of the VP1 protein; (A) PCR amplification of the FCV VP1 fragment; (B) Recombinant pMAL C5x The VP1 vector was double-digested with NdeI and EcoRI. (C) SDS-PAGE analysis of purified recombinant VP1 protein. (D) Western blot analysis of recombinant VP1 protein using FCV-positive serum; Figure 2. Serum antibody titers in mice after three FCV VP1 protein immunizations (measured by indirect ELISA). Mice 1–5: immunized with recombinant VP1 protein; blank control: not immunized. Figure 3. IFA detection of monoclonal antibody reactivity: positive control: serum from VP1-immunized mice; negative control: DMEM medium; others: supernatant from nine monoclonal hybridoma cell lines; Figure 4. Western blot analysis of nine monoclonal antibodies against recombinant FCV VP1 protein; Figure 5. Subclass analysis of nine monoclonal antibodies using a commercial antibody typing kit; Figure 6. Blocking activity of nine monoclonal antibodies against FCV-positive, positive, and weakly positive serum samples; Figure 7. Purification and HRP labeling analysis of monoclonal antibody 8F9; (A) SDS-PAGE of antibody purified by rProtein G Beads gravity column; (B) Direct ELISA analysis of HRP-conjugated 8F9 monoclonal antibody titer. Figure 8. Optimization results of reaction conditions for the blocking ELISA method; (A) Optimal antigen coating concentration: 1 μg / mL; (B) Optimal blocking agent: 1% BSA; (C) Optimal blocking time: 90 minutes; (D) Optimal dilution of the test serum: 1:8; (E) Optimal incubation time of the serum: 30 minutes; (F) Optimal dilution of HRP-mAb 8F9: 1:400; (G) Optimal incubation time of HRP-mAb 8F9: 30 minutes; (H) Optimal color development time: 15 minutes. Figure 9. Detection and ROC analysis of cat serum samples; (A) Blocking ELISA results of 86 FCV-negative and 78 FCV-positive serum samples; (B) ROC curve analysis of 164 serum samples. Figure 10. Assessment of specificity and analytical sensitivity of blocking ELISA; (A) assessment of cross-reactivity with positive sera of other common feline pathogens; (B) determination of the maximum detectable dilution of FCV strongly positive, FCV positive and FCV weakly positive sera. Detailed Implementation

[0017] Example 1. Expression and purification of recombinant FCV VP1 protein 1. Viral RNA was extracted from feline calicivirus (FCV) using a commercially available viral RNA extraction kit (Tiangen, China) and reverse transcribed into cDNA using the HiScript IV first-strand cDNA synthesis kit (+gDNA wiper) (Novizan, China). Primers designed for the VP1 gene (GenBank ID: OQ718383.1) are as follows: Upstream primer: 5′-gagggaaggatttcacatatgATGTGCTCAACCTGCGCTAAC-3′ (SEQ ID NO.1); Downstream primer: 5′-acctgcagggaattcggatccTTATACCGCTCCTAATATTTGAGGC-3′ (SEQ ID NO.2). The VP1 gene fragment was amplified using PrimeSTAR® MaxDNA Polymerase Ver.2 (Takara, China) and verified by 1% agarose gel electrophoresis. pMAL The C5x(+) vector was double-digested with NdeI and EcoRI (NEB, USA). Both the PCR product and the linearized vector were gel-extracted (Tiangen, China) and assembled using DNA Assembly Mix Ultra (Yisheng Biotechnology, China) to generate the recombinant plasmid pMAL. C5x VP1. After transformation into E. coli BL21(DE3) competent cells, transformants were screened on LB agar plates supplemented with ampicillin and cultured overnight at 37°C. Single colonies were selected for colony PCR and DNA sequencing to confirm correct construction. For protein expression, positive clones were inoculated 1:100 into LB medium containing ampicillin and cultured at 37°C with shaking until OD600 reached 0.5–0.6. Transformation was performed using 1 mM isopropyl β-D-coated lysate. D 1 Thiogalactoside (IPTG) induced protein expression for 5 hours at 37°C and 200 rpm. Cells were collected by centrifugation and resuspended in lysis buffer (50 mM Tris) containing 0.5 mM benzyl sulfonyl fluoride (PMSF). The protein was lysed in HCl (100 mM NaCl, 5% glycerol, pH 8.0) and sonicated. After centrifugation, the supernatant was collected and loaded onto a Dextrinin Beads 6FF gravity column (Huide Biotech, China) to purify the VP1 fusion protein with the MBP tag. The MBP tag was then cleaved using Factor Xa protease (NEB, USA). The protein was purified by SDS-PAGE. PAGE and Western blot analyses confirmed protein purity and characteristics.

[0018] Results: To simultaneously obtain the immunogen and coating antigen, the VP1 protein of feline calicivirus (FCV) was expressed using a prokaryotic system. The VP1 gene was amplified from FCV cDNA, yielding a product of approximately 1992 bp, which was confirmed by 1% agarose gel electrophoresis (Figure 1A) to be consistent with the expected size. The amplified fragment was cloned into pMAL. The C5x(+) expression vector was used. After double digestion with NdeI and EcoRI, the vector backbone and insert fragment were released (Figure 1B), confirming successful plasmid construction. Following transformation, induced expression, and purification, SDS-PAGE analysis showed that the recombinant protein had a molecular weight of approximately 73.4 kDa and a purity of approximately 90%. Figure 1 C). Western blot analysis using FCV-positive serum revealed specific bands at the same molecular weight (C). Figure 1 (D) indicates that the purified recombinant VP1 protein retained its antigenicity.

[0019] 2. Screening of monoclonal antibodies and evaluation of their blocking efficacy VP1 protein was emulsified with MONTANIDE ISA 201 VG adjuvant (Sypico (Shanghai) Specialty Chemicals Co., Ltd., China) at a 1:1 (v / v) ratio. Each mouse received 50 µg of VP1 protein per immunization, administered via intramuscular injection at 14-day intervals. Serum samples were collected after the third immunization, and antibody titers were assessed by indirect ELISA (using purified VP1 as the coating antigen) and indirect immunofluorescence assay (IFA). Mice with satisfactory serum titers received a final booster immunization via intraperitoneal injection of 50 µg of protein 3–5 days before cell fusion. Spleen cells harvested from immunized mice were fused with SP2 / 0 myeloma cells according to a pre-defined protocol [MENG L et al., 2025]. Approximately 10 days post-fusion, antigen-specific antibodies were screened for in the supernatant of wells containing hybridoma cell clusters using IFA. Positive wells were then sorted by flow cytometry. After clone amplification, the culture supernatant of positive hybridomas was collected for further identification. To evaluate the blocking activity of each monoclonal antibody (mAb), VP1 protein was coated onto an ELISA plate. Feline calicivirus positive or negative reference serum was added to each well as a primary antibody for incubation, followed by the corresponding mAb supernatant and goat anti-mouse IgG-HRP as secondary and third antibodies, respectively. Absorbance at 450 nm was measured, and the blocking rate of each mAb was calculated. The clone exhibiting the strongest blocking effect was selected for subsequent experiments.

[0020] Results: To assess the immunogenicity of recombinant VP1 protein, mouse serum was collected 14 days after the third immunization and analyzed by indirect ELISA and IFA. The results showed that the serum titer by indirect ELISA exceeded 1:51,200 (…). Figure 2The IFA titer exceeded 1:1,600, confirming its strong immunogenicity and suitability for hybridoma preparation. Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells. Nine hybridoma cell lines stably secreting anti-FCV VP1 monoclonal antibodies (MAb) were identified through IFA screening (Figure 3). Western blot analysis using the supernatant of the nine MAb lines as primary antibodies showed that all antibodies recognized the denatured recombinant VP1 protein, producing clear bands (Figure 4). Combining the Western blot and IFA results, it was shown that the selected MAb recognized both linear and conformational epitopes. Subclass analysis using a commercial MAb genotyping kit revealed that three MAb lines were IgG1 / κ, two were IgG2a / κ, and one each were IgG1 / λ, IgG2b / κ, IgG2b / λ, and IgM / κ (Figure 5). The blocking activity of each MAb was evaluated in a competitive ELISA format: recombinant VP1 was coated as the antigen, FCV-positive serum was used as the primary antibody, the corresponding MAb was used as the secondary antibody, and goat anti-mouse IgG-HRP was used as the triad antibody. Seven of the nine MAbs showed blocking activity, with MAb 8F9 showing the highest blocking rate (Figure 6). Therefore, MAb 8F9 was selected for the establishment of subsequent detection methods.

[0021] Complete heavy chain (447aa in total length) (SEQ ID NO.3): QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDVKRYNPALKSRLTISKDTSSSQVFLKIASVDTADTATYYCARIDRGNYGFDYWG QGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK; Complete light chain (219aa in total length) (SEQ ID NO.4): DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSNQNKYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYNSYPWTFGGGTKLE IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0022] 3. Monoclonal antibody purification and labeling Ascites was produced using 7-8 week old female BALB / c mice. Mice were first primed via intraperitoneal injection of 0.5 mL of mineral oil (Alphabio, China). Three to five days later, each mouse was injected intraperitoneally with approximately 1.5 × 10⁻⁶ ppm of mineral oil. 6Hybridoma cells were collected. Ascites fluid was collected 5–7 days after inoculation, clarified by centrifugation (10,000 rpm, 10 min, 4°C), and the supernatant was stored at -80°C for later use. Monoclonal antibodies (MAb) were purified from the ascites fluid using an rProtein G Beads gravity column (Huide Biotechnology, China) according to the manufacturer's instructions. Indirect ELISA was performed to determine the MAb titer: purified VP1 protein was coated as the capture antigen; serially diluted MAb (starting from 1:200, 2-fold dilution) was used as the primary antibody; mouse ascites fluid injected with SP2 / 0 cells was used as a negative control; and goat anti-mouse IgG-HRP was used as the secondary antibody. Finally, the purified MAb was conjugated with horseradish peroxidase (HRP) using a commercial labeling kit (Abcam) according to the manufacturer's protocol.

[0023] 4. Indirect immunofluorescence assay (IFA) Cat kidney cells (F81) were seeded into 96-well plates. When the cells reached approximately 80% confluence, the culture medium was removed and replaced with feline calicivirus (FCV) inoculum. The plates were incubated at 37°C and 5% CO2 for 4 hours until visible cytopathic effect (CPE) appeared. The viral supernatant was discarded, and the cells were gently washed three times with PBS. 100 µL of pure ethanol (on ice) was added to each well, and the cells were fixed at 4°C for 30 minutes. After fixation, the ethanol was removed, and the cells were washed three times with PBS. Then, 50 µL of hybridoma cell culture supernatant was added to each well, and the cells were incubated at 37°C for 60 minutes, followed by washing three times with PBST. Subsequently, 50 µL of FITC-labeled goat anti-mouse IgG diluted in PBS was added to each well, and the cells were incubated at 37°C in the dark for 60 minutes. Finally, the cells were washed three times with PBST, and the nuclei were counterstained with DAPI. Fluorescence signals were observed and recorded using a fluorescence microscope.

[0024] 5. Western Blot Analysis Recombinant VP1 protein was mixed with 6× SDS-PAGE loading buffer and boiled at 100°C for 10 minutes. 10 µL of denatured sample was loaded into each well. Electrophoresis was performed on a polyacrylamide gel at 80 V for 30 minutes, followed by electrophoresis at 120 V for 60 minutes. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using standard wet transfer conditions. The membrane was blocked in PBST containing 5% skim milk powder at room temperature for 1 hour, followed by washing three times with PBST (10 minutes each time). The membrane was then incubated with monoclonal antibody (primary antibody) diluted with blocking buffer at room temperature for 1 hour, followed by washing three times with PBST. Next, the membrane was incubated with goat anti-mouse IgG (H+L)-HRP conjugate (secondary antibody) at room temperature in the dark for 1 hour, and finally washed three times with PBST. Protein bands were visualized using a chemiluminescence imaging system.

[0025] Results: To scale up antibody production, female BALB / c mice (7-8 weeks old) were first primed via intraperitoneal injection of mineral oil. One week later, they were injected intraperitoneally with the 8F9 hybridoma cell line. Ascites fluid was harvested 5-7 days post-inoculation and purified using a gravity column with rProtein G Beads, followed by conjugation with horseradish peroxidase (HRP). SDS-PAGE analysis of the purified antibody under denaturing conditions showed high purity, displaying clear bands corresponding to the heavy chain (~55 kDa) and light chain (~25 kDa) (Figure 7A). The titer of the HRP-conjugated monoclonal antibody was determined by direct ELISA, reaching 1:6,400 (Figure 7B).

[0026] Example 2. Establishment and optimization of blocking ELISA 1. Optimal assay conditions were determined using a checkerboard titration design. Purified VP1 protein was diluted to concentrations ranging from 0.5 to 5 μg / mL with carbonate-bicarbonate buffer (pH 9.6), and 100 μL of each concentration was coated onto each well of a 96-well ELISA plate and incubated overnight at 4°C. The plates were then washed three times with PBST. For blocking, different blocking buffers were evaluated, including 1% BSA, 1–5% skim milk powder, and 1% gelatin in PBST. Blocking was performed at 37°C for 30–150 minutes, followed by three washes with PBST. Feline calicivirus positive and negative reference sera were serially diluted (from 1:2 to 1:64), and 100 μL was added to each well, incubating at 37°C for 15–150 minutes. After washing, add 100 μL / well of HRP-labeled monoclonal antibody diluted 1:100 to 1:3200 and incubate at 37°C for 15–150 min. Add 100 μL of TMB substrate to each well and incubate at room temperature for 5–30 min to initiate the colorimetric reaction. Stop the reaction by adding 50 μL of 2 M sulfuric acid to each well. Measure the absorbance of the positive control (P) and negative control (N) at 450 nm. Select the conditions that produce the lowest P / N ratio as the optimal conditions for subsequent assays.

[0027] 2. Determination of critical values ​​and evaluation of specificity, sensitivity, and repeatability. Eighty-six IFA-certified feline calicivirus (FCV) negative serum samples and 78 FCV positive serum samples were analyzed using an optimized blocking ELISA. The sample / negative control ratio (P / N) and inhibition rate (PI) were calculated for each sample. Receiver operating characteristic (ROC) curve analysis was performed using GraphPad Prism 8 and SPSS Statistics 17.0 to determine the cutoff value for the assay. The Youden index was derived from the specificity and sensitivity data, and the PI value corresponding to the highest Youden index was selected as the diagnostic cutoff value. To assess specificity, the method was applied to known positive sera for other common feline pathogens, including feline herpesvirus type I, feline panleukopenia virus, Salmonella, Escherichia coli, and Mycoplasma, using FCV positive and FCV negative sera as controls. To assess analytical sensitivity, serial dilutions (1:2 to 1:1024) of strongly positive, positive, weakly positive, and negative FCV sera were tested to determine the highest detectable dilution. For reproducible testing, three independent batches of ELISA plates (10 plates per batch) were prepared. Five plates from each batch were randomly selected and tested with the same FCV serological group (strongly positive, positive, weakly positive, and negative) in intra-batch and inter-batch runs. The coefficient of variation (CV) was calculated to assess the reproducibility of the assays.

[0028] 3. Clinical Sample Analysis The optimized blocking ELISA established in this study and the DB22 / T 3035-based method were used. The indirect ELISA method described in the 2019 document "ELISA Method for Detection of Feline Calicivirus in Experimental Use" was used to test a total of 105 clinical serum samples from cats in parallel. The results obtained by the two methods were compared, and the overall concordance rate between the two methods was calculated.

[0029] 4. Statistical Analysis Statistical analyses were performed using GraphPad Prism 8 (San Diego, CA, USA) and SPSS Statistics 17.0. Cutoff values ​​for blocking ELISA were determined based on sensitivity, specificity, Youden index, and area under the receiver operating characteristic (AUC) curve. Repeatability of assays was assessed using mean, standard deviation, and coefficient of variation. Comparability between blocking ELISA and commercially available indirect ELISA kits was evaluated using Cohen's kappa coefficient.

[0030] Results: To improve the performance of the blocking ELISA, key reaction parameters were optimized. The following conditions were selected based on obtaining the lowest positive to negative control (P / N) ratio: Coating antigen concentration: 1.0 μg / mL recombinant VP1 (Figure 8A); Blocking agent: 1% BSA (Figure 8B); Blocking time: 90 min (Figure 8C); Serum dilution: 1:8 (Figure 8D); Serum incubation time: 37°C for 30 min (Figure 8E); HRP-labeled 8F9 MAb dilution: 1:400 (Figure 8F); MAb incubation time: 37°C for 30 min (Figure 8G); TMB substrate incubation time: 15 min at room temperature (Figure 8H).

[0031] (1) Determination of the diagnostic threshold for blocking ELISA Under optimized conditions, blocking ELISA was applied to 86 feline calicivirus (FCV) negative serum samples and 78 FCV positive serum samples. Diagnostic performance was assessed by receiver operating characteristic (ROC) curve analysis (SPSS software). The optimal cutoff value for inhibition rate (PI) was determined to be 23.5%, at which point the sensitivity was 94.9%, the specificity was 93.02%, and the corresponding Youden index was 0.879. The area under the ROC curve (AUC) was 0.991 (95% CI: 0.9817–0.9994, P<0.0001) (Figures 9A, 9B). Therefore, under effective assay conditions, serum samples with a PI value <23.5% were considered FCV antibody negative, while serum samples with a PI value ≥23.5% were considered FCV antibody positive.

[0032] (2) Performance evaluation of blocking ELISA To evaluate the specificity of the established blocking ELISA, positive sera against other common feline pathogens (feline herpesvirus type 1 (FHV-1), feline panleukopenia virus (FPV), Salmonella, Escherichia coli, and Mycoplasma feli) were tested under optimized assay conditions. FCV-positive and FCV-negative sera served as controls. The assay specifically detected FCV antibodies and showed no cross-reactivity with positive sera against the other five pathogens (Figure 10A), confirming its high specificity. Analytical sensitivity was assessed by testing a series of two-fold dilutions of FCV-strongly positive, FCV-positive, FCV-weakly positive, and FCV-negative sera. The highest detectable dilution for strongly positive sera was 1:256, for positive sera 1:128, for weakly positive sera 1:32, while negative sera showed no reactivity (Figure 10B). To assess assay reproducibility, three independent batches of ELISA plates were prepared. Five plates were randomly selected from each batch, and the same FCV serological group (strongly positive, positive, weakly positive, and negative) was tested in intra-batch and inter-batch runs. The coefficients of variation (CV) for intra-batch tests were 0.96%, 0.83%, 2.88%, and 5.38%, respectively; the CVs for inter-batch tests were 0.79%, 0.71%, 3.14%, and 7.05%, respectively (Table 1). All CV values ​​were below 10%, indicating that the established blocking ELISA had good reproducibility and stability.

[0033] Table 1. Reproducibility validation of the blocking ELISA method

[0034] Example 3. Clinical Application To evaluate the clinical applicability of the established blocking ELISA, 105 feline clinical serum samples were tested in parallel using the optimized blocking ELISA and the indirect ELISA antibody detection method described in DB22 / T3035–2019 "ELISA Method for Detection of Feline Calicivirus Antibodies in Laboratory Use". The concordance between the two detection methods was assessed. Results showed that the seropositivity rate for feline calicivirus (FCV) in the tested population was 24.76% (26 / 105). The overall concordance rate between the blocking ELISA and the reference indirect ELISA was 96.55% (Table 2), with a Cohen's kappa value of 0.9, indicating excellent concordance. These findings demonstrate that the blocking ELISA developed in this study is a reliable tool for FCV antibody detection and assessment of vaccine immune responses.

[0035] Table 2 Comparison of clinical sample cat serum detection and indirect ELISA antibody detection methods.

[0036]

Claims

1. A monoclonal antibody to feline calicivirus, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 2, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.

3.

2. A gene, characterized in that, The gene of claim 2 encodes the monoclonal antibody of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector comprises the gene of claim 2.

4. A recombinant host cell, characterized in that, The host cell contains the recombinant vector of claim 3.

5. The monoclonal antibody of claim 1 for use in the preparation of a kit for detecting feline calicivirus, detecting antibodies against feline calicivirus, or detecting the effect of a vaccine against feline calicivirus infection.

6. The gene of claim 2 for use in the preparation of a kit for detecting feline calicivirus, detecting antibodies against feline calicivirus, or detecting the effect of a vaccine against feline calicivirus infection.

7. The recombinant vector of claim 3 for use in the preparation of a kit for detecting feline calicivirus, detecting antibodies against feline calicivirus, or detecting the effect of a vaccine against feline calicivirus infection.

8. The host cell of claim 4 for use in the preparation of a kit for detecting feline calicivirus, detecting antibodies against feline calicivirus, or detecting the effect of a vaccine against feline calicivirus infection.

9. Use according to claim 8, characterized in that, The kit is a blocking ELISA kit.

10. A kit for detecting feline calicivirus, characterized by, The kit comprises the monoclonal antibody of claim 1.

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