Application of VP1 monoclonal antibody and polyclonal antibody and latex microsphere immunochromatography based on the same in detection of feline calicivirus

Abstract

The application relates to a VP1 monoclonal antibody and a polyclonal antibody and application of latex microsphere immunochromatography based on the same in detection of feline calicivirus. The VP1 protein is expressed through a prokaryotic expression system, and corresponding polyclonal antibodies and monoclonal antibodies are prepared. The feline calicivirus latex microsphere immunochromatography detection test paper constructed by the application has the characteristics of simple operation, rapid reaction, strong specificity and good stability, can be used for early diagnosis of feline calicivirus infection, and has important significance for rapid screening of feline calicivirus.

Description

Technical Field

[0001] This invention relates to the field of feline calicivirus technology, and in particular to a VP1 monoclonal antibody and a polyclonal antibody and their application in the detection of feline calicivirus based on latex microsphere immunochromatography. Background Technology

[0002] Feline calicivirus (FCV) belongs to the family Caliciviridae and the genus Vesivirus. It is a non-enveloped single-stranded positive-sense RNA virus with an icosahedral symmetry structure, a diameter of about 30-40 nm, and 32 cup-shaped depressions on its surface. It is prone to genetic mutation and antigenic variation. The FCV genome is about 7.7 kb in length and contains three main open reading frames (ORFs). Among them, the capsid protein VP1 encoded by ORF2 is the main structural protein of the virus and plays an important role in viral replication, infection and induction of neutralizing antibodies. The structure of this protein is mainly composed of six regions: A, B, C, D, E and F. Regions A, B, D and F of the VP1 genome are relatively conserved, while regions C and E are hypervariable. Therefore, there are significant antigenic differences between different strains, which poses a great challenge to vaccine development [3]. Research on VP1 and its truncated recombinant protein has provided a basis for vaccine development.

[0003] Feline calicivirus (FCV) has become a major infectious pathogen threatening the health of felines worldwide. In the early stages of FCV infection, clinical manifestations often include upper respiratory symptoms such as sneezing, runny nose, conjunctivitis, and lethargy. This is followed by the most typical symptom of FCV infection: oral ulcers (commonly on the tongue and hard palate), leading to drooling, anorexia, and difficulty chewing. Certain virulent strains can further trigger severe systemic reactions, causing widespread vascular endothelial cell damage and severe necrotizing vasculitis, while also leading to increased vascular permeability, tissue edema, interstitial pneumonia, and hemorrhagic necrosis in multiple organs, including the liver and pancreas, ultimately resulting in death due to disseminated intravascular coagulation and multiple organ failure. The virulence varies greatly among different FCV strains; some highly pathogenic strains already identified in my country, such as FCV-VSD, can cause severe systemic clinical symptoms. The high morbidity and mortality rates caused by virulent strains pose a serious threat to the health of kittens. Furthermore, the high mutation rate of feline calicivirus poses a direct challenge to its detection.

[0004] Currently, laboratory diagnosis of feline calicivirus still heavily relies on molecular biology techniques such as PCR. However, the rapid evolution of the viral genome, especially the hypervariable region encoding the capsid protein, often leads to mutations in the primer binding sites used for PCR detection, significantly reducing detection sensitivity. Furthermore, these detection methods are time-consuming, require sophisticated equipment, and are complex to operate, making them difficult to rapidly implement in grassroots veterinary hospitals, animal shelters, or at outbreak sites. Immunochromatographic detection methods for feline calicivirus are relatively few; colloidal gold and fluorescent microspheres are commonly used as signal markers in immunochromatographic test strips both domestically and internationally. Summary of the Invention

[0005] To address the aforementioned technical challenges, this invention provides a rapid detection method for feline calicivirus (FCV) antigens using latex microsphere-based immunochromatographic test strips. This method enables simple, rapid, and visual detection of FCV antigens. The invention utilizes a prokaryotic expression system to express the VP1 protein and prepare corresponding polyclonal and monoclonal antibodies. This latex microsphere-based immunochromatographic detection method maintains high accuracy while significantly improving convenience and applicability, providing strong technical support for early diagnosis, timely intervention, and effective control of virus transmission. It also holds significant practical importance for enhancing grassroots FCV prevention and control capabilities.

[0006] The first object of the present invention is to provide a VP1 antigen, the amino acid sequence of which is shown in SEQ ID NO 1.

[0007] A second objective of the present invention is to provide a polyclonal antibody pAb-VP1 and a monoclonal antibody 2B9 against the said VP1 antigen.

[0008] A third objective of this invention is to provide a test strip comprising the aforementioned polyclonal antibody pAb-VP1 and monoclonal antibody 2B9.

[0009] In some embodiments of the present invention, the polyclonal antibody pAb-VP1 is coupled with red latex microspheres as an immune probe, and the monoclonal antibody 2B9 is used as a detection line antibody. In some embodiments of the present invention, the polyclonal antibody pAb-VP1 conjugated red latex microspheres are prepared by the following method: A latex microsphere suspension containing a coupling buffer is provided; EDC solution and NHS solution were added to the latex microsphere suspension, and the mixture was ultrasonically mixed and then activated by rotation. After activation, the solid phase was separated and washed with coupling buffer. Coupling buffer was added and the microspheres were ultrasonically mixed to obtain a microsphere solution. The polyclonal antibody pAb-VP1 was dispersed in a coupling buffer to obtain a coupling solution containing the antibody. The antibody-containing conjugation solution was mixed with the microsphere solution and then rotated for conjugation. After the reaction was complete, the solid phase was separated and washed to obtain polyclonal antibody pAb-VP1 conjugated red latex microspheres.

[0010] In some embodiments of the present invention, the concentration of the antibody-containing conjugate is 0.1-0.4 μg / μl, preferably 0.2 μg / μL.

[0011] In some embodiments of the present invention, the conditions for rotational activation are: rotational activation at 37°C and 180 r / min for 45 min; the conditions for rotational coupling are: rotational coupling at 37°C and 180 r / min for 2 h.

[0012] A fourth objective of this invention is to provide a kit comprising the aforementioned polyclonal antibody and / or monoclonal antibody, or the aforementioned test strip.

[0013] In some embodiments of the present invention, the double-antibody sandwich ELISA kit is described.

[0014] A fifth object of the present invention is to provide the use of the polyclonal antibody or monoclonal antibody, the test strip or the kit in the preparation of a test for feline calicivirus.

[0015] The technical solution of the present invention has the following advantages compared with the prior art: This invention is based on the double-antibody sandwich principle, using polyclonal antibody pAb-VP1 conjugated with red latex microspheres as an immunoprobe and monoclonal antibody 2B9 as a detection line antibody. Performance verification of the test strip with optimized process parameters showed that the test strip had a detection sensitivity of 22.66 ng / mL for purified VP1 protein; it showed no cross-reactivity with feline herpesvirus, feline parvovirus, feline infectious peritonitis virus, and normal cell lysates, exhibiting good specificity; intra- and inter-batch repeatability was stable; and accelerated degradation experiments showed that it could be stably stored at 37°C for more than 25 days. Using this test strip and RT-PCR to test 96 clinical swab samples in parallel, the overall concordance rate was 90.6% (87 / 96), with a positive concordance rate of 85.3% (29 / 34) and a negative concordance rate of 93.5% (58 / 62). The results show that the feline calicivirus latex microsphere immunochromatographic test strip constructed in this invention has the characteristics of simple operation, rapid reaction, strong specificity and good stability. It can be used for the early diagnosis of feline calicivirus infection and is of great significance for rapid screening of feline calicivirus. Attached Figure Description

[0016] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This invention relates to PCR amplification of the VP1 gene.

[0017] Figure 2 This invention relates to the purification and Western blotting (WB) identification of VP1 protein; wherein, A: VP1 protein purification and staining results, M is the protein marker, 1 is the precipitated protein, 2 is the washing buffer, and 3 is the elution buffer; B: Western blotting identification results, M is the protein marker, and 1 is the VP1 protein.

[0018] Figure 3 This invention relates to the identification and titer detection of polyclonal antibodies; wherein, A: IFA identification result; 1: serum of mouse No. 1; 2: serum of mouse No. 2; 3: serum of mouse No. 3; NC: negative mouse serum; Scale Bar = 100 μm; B: Western-Blot identification result; M: marker; 1: antigen protein VP1; 2: negative serum; C: polyclonal antibody titer.

[0019] Figure 4 This invention relates to the identification and titer detection of monoclonal antibody ascites fluid; wherein, A: IFA identification results; 1, 2, and 3 are purified monoclonal antibody ascites fluid samples 2B9, 2F7, and 3C6, respectively; 4 is positive serum; NC is negative mouse serum; Scale Bar = 100 μm; B: Western-Blot identification results; M: marker; 1, 2, and 3 are purified monoclonal antibody ascites fluid samples 2B9, 2F7, and 3C6 incubated as primary antibodies; C: Indirect ELISA detection results.

[0020] Figure 5 This invention relates to test strips with different antibody combinations; wherein, A: polyclonal antibody pAb-VP1 conjugated with latex microspheres as an immunoprobe; 1, 2, and 3 are monoclonal antibodies 2F7, 2B9, and 3C6 as detection lines, respectively; B: monoclonal antibody 2B9 conjugated with latex microspheres as an immunoprobe; 1, 2, and 3 are monoclonal antibodies 2F7, 3C6, and polyclonal antibody pAb-VP1 as detection lines, respectively; C: monoclonal antibody 2F7 conjugated with latex microspheres as an immunoprobe; 1, 2, and 3 are monoclonal antibodies 2B9, 3C6, and polyclonal antibody pAb-VP1 as detection lines, respectively; D: monoclonal antibody 3C6 conjugated with latex microspheres as an immunoprobe; 1, 2, and 3 are monoclonal antibodies 2B9, 2F7, and polyclonal antibody pAb-VP1 as detection lines, respectively. Figure 6 The amount of antibody conjugated to different immune probes in this invention.

[0021] Figure 7 These are the different C-line antibody concentrations of this invention.

[0022] Figure 8 These are the different T-line antibody concentrations of this invention.

[0023] Figure 9 This invention relates to the sensitivity testing of test strips.

[0024] Figure 10 This invention relates to the test strip specificity test.

[0025] Figure 11 This invention relates to the repeatability test of test strips; wherein, A: intra-batch repeatability test results; 1, 2, and 3 are test results of the same batch of test strips; B: inter-batch repeatability test results; 1, 2, and 3 are test results of different batches of test strips.

[0026] Figure 12 This invention relates to an accelerated destruction experiment of the test strip. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0028] Example 1

[0029] The materials and methods used in this embodiment are as follows: I. Main Materials and Instruments 1. Main experimental materials The feline calicivirus positive isolate FCV / SHsj / 2024 was purchased commercially and stably passaged and preserved in our laboratory for a long time. CRFK cells, pET-28a plasmid, and myeloma cells SP2 / 0 were purchased commercially and preserved in our laboratory. Escherichia coli DH5α (catalog number: CD201-01) and BL21 (DE3) (catalog number: CD601-02) competent cells were purchased from Beijing TransGen Biotech Co., Ltd. Ten 6-week-old SPF-grade BALB / c female mice were purchased from Shengchang Biotechnology Co., Ltd.

[0030] 2. Main reagents and instruments Viral genomic DNA extraction kit and reverse transcription PrimerScript RT reagent kit were purchased from TaKaRa Biotechnology Co., Ltd.; TransStart FastPfu Fly DNA Polymerase was purchased from TransGenBiotech Co., Ltd.; Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) antibody was purchased from Yeasen Co., Ltd.; red latex microspheres were purchased from Suzhou Weidu Biotechnology Co., Ltd.; MES, EDC, ProClin 300 and ethanolamine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; biological transmission electron microscope (model Tecnai G2) was purchased from Thermo Fisher Scientific Co., Ltd.; XP cycler PCR instrument was purchased from Hangzhou Borui Technology Co., Ltd.; constant temperature water bath was purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.; non-contact ultrasonic instrument (model SCIENTZ08-III) was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; gold labeling machine (model HGS510) and programmable strip cutter (model HGS201) were purchased from Hangzhou Fenghang Technology Co., Ltd.

[0031] 3. Solution Microsphere blocking solution (50 mL system): Sodium borate buffer system containing BSA, ethanolamine blocking agent, Tween-20, and preservative ProClin 300, pH adjusted to 9.0±0.05: 5.6 mL sodium tetraborate decahydrate solution, 2.5 mL sodium borate solution, 0.5 g BSA, 0.12 mL ethanolamine, 0.025 mL Tween-20, and ultrapure water to a final volume of 50 mL.

[0032] Microsphere washing solution (50 mL system): Tris buffer system, used for washing before and after sealing: Tris-Base 0.151 g, NaCl 0.438 g, trehalose 2.5 g, BSA 0.5 g, Tween-20 0.025 mL, ProClin 300 0.05 mL, ultrapure water to a final volume of 50 mL, pH 7.2±0.05.

[0033] Microsphere preservation solution: i.e., the microsphere washing solution mentioned above.

[0034] II. Preparation of Monoclonal Antibodies 1. Construction of recombinant plasmids Using software analysis, a fragment with high immunogenicity in the coding region of the VP1 gene of the FCV / SHsj / 2024 strain (as shown in SEQ ID NO1) was selected, and specific primers for homologous recombination with the pET-28a vector were designed. The primers are shown in Table 1. After PCR amplification, the product was recovered, purified, and ligated into the pET-28a vector. The recombinant vector was then transformed into the DH5α cloning host bacteria. Transformed bacteria were plated on kanamycin-resistant LB agar plates. Single colonies were picked for PCR identification. Plasmids that tested positive were sent to the company for sequencing. The positive plasmids returned by the biotechnology company were transformed into prokaryotic expression competent cells BL21(DE3). Single colonies were picked from overnight culture plates and inoculated into kanamycin-resistant LB liquid medium. The cells were cultured at 37°C and 180 r / min for more than 3 h with shaking. When the OD value of the bacterial culture reached about 0.6, 0.1 mol / L IPTG inducer was added, and the culture conditions were adjusted to 16°C and 160 r / min to induce expression for 16 h. After expression, the bacterial culture was centrifuged at 4°C and 8000 r / min for 10 min, the medium was discarded, and all bacterial pellets were collected. The pellets were resuspended in PBS and sonicated in an ice-water bath. The supernatant and pellets were analyzed by SDS-PAGE. The target protein was an inclusion body protein (as shown in SEQ ID NO1, recombinant VP1 antigen protein), which was expressed in a precipitate and purified by nickel column chromatography before being verified by Western blot.

[0035] Table 1 Primers for VP1 gene homologous recombination

[0036]

[0037] 2. Immunize mice with antigen protein Mice were immunized subcutaneously using the successfully identified recombinant VP1 antigen protein according to the established immunization protocol. Blood samples were collected periodically after immunization, serum was separated, and the immunization effect was evaluated. The specific mouse immunization program is shown in Table 2.

[0038] Table 2. Immunization schedule for mice

[0039] 3. Preparation of monoclonal antibodies BALB / c mice with sufficiently high immunogenicity were used to aseptically isolate spleen cells, which were then fused with resuscitated SP2 / 0 myeloma cells. Polyethylene glycol 1450 (PEG-1450) was used as a fusion inducer to induce cell membrane fusion. Cell fusion was monitored closely, and unfused cells were discarded using HAT selective medium. Hybridoma cells were tested approximately 10 days post-fusion. Cell supernatant was collected via a half-medium change, and antibody detection was performed using ELISA and IFA methods. Both methods used the hybridoma cell supernatant as the primary antibody. Positive and negative controls were established for each assay. The ELISA criterion was a P / N ratio greater than 2, and the IFA criterion was significant fluorescence, with no fluorescence in the negative control. Positive hybridoma cells were then selected.

[0040] 4. Purification of monoclonal antibodies Monoclonal antibodies were purified using Protein G affinity chromatography: Monoclonal antibody ascites fluid was diluted with equilibration buffer and filtered through a 0.45 μm filter membrane; Protein G chromatography media were packed into a column, and the column was equilibrated 2-3 times with 5 column volumes of equilibration buffer beforehand; the treated sample was loaded and incubated overnight on a rotating disc to ensure complete binding; the column was washed with 10-15 column volumes of washing buffer to remove non-specific proteins; elution was performed with 5-10 column volumes of elution buffer, and the eluted fractions were collected in fractions; the eluted fractions were immediately adjusted to neutral with 1 / 10 volume of neutralizing buffer; after purification, the packing material was equilibrated sequentially with 3-5 column volumes of equilibration buffer and 5 column volumes of filtered pure water, and finally washed with 20% ethanol and stored at 4°C. The titers were detected by indirect ELISA and indirect fluorescent immunoassay, and antibody typing was performed using a mouse monoclonal antibody Ig / subclass identification kit from Bio-Rad Laboratories.

[0041] III. Preparation of Immunochromatographic Test Strips 1. Latex microspheres conjugated with antibodies Take 12.5 μL of latex microsphere suspension and add it to 1 mL of MES coupling buffer (2-(N-morpholino)ethanesulfonic acid buffer). Sonicate at 700 W for 1 min to mix the microspheres. After mixing, centrifuge at 15℃ and 15000 g for 10 min, discarding the supernatant. Repeat the washing three times, add 5 μL of freshly prepared 10 mg / mL LEDC solution, vortex to mix, then add 33 μL of 10 mg / mL NHS solution and sonicate to mix. Activate by rotation at 37℃ and 180 r / min for 45 min. After activation, wash the precipitate twice with 1.5 mL of coupling buffer to remove unreacted reagents. Add 0.75 mL of coupling buffer, sonicate at 700 W for 1 min to mix the microspheres, obtaining the microsphere solution. Add 50 μg of the antibody to be coupled (polyclonal antibody pAb-VP1) to 250 mL of the solution. The antibody-containing conjugated solution was prepared by adding μL of conjugation buffer to the microsphere solution, vortexing, and then centrifuging the centrifuge tube at 37℃ and 180 r / min for 2 h. After conjugation, the precipitate was washed twice with 1.5 mL of conjugation buffer to remove unreacted reagents. After centrifugation, the conjugated product was precipitated and 500 μL of microsphere blocking solution was added, vortexed, and blocked at 37℃ and 180 r / min for 1 h. After blocking, the precipitate was washed twice with microsphere washing solution. The washed microspheres were resuspended in 0.5 mL of microsphere preservation solution, sonicated, and the final concentration of microspheres was adjusted to 1 mg / mL. The microspheres were then stored at 2-8℃ in the dark for later use. 2. NC film marking The NC membrane was scribing using a gold-labeled membrane scribing machine. The opposite end of the membrane was fixed to the surface of the scribing machine with a magnet. The following solutions were prepared in advance: T-line monoclonal antibody 2B9 gradient dilutions (0.5, 1.0, 1.5, 2.0, 2.5 mg / mL) and C-line goat anti-mouse IgG gradient dilutions (0.5, 0.75, 1.0, 1.25, 1.5 mg / mL). The corresponding antibody solutions were aspirated into the scribing machine's infusion tubing using a back-aspiration technique. The scribing parameters were set to 1 μL / cm. The T-line was started 2 cm from the edge of the NC membrane, and the C-line was drawn 0.5 cm behind the T-line. After scribing, the NC membrane was dried at 37°C for 16 h. After drying, it was sealed in a plastic bag with desiccant and stored at room temperature in a dark environment.

[0042] 3. Assembly of test strips Glass fiber was cut to make a sample pad; absorbent paper was cut to make an absorbent pad; an immunoprobe solution coupled with latex microspheres was uniformly sprayed onto the surface of the treated conjugate pad at a volume of 300 μL / piece; the NC membrane was pasted onto the center of the PVC backing plate with the T-line facing forward and the C-line facing backward; then the conjugate pad was attached to the side near the T-line, overlapping the NC membrane by 2 mm, and the sample pad was attached to the outside of the conjugate pad, maintaining a 2 mm overlap; the absorbent pad was attached to the side near the C-line, also overlapping the NC membrane by 2 mm; finally, the assembled test strip was placed into the test strip holder and the holder was pressed tightly, and the whole strip was stored in a dry and light-protected condition.

[0043] 4. Testing procedures and result interpretation Add 100 μL of VP1 standard protein diluted with PBS to the wells of the test strip. After reacting for 10-15 minutes, interpret the results: if both the T and C lines show color, the result is positive; if only the C line shows color and the T line does not, the result is negative; if neither the T nor C lines show color, or only the T line shows color, the result is invalid. In a positive result, the intensity of the T line reflects the antigen level; a lighter color indicates a weak positive, and a darker color indicates a strong positive.

[0044] IV. Condition Optimization 1. Optimal concentration of immune probe Different antibody amounts (10-90 μg, 10, 30, 50, 70, 90 μg) were conjugated with latex microspheres to prepare immunoprobes, which were then uniformly sprayed onto the conjugate pad. Test strips were assembled and the VP1 antigen protein solution was detected using the recommended antibody concentration (1 mg / mL) for both the detection and control lines. The optimal antibody conjugation amount was determined by comparing the immunochromatographic colorimetric effects under different conditions, based on the most significant positive / negative differentiation.

[0045] 2. Optimal control line (C line) antibody concentration Goat anti-mouse IgG immobilized on the C-line of the NC membrane is used to capture free immunoprobes, thus forming a control line. Too low a concentration of antibody in the C-line will result in a faint band color, affecting interpretation; too high a concentration will lead to reagent waste. To optimize the system, five concentration gradients of goat anti-mouse IgG solutions (1.5, 1.25, 1.0, 0.75, and 0.5 mg / mL) were prepared for C-line streaking. Test strips were assembled under the conditions of 50 μg of immunoprobe antibody conjugation and a recommended detection line antibody concentration of 1 mg / mL. The color development effect of the C-line was detected using VP1 antigen protein solution, and the optimal working concentration was selected based on the detection results.

[0046] 3. Optimization of antibody concentration in the detection line (T line) Antibodies immobilized on the T-line of the NC membrane are used to capture antigens in the sample, thereby intercepting the ternary complex composed of latex microspheres, polyclonal antibodies, and antigens. Low T-line antibody concentrations result in weak band development, affecting result interpretation; excessively high concentrations lead to reagent waste. To determine the optimal coating concentration, five concentration gradients of monoclonal antibody solutions (0.5, 1, 1.5, 2, and 2.5 mg / mL) were prepared and used for T-line streaking. Test strips were assembled under conditions of 50 μg of immunoprobe antibody conjugation and 1.25 mg / mL of control line antibody concentration. The T-line color development effect was detected using VP1 antigen protein solution, and the optimal working concentration was selected based on the color development effect.

[0047] 4. Performance testing of latex microsphere test strips (1) Sensitivity test The VP1 antigen protein solution with an initial concentration of 0.58 mg / mL was serially diluted with PBS buffer at dilution ratios of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and 1:51200, yielding samples with corresponding concentrations of 2900, 1450, 725, 362.5, 181.25, 90.63, 45.31, 22.66, and 11.33 ng / mL. Each component of the test strip was tested under the optimal conditions obtained in the above experiments at each dilution. The lowest antigen concentration that still produced a clear positive band was taken as the sensitivity of the detection system.

[0048] (2) Specificity test Specificity is one of the key indicators for evaluating the detection performance of immunochromatographic test strips. To verify its specificity, FCV virus solution was used as the positive group, and CRFK cell solution, FPV virus solution, FHV virus solution, and FIPV virus solution were used as control groups, with the same volume and processing conditions. If the test strip shows a T-line positive reaction only in the FCV virus solution sample and is negative in all control groups, it indicates that the established method has good specificity and can effectively distinguish the target virus from other potential interfering pathogens or matrix.

[0049] (3) Repeatability test The intra-batch repeatability of the immunochromatographic test strips prepared in the same batch was evaluated by repeatedly testing the same sample three times and using the consistency of the results. Then, the intra-batch repeatability of the test strips was evaluated by testing the same sample with immunochromatographic test strips prepared in different batches and using the degree of difference in the results.

[0050] (4) Accelerated destruction experiment To evaluate the storage stability of the immunochromatographic test strips, an accelerated degradation experiment was conducted: test strips prepared in the same batch were sealed in plastic bags and placed in a 37°C constant temperature incubator. They were removed on days 5, 10, 15, 20, and 25, and after being brought to room temperature, they were tested using standard positive and negative samples. The performance stability of the test strips under accelerated aging conditions was evaluated by comparing the color intensity of the T / C line and the consistency of the positive and negative interpretation results at each time point.

[0051] (5) Clinical sample testing The latex microsphere immunochromatographic test strip developed in this study was used to test 96 clinical samples from suspected FCV infection cases in Shanghai. These clinical samples included conjunctival swabs, nasal swabs, pharyngeal swabs, and anal swabs. RT-PCR was also used to identify these samples, and the consistency of the results from the two methods was compared. The concordance rate was calculated to evaluate the clinical sample detection efficacy of the test strip developed in this study.

[0052] V. Results 1. Expression of recombinant antigen protein Using the cDNA of the FCV / SHsj / 2024 strain as a template, PCR amplification was performed using the specific primers designed in Table 1. The target gene fragment was 1680 bp. The PCR amplification results are shown in [Table 1]. Figure 1 The size of the PCR amplification product was consistent with the expectation, indicating that the target gene fragment was successfully amplified.

[0053] The target gene fragment was recovered and sent to the company for sequencing. The sequencing results showed it was correct. The gene fragment was then cloned into the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-VP1. This plasmid was then transformed into BL21(DE3) for protein expression. Since protein expression was primarily concentrated in the precipitate, inclusion body purification, refolding, and ultrafiltration concentration were employed to obtain high-purity recombinant protein VP1. The purification results are shown below. Figure 2 The protein concentration of VP1 (A) was determined to be 1.74 mg / mL using the BCA method. VP1 protein was identified by Western blotting. The protein expressed on the pET-28a vector carried a His tag; therefore, mouse anti-His antibody was used as the primary antibody for identification. The WB results are shown below. Figure 2 In the middle B, VP1 was a single band with a protein size of approximately 60.1 kDa, indicating successful expression of the FCV-VP1 antigen protein. After fusing mouse spleen cells with SP2 / 0 myeloma cells, three hybridoma cell lines that stably secreted anti-VP1 monoclonal antibodies were obtained through indirect ELISA detection and screening, and were named 2B9, 2F7, and 3C6, respectively.

[0054] 2. Identification and titer testing of polyclonal antibodies Blood was collected from the eyes of all immunized mice, and serum was separated to obtain polyclonal antibodies. Antibody specificity was then identified by Western blotting and IFA experiments, and antibody titer was determined by indirect ELISA. Results are shown below. Figure 3 .

[0055] IFA results showed that all positive serum groups exhibited clear, specific fluorescent signals, significantly differentiable from the negative serum control group. Western blotting further confirmed that the positive serum showed a single protein band at approximately 60.1 kDa, consistent with the expected molecular weight of the target protein. Indirect ELISA assays indicated that the prepared polyclonal antibody achieved a titer of 1:1.024×10⁻⁶. 5 The concentration of the polyclonal antibody pAb-VP1 was determined to be 5.27 mg / mL using a BCA kit.

[0056] 3. Monoclonal antibody titer detection IFA results showed that all three purified monoclonal antibodies produced specific fluorescent signals. The signal intensities of 2B9 and 2F7 were comparable to those of the polyclonal antibody, while the signal of 3C6 was weaker. Western blot analysis further confirmed that all three monoclonal antibodies specifically recognized the VP1 antigen, exhibiting a single band at approximately 60.1 kDa, consistent with the expected protein size.

[0057] The titers of the purified monoclonal antibodies were determined by indirect ELISA. The results showed that the titers of 2B9, 2F7, and 3C6 were 1:1.024×10⁻⁶. 5 1:5.12×10 4 And 1:1.28×10 4 This indicates that all three monoclonal antibodies possess high antibody-binding activity. The concentrations of 2B9, 2F7, and 3C6, measured using the BCA kit, were 1.38 mg / mL, 1.69 mg / mL, and 0.94 mg / mL, respectively.

[0058] 4. Identification of monoclonal antibodies Using a commercially available antibody subtype identification kit, the subtypes of three purified ascites monoclonal antibodies (2B9, 2F7, and 3C6) were identified by indirect ELISA. The results are shown in Table 3. The identification results showed that the heavy chains of monoclonal antibodies 2B9 and 2F7 were both IgG1 type, while the heavy chain of monoclonal antibody 3C6 was IgG2a type; the light chains of all three monoclonal antibodies were κ chains. In summary, 2B9 and 2F7 are IgG1 / κ type antibodies, and 3C6 is an IgG2a / κ type antibody.

[0059] Table 3. Identification of Monoclonal Antibody Subtypes

[0060] 5. Optimal antibody combination Immunochromatographic techniques generally employ a double-sandwich method with paired antibodies for virus detection. Therefore, before optimizing the components of the test strip, this experiment first performed antibody pairing to select the optimal antibody combination for subsequent experiments. The three monoclonal antibodies (2B9, 2F7, 3C6) and one polyclonal antibody (pAb-VP1) prepared in the previous experiment were paired in pairs as immunoprobe and detection antibody combinations, and the optimal combination was selected for subsequent experiments. The specific combinations are as follows: pAb-VP1 as the immunoprobe, 2B9, 2F7, and 3C6 as detection antibodies; 2B9 as the immunoprobe, 2F7, 3C6, and pAb-VP1 as detection antibodies; 2F7 as the immunoprobe, 2B9, 3C6, and pAb-VP1 as detection antibodies; 3C6 as the immunoprobe, 2B9, 2F7, and pAb-VP1 as detection antibodies. Twelve paired combinations were formed. Test strips were assembled under the recommended concentrations (50 μg, 1.0 mg / mL, and 1.0 mg / mL, respectively) for antibody conjugation, C-line concentration, and T-line concentration. VP1 antigen protein solution was used for detection. Results were viewed after 15 minutes of reaction. The results are as follows: Figure 5 As shown in the diagram, among the 12 paired antibody combinations, the following three pairs showed good detection results: polyclonal antibody pAb-VP1 as the immunoprobe and monoclonal antibody 2F7 as the detection line; polyclonal antibody pAb-VP1 as the immunoprobe and monoclonal antibody 2B9 as the detection line; and monoclonal antibody 2B9 as the immunoprobe and pAb-VP1 as the detection line. These three sets of results showed clean backgrounds without any discoloration, consistent positive and negative control lines, and clear and distinct detection lines, clearly distinguishing between positive and negative results. The pairing of polyclonal antibody pAb-VP1 and monoclonal antibody 2B9 showed the best results, and even after swapping their positions, both showed good detection results. Considering that polyclonal antibodies can bind to more antigenic epitopes than monoclonal antibodies and have a stronger ability to capture antigens as immunoprobes, polyclonal antibody pAb-VP1 was chosen as the immunoprobe and monoclonal antibody 2B9 as the detection antibody for subsequent experiments.

[0061] 6. Optimal dosage of immune probes Further optimization of the conjugation amount between latex microspheres and the polyclonal antibody pAb-VP1 was performed. Gradient conjugation amounts were set according to the method in section 1.8.1 for detection, and the results are shown below. Figure 6 The experimental group with an antibody conjugation amount of 50 μg showed the best detection performance, with clear detection lines, a clean and unstained background, and the ability to effectively distinguish between positive and negative results. Therefore, the optimal conjugation amount of the polyclonal antibody pAb-VP1 to latex microspheres was determined to be 50 μg.

[0062] 7. Optimal C-line antibody concentration The detection was performed according to the C-line antibody concentration gradient set in step 4, section 2. The detection results are shown below. Figure 7Both 1.25 mg / mL and 1.5 mg / mL C-line antibodies produced clear and distinct bands, demonstrating good colorimetric performance. Considering the need to conserve antibody dosage while ensuring the reliability of the detection system, 1.25 mg / mL was ultimately selected as the working concentration for the C-line antibody.

[0063] 8. Optimal T-line antibody concentration The detection was performed according to the T-line antibody concentration gradient set in step 4, section 3. The detection results are shown below. Figure 8 Both 2.0 mg / mL and 2.5 mg / mL concentrations of the T-line antibody produced clear and distinct bands, demonstrating good colorimetric performance. Considering the need to conserve antibody dosage while ensuring the reliability of the detection system, 2.0 mg / mL was ultimately selected as the working concentration for the T-line antibody.

[0064] 9. Test strip sensitivity Dilute the VP1 antigen protein solution according to the method in step 4, section 4 (1), and use a test strip to detect the lowest antigen concentration. The test results are shown in [the table below]. Figure 9 The test line was still visible when the antigen protein was diluted 1:25600 to 22.66 ng / mL, but almost invisible when diluted 1:51200 to 11.33 ng / mL. This indicates that the minimum detectable concentration of the VP1 antigen protein for this test strip is 22.66 ng / mL, and the test strip has good sensitivity.

[0065] 10. Test strip specificity Following the method in step 4, section 4 (2), use the test strip to detect different viral solutions to determine whether the test strip is specific. The test results are shown in [link to test results]. Figure 10 The test results showed that only the FCV virus solution was positive, while the FHV-1, FIPV, FPV virus solutions and the uninoculated CRFK cell solution (used as a negative control) were all negative. This indicates that the test strip specifically recognizes the FCV antigen and does not cross-react with FHV-1, FIPV, or FPV, demonstrating good specificity.

[0066] Test strip repeatability According to the intra-batch and inter-batch repeatability experiments set in step 4, section 4 (3), the repeatability of the test strips was tested, and the test results are as follows: Figure 11 As shown, the test strips from the same batch yielded consistent results, with clear distinction between positive and negative results. The C-line and T-line were clear and uniform. The test strips from different batches also showed consistent results, with slight background variations due to different storage times, but the C-line and T-line remained clear, and the positive and negative results were easily distinguishable. This indicates that the test strip exhibits good intra-batch and inter-batch repeatability.

[0067] Test strip accelerated destruction experiment Following the experimental conditions in step 4, section 4 (4), the same batch of test strips were placed into sealed bags and placed in a 37°C environment. The changes in the test strip detection effect were then tested at different times. The test results are shown in […]. Figure 12 As shown in the figure, the test strips stored at 37℃ for 5, 10, 15, 20, and 25 days showed no significant difference in detection performance. The positive and negative results were clearly distinguishable, with the C and T lines clearly visible, and the detection results were uniform. Only after 15 days did a slight yellowing of the background due to oxidation occur, but this did not affect the interpretation of the results. Therefore, the results indicate that this test strip can be stored at 37℃ for at least 25 days.

[0068] Clinical sample testing results The test strip detection method established in this study and the PCR identification method established in step 4(5) were used to test 96 clinical samples from suspected FCV infection cases in Shanghai. The test results are shown in Table 4. The consistency of positive samples was 85.3% (29 / 34), the consistency of negative samples was 93.5% (58 / 62), and the overall consistency of samples was 90.6% (87 / 96). The results indicate that the latex microsphere immunochromatographic antigen detection test strip established in this study can be applied to clinical testing.

[0069] Table 4 Comparison of Clinical Sample Test Results

[0070] discuss This invention utilizes latex microspheres, ensuring high sensitivity and stability while also considering ease of naked-eye interpretation and cost control, making it suitable for grassroots veterinary settings. This aligns with the development strategy of many point-of-care testing (POCT) strips for economically important animals. Compared to some high-sensitivity test strips in human medicine that employ fluorescent labeling, this study prioritizes rapid on-site screening rather than precise quantification; therefore, latex microspheres represent a more balanced choice. In terms of methodological comparison, the core processes of this study, including antibody pairing, labeling technology, membrane system optimization, and performance evaluation, share commonalities with the development of various animal virus immunochromatographic test strips, such as those shown by Zhang et al. for canine parvovirus and Wang et al. for classical swine fever virus, demonstrating the versatility of this technology platform. However, the biological characteristics, antigenic properties, and sample matrix of different pathogens vary significantly, directly impacting antibody screening strategies and performance parameters. For example, test strips for highly variable viruses like feline calicivirus, feline coronavirus, and influenza virus emphasize broad-spectrum antibody activity; while those for enteric diseases need to address complex sample interference. The performance results of this study demonstrate the specific implementation of customized optimization of a general process for FCV-VP1 antigen and cat swab samples.

[0071] This invention successfully prepared high-titer polyclonal antibodies and three specific monoclonal antibodies using the VP1 protein of a prevalent feline calicivirus strain FCV / SHsj / 2024 as an immunogen, laying the material foundation for the establishment of a detection system. A feline calicivirus latex microsphere immunochromatographic detection method based on a double-antibody sandwich method was successfully established, exhibiting good sensitivity, specificity, and stability. In terms of sensitivity, the detection limit for purified VP1 protein reaches 22.66 ng / mL. While this value is lower than that of nucleic acid amplification techniques such as PCR, it is comparable to the sensitivity level of most commercially available rapid viral antigen detection reagents, sufficient to meet the detection needs of common viral loads in clinical samples. Regarding specificity, the test strip can accurately distinguish FCV from FHV-1, FIPV, and FPV, indicating that the selected antibodies have high specificity for the FCV VP1 antigen, which is crucial for differential diagnosis among various feline diseases with similar clinical symptoms. Furthermore, the test strip also exhibits good intra-batch and inter-batch repeatability, reflecting the stability and controllability of the production process, which is the basis for product standardization and large-scale production. Furthermore, this study conducted an accelerated degradation experiment on the test strip, showing no significant performance degradation after 25 days of storage at 37℃. This indicates that the test strip possesses certain storage stability, suggesting a potentially longer storage time at room temperature, which is beneficial for its practical storage and distribution after commercialization. Finally, this study also compared and validated the constructed test strip with the gold standard PCR method in clinical sample testing. The results of testing 96 suspected clinical samples showed an overall concordance rate of 90.6% between the test strip and PCR, demonstrating its suitability for on-site testing and promising application prospects.

[0072] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A VP1 antigen, characterized in that, The amino acid sequence of the VP1 antigen is shown in SEQ ID NO 1.

2. A polyclonal antibody pAb-VP1 and a monoclonal antibody 2B9 against the VP1 antigen of claim 1.

3. A test strip, characterized in that, Includes the polyclonal antibody pAb-VP1 and monoclonal antibody 2B9 as described in claim 2.

4. The test strip according to claim 3, characterized in that, The polyclonal antibody pAb-VP1 is conjugated with red latex microspheres as an immune probe, and the monoclonal antibody 2B9 is used as a detection line antibody.

5. The test strip according to claim 4, characterized in that, The polyclonal antibody pAb-VP1 conjugated with red latex microspheres was prepared by the following method: A latex microsphere suspension containing a coupling buffer is provided; EDC solution and NHS solution were added to the latex microsphere suspension, and the mixture was ultrasonically mixed, followed by rotational activation. After activation, the solid phase was separated from the liquid phase and washed with coupling buffer. Add coupling buffer and sonicate to mix the microspheres to obtain a microsphere solution; The polyclonal antibody pAb-VP1 was dispersed in a coupling buffer to obtain a coupling solution containing the antibody. The antibody-containing conjugate solution is mixed with the microsphere solution and then rotated for conjugation. After the reaction is complete, the solid phase is separated and washed to obtain polyclonal antibody pAb-VP1 conjugated red latex microspheres.

6. The test strip according to claim 5, characterized in that, The concentration of the antibody-containing conjugate is 0.1-0.4 μg / μl.

7. The test strip according to claim 5, characterized in that, Rotational activation conditions: 37℃, 180 r / min for 45 min; rotational coupling conditions: 37℃, 180 r / min for 2 h.

8. A reagent kit, characterized in that, Includes the polyclonal antibody and / or monoclonal antibody as described in claim 2, or the test strip as described in any one of claims 3 to 7.

9. The reagent kit according to claim 8, characterized in that, The double-antibody sandwich ELISA kit.

10. The use of a polyclonal antibody or monoclonal antibody as described in claim 2, a test strip as described in any one of claims 3 to 7, or a kit as described in claim 8 or 9 in the preparation of a test for detecting feline calicivirus.

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