Competitive ELISA kit for accurately and quantitatively detecting feline panleucopenia virus antigen

Abstract

The invention discloses a competitive ELISA (Enzyme-Linked Immunosorbent Assay) kit capable of accurately quantifying feline panleucopenia virus antigen. The kit comprises an elisa plate, cat panleucopenia virus negative / positive serum, a cat panleucopenia virus standard antigen and an elisa secondary antibody. Wherein the elisa plate is a recombinant protein coated with feline panleucopenia virus VP2, and the protein is a protein shown as a sequence 4 in a sequence table. A competitive ELISA method is adopted, the feline panleukopenia virus VP2 protein expressed by an escherichia coli expression system is small in antigen dosage and high in sensitivity and specificity, whether feline panleukopenia virus antigen exists or not can be efficiently detected, and the kit is high in specificity and specificity. Meanwhile, different standard substances can be used for accurately quantifying the content of the feline panleucopenia VP2 protein or the feline panleucopenia virus titer in the antigen or the vaccine. The kit disclosed by the invention is high in sensitivity, good in specificity and convenient to operate, and has a good market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, this invention relates to a competitive ELISA method for accurate quantitative detection of feline panleukopenia virus antigen. Background Technology

[0002] Feline panleukopenia virus (FPV) is a genus of parvovirus in the family Parvoviridae, commonly causing acute gastroenteritis and leukopenia in kittens. FPV was first discovered in 1928 and isolated from tissue culture in 1964. FPV causes feline panleukopenia. This virus has been shown to infect all felines. FPV is a 5,000 bp long single-stranded DNA virus with two open reading frames (ORFs), ORF1 and ORF2. ORF1 encodes two non-structural proteins, non-structural protein 1 (NS1) and non-structural protein 2 (NS2). The ORF2 region encodes two capsid proteins, VP1 and VP2. These proteins are splice variants, almost identical in sequence except for a unique 143-amino acid N-terminal extension in VP1. VP2 is the major capsid protein, comprising approximately 90% of the entire viral capsid, and is the primary target of neutralizing antibodies, thus determining host range. Specific amino acids in the VP2 protein are crucial for determining the differences in host range between feline panleukopenia virus (FPV) and canine parvovirus type 2 (CPV-2).

[0003] Currently, the development of vaccines and drugs for feline panleukopenia (FPV) has gradually gained attention from animal health companies both domestically and internationally. Besides the widely marketed "Fantastic Bacteria" vaccine, significant progress has also been made in the development of domestically produced vaccines. However, in the vaccine production process, the detection of FPV titer still relies on cumbersome and time-consuming methods for determining the median lethal dose (LD50), and these methods cannot detect inactivated viruses. Furthermore, the detection of related antigenic proteins largely relies on the grayscale method after PAGE staining, which remains time-consuming and labor-intensive.

[0004] Although only one subtype of FPV is currently known, it is still slowly mutating, and its presence seriously affects the health of cats. Given the intensive research and development of products for FPV prevention and treatment both domestically and internationally, establishing a rapid, accurate, and efficient method for the quantitative detection of FPV-related antigens is urgently needed. This study presents a competitive ELISA method for the accurate quantitative detection of FPV-related antigens. This method has been validated and can accurately quantify the viral load of live and inactivated FPV, as well as the content of the related structural protein VP2, aiming to provide reliable assistance for the research and development of products related to FPV prevention and treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a competitive ELISA method that can accurately quantify FPV antigen. This method uses feline panleukopenia virus VP2-1 protein as the coating antigen to establish a competitive ELISA method with good specificity, sensitivity and reproducibility, which can be used to quantitatively detect the protein concentration of vaccines, intermediate products, vector expression products or the content of FPV antigen in cell cultures.

[0006] To achieve the above objectives, this invention first screened and obtained a high-performance feline panleukopenia virus (FPV) VP2 protein. Then, using biological software, it analyzed the antigenic epitope information of the FPV VP2 protein, predicted the dominant antigenic region, and tandemly generated it using a linker to obtain the recombinant FPV VP2 protein (VP2-1 protein). The FPV VP2-1 protein provided by this invention is an FPV VP2-1 protein expressed using an E. coli expression system. The molecular weight of the expressed protein is approximately 60 kDa, and it is highly expressed in the precipitate after bacterial cell disruption. The nucleotide sequence shown is SEQ ID No. 3, and its corresponding amino acid sequence is SEQ ID No. 4.

[0007] This invention discloses a competitive ELISA kit for accurate quantification of FPV antigen. The kit comprises an enzyme-linked reaction plate coated with feline panleukopenia virus VP2-1 protein as antigen, FPV positive serum, FPV negative serum, purified FPV standard antigen, and enzyme-labeled secondary antibody. The amino acid sequence of the feline panleukopenia virus VP2-1 protein is shown in Sequence 4 of the sequence listing.

[0008] The competitive ELISA kit for accurately quantifying feline panleukopenia virus antigen includes a removable 96-well ELISA plate; the feline panleukopenia virus VP2-1 protein is a prokaryotic system expression protein.

[0009] The optimal preparation method and conditions for the enzyme-linked reaction plate are as follows: dissolve the feline panleukopenia virus VP2-1 protein in a carbonate solution at pH 9.6, and then add 100 μl (8 μg / mL) of feline panleukopenia virus VP2-1 protein to each well of a 96-well polystyrene enzyme-linked reaction plate. Incubate at 2-8°C for 8-12 hours to allow the feline panleukopenia virus VP2-1 protein to fully bind to the enzyme-linked reaction plate. Then, add 300 μl of PBS buffer containing 1% (g / ml) bovine serum albumin (BSA) at pH 7.4 to each well. Block at 37°C for 2-3 hours, spin dry, and store at 4°C after the enzyme-linked reaction plate has dried.

[0010] The FPV-positive serum is rabbit or guinea pig serum collected after immunization with one or two of the feline panleukopenia virus inactivated virus and Escherichia coli expression system expressing feline panleukopenia virus VP2 protein; the FPV-negative serum is SPF guinea pig or rabbit serum.

[0011] The FPV purification standard antigen is an antigen that has been purified by sucrose density gradient centrifugation, and then inactivated after virus content determination. Specifically, it can be the feline panleukopenia virus VP2-1 protein expressed by Escherichia coli, which has been purified by a protein purification column and its concentration has been determined.

[0012] The enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-rabbit IgG antibody or goat anti-guinea pig IgG antibody.

[0013] The method further includes substrate solution A, substrate solution B, and a stop solution. Substrate solution A is a citrate phosphate buffer containing 0.6 mg / ml hydrogen peroxide urea, and substrate solution B is a 0.2 mg / ml tetramethylbenzidine solution. The two are mixed in a 1:1 ratio before use. The stop solution is a 2 mol / L sulfuric acid solution.

[0014] The method further includes a sample dilution buffer and a concentrated wash buffer (20-fold); the sample dilution buffer is a 0.01M phosphate buffer containing 0.5% (g / ml) casein at pH 7.4; the concentrated wash buffer is a 0.01M phosphate buffer containing 0.8%~1.2% (ml / ml) Tween-20 at pH 7.4.

[0015] The detection procedure of the method of this invention is as follows: 1. Equilibration: Remove all relevant samples from the refrigerated environment and allow them to equilibrate at room temperature for 30 minutes before use; mix liquid reagents thoroughly before use.

[0016] 2. Solution preparation: Dilute the concentrated washing solution 20 times with distilled water or deionized water to obtain the washing buffer solution; 3. Setup: 2 negative control wells and 2 positive control wells, with the rest being sample wells to be tested.

[0017] 4. Pre-dilution of the sample to be tested: Dilute the inactivated FPV antigen, the feline panleukopenia virus VP2 protein, the treated vaccine, etc. in the sample to be tested in an appropriate ratio using sample diluent, and then perform serial dilution at a ratio of 2.

[0018] 5. Dilution of standards: Select appropriate standards (i.e., inactivated antigen standards for inactivated antigens and expressed protein standards for expressed proteins) according to the different sources of antigens and perform serial dilutions of 2-fold.

[0019] 6. Sample addition: Add 50µl of positive serum to each well according to the pre-set instructions, followed by 50µl of the proportionally diluted test sample. The sample addition process should be as short as possible.

[0020] 7. Incubation: Shake well and place in a 37°C incubator for 30 minutes.

[0021] 8. Washing the plate: Discard the reaction solution, add 300 μl of diluted washing buffer to each well, soak for 15 seconds, discard the washing solution, wash the plate 4 times and then pat dry.

[0022] 9. Add enzyme: Add 100 μl of the corresponding horseradish peroxidase-labeled IgG antibody to each well (add goat anti-rabbit IgG antibody to rabbit serum, and add goat anti-guinea pig IgG antibody to guinea pig serum).

[0023] 10. Incubation: Place in a 37℃ incubator and react for 30 minutes.

[0024] 11. Washing the plate: Discard the reaction solution, add 300µl of diluted washing buffer to each well, soak for 15s, discard the washing solution, wash the plate 4 times and then pat dry.

[0025] 12. Add 100µl of substrate working solution to each well for color development (the substrate working solution is obtained by mixing equal volumes of substrate solution A and substrate solution B, and should be prepared fresh before use), shake to mix well, and place in a 37℃ incubator to react for 15 min in the dark.

[0026] 13. Add 50µl of colorimetric stop solution to each well and shake to mix and terminate the reaction.

[0027] 14. Measure the OD of each well. 450nm The OD value (the reaction plate after adding the stop solution should be read within 15 minutes) 450nm value).

[0028] Interpretation of test results: 1. Negative control OD 450nm The average value should be less than 0.3; otherwise, it is invalid.

[0029] 2. The positive control should have a test value between 0.8 and 2.0; otherwise, it is invalid.

[0030] 3. Establishment of the standard curve: using the OD of the standard product... 450nm A standard curve is constructed using the values ​​on the x-axis (x-axis) and the concentration of standard protein on the y-axis (y-axis). R0 2 The value should be ≥0.98. 4. Determination of the concentration of the sample to be tested: Measure the OD of the sample to be tested... 450nm Substitute two sets of data with values ​​close to 1 into the standard curve, calculate the original concentration, and then take the average value to obtain the concentration of the sample to be tested.

[0031] The detection method described above in this invention can be used to detect feline panleukopenia virus antigens or vaccines from different sources.

[0032] The positive effects of this invention are as follows: This invention uses the feline panleukopenia virus VP2-1 protein expressed by Escherichia coli, which makes the method have the advantages of high sensitivity and strong specificity.

[0033] Furthermore, when the present invention uses feline panleukopenia virus VP2-1 protein as the coating agent, its effect is significantly better than using feline panleukopenia virus inactivated virus and VP2 protein.

[0034] This method has good specificity and shows no cross-reactivity with common feline pathogenic viruses such as feline rhinotracheitis virus, feline infectious peritonitis virus, feline calicivirus, and rabies virus. This method can detect FPV antigens from different sources, such as live FPV, inactivated FPV, E. coli expressing VP2 protein, and baculovirus expressing VP2 protein. The limit of detection of this method can reach 10. 3.5 TCID 50 / mL or 0.2ug / ml; it can be applied to the entire process of corresponding vaccine preparation; In summary, this method uses E. coli expressing feline panleukopenia virus (FPV) VP2-1 protein to coat an enzyme-linked reaction (ELISA) plate. It requires a small amount of antigen, exhibits high sensitivity and specificity, and can effectively detect FPV antigens or vaccines from different sources. Experimental results show that the detection method of this invention has good repeatability, high specificity, and high sensitivity. It can meet the needs of different levels of personnel and has broad market prospects and good economic and social benefits. Attached Figure Description

[0035] Figure 1 For antigen epitope prediction results and preferred regions; Figure 2 SDS-PAGE results of VP2-1 protein expression in BL21 cells; M1: protein marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: uninduced whole cell lysate; 1: whole cell lysate induced at 15℃ for 16 hours; 2: whole cell lysate induced at 37℃ for 4 hours; NC1: uninduced cell lysate supernatant; NC2: uninduced cell lysate pellet; 3: cell lysate supernatant induced at 15℃ for 16 hours; 4: cell lysate supernatant induced at 37℃ for 4 hours; 5: cell lysate pellet induced at 15℃ for 16 hours; 6: cell lysate pellet induced at 37℃ for 4 hours.

[0036] Figure 3 SDS-PAGE image of purified VP2-1 protein; Figure 4Results of Western blot (anti-His antibody) for purifying VP2-1 protein; Figure 5 Serological antigenicity analysis of VP2 and VP2-1 proteins; Figure 6 The results of SDS-PAGE analysis of purified rabbit anti-FPV positive serum; Figure 7 Western blot results of purified VP2-1 protein and rabbit anti-FPV positive serum; Figure 8 Western blot results of purified VP2-1 protein with rabbit negative serum; Figure 9 For different P / N values ​​of the sealing liquid; Figure 10 P / N values ​​for different closure times; Figure 11 P / N values ​​for different dilutions and reaction times of anti-rabbit enzyme-labeled secondary antibody; Figure 12 OD of rabbit anti-FPV positive serum 450nm Value distribution chart; Figure 13 Rabbit negative serum OD 450nm Value distribution chart; Figure 14 Competitive ELISA standard curve. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0039] It should be noted that, unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0040] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, or article that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, or article.

[0041] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0042] Example 1: Expression and identification of feline panleukopenia virus VP2 antigen epitope tandem recombinant protein (VP2-1 protein). 1. Prediction of the dominant region of VP2 protein antigen Biological software was used to analyze antigenic epitope information of the FPV virus VP2 protein. This analysis primarily focused on the protein's hydrophilicity, surface-probability, flexibility, and antigenic index to preliminarily predict the dominant antigenic regions of the VP2 protein. The predicted nucleotide fragments were then tandemly linked using a linker, and the tandem nucleotide sequences were optimized and artificially synthesized according to codons preferred by *E. coli*. The antigenic epitope prediction results and optimized regions are shown below. Figure 1 .

[0043] The nucleotide sequence of the VP2 protein is SEQ ID No. 1, and the amino acid sequence of the VP2 protein is SEQ ID No. 2; The nucleotide sequence of the VP2-1 recombinant protein with the dominant region of the VP2 antigen is SEQ ID No. 3, and the amino acid sequence of the VP2-1 recombinant protein with the dominant region of the VP2 antigen is SEQ ID No. 4.

[0044] The two proteins are labeled VP2 and VP2-1, respectively.

[0045] Table 1. Recombinant expression of VP2-1 protein protein carrier Size (kDa) Cloning strategy VP2-1 pET-30a(+) Approximately 60 NdeI--ATG--His tag--VP2-1 --Stop codon --HindIII The protein gene fragment synthesized in vitro was inserted into the pET-30a(+) expression vector using NdeⅠ and HindⅢ to obtain the recombinant vector. The obtained recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and positive clones were screened. The recombinant plasmids identified as positive were transformed into Escherichia coli BL21(DE3) competent cells. Positive single colonies containing specific protein expression plasmids were picked and inoculated into LB medium containing kanamycin. After incubation at 37°C with shaking overnight, the recombinant bacteria expressing VP2-1 were named pET30-VP2-1, and the recombinant bacteria expressing VP2 were named pET30-VP2.

[0046] 2. Identification of Expression Results SDS-PAGE expression identification of VP2-1 protein showed that, compared with the pre-induction control, the recombinant bacteria expressed mainly soluble protein after IPTG induction. The optimal induction conditions for VP2-1 protein were: induction at 37℃ for 4 hours, followed by harvesting of cells and supernatant. SDS-PAGE gel scanning analysis stained with Coomassie Brilliant Blue estimated the size of the recombinant protein to be approximately 60 kDa, consistent with expectations. See results below. Figure 2 .

[0047] 3. Purification results and specificity identification Protein purification was performed according to the protein purification method of the BeyoGold™ His-tag Purification Resin kit. The expressed VP2-1 protein was purified by ultrafiltration (ultrafiltration tube), and the collected samples were concentrated to a purity of approximately 90% (see results). Figure 3 ); VP2-1 protein-specific WB is shown in Figure 4 .

[0048] 4. Serological antigenicity analysis of VP2 and VP2-1 proteins VP2 and VP2-1 proteins were used as coating antigens, respectively, at the same concentration (8 μg / ml), 100 μl / well for coating the ELISA plate. A 1:800 dilution of FPV positive control serum was used as the primary antibody, and a 1:10000 dilution of HRP-labeled goat anti-rabbit IgG was used as the secondary antibody. Serum negative control, empty vector induction negative control, and blank control were included. Two parallel controls were set up for each well, and OD was measured. 450nm The P / N ratio is used to determine the antigenicity of VP2 and VP2-1 proteins. Serological antigenicity analysis results show that the P / N (OD) ratio of VP2-1 protein expressed after tandem expression of the dominant antigenic region of VP2 protein is significantly higher. 450nm The value of ) was much higher than that of VP2 protein, and the difference between the two was significant (P<0.05). See the comparison results below. Figure 5Based on the results of bioinformatics software and Western blotting antigenicity analysis, the dominant antigenic fragment of FPV VP2-1 was finally identified. The results are shown in Tables 2 and 3.

[0049] Table 2. Ratios (P / N) of VP2 and VP2-1 protein coatings compared to serum negative controls. Coated protein VP2 protein VP2-1 protein p-value 0.968 1.112 N value 0.091 0.083 P / N value 10.64 13.40 Table 3. Ratios (P / N) of VP2 and VP2-1 protein coated with empty vector versus negative control. Coated protein VP2 protein VP2-1 protein p-value 0.968 1.112 N value 0.089 0.081 P / N value 10.88 13.73 Example 2: Preparation of a competitive ELISA kit for accurate quantitative detection of feline panleukopenia virus antigen The competitive ELISA kit for accurate quantification of feline panleukopenia virus of the present invention comprises: (1) 96-well detachable polystyrene enzyme-linked reaction plate coated with recombinant feline panleukopenia virus VP2 protein VP2-1; 2×96 wells.

[0050] (2) Positive control serum: FPV positive serum (1 tube, 1.0 ml / tube).

[0051] (3) Negative control serum: Specific pathogen-free (SPF) rabbit serum or guinea pig serum is used as the negative control serum for the kit (1 tube, 1.5 ml / tube).

[0052] (4) Enzyme-labeled secondary antibody: Horseradish peroxidase-labeled goat anti-rabbit IgG antibody or goat anti-guinea pig IgG antibody. Purchased from EarthOx.

[0053] (5) Sample dilution solution: 0.01M phosphate buffer containing 0.5% (g / ml) casein, pH 7.4, 1 bottle (24ml / bottle).

[0054] (6) Substrate solution A: citrate phosphate buffer containing 0.6 mg / ml hydrogen peroxide urea (1 bottle, 12 ml / bottle).

[0055] (7) Substrate solution B: 0.2 mg / ml tetramethylbenzidine (TMB) solution (1 bottle, 12 ml / bottle).

[0056] (8) Termination solution: 2 mol / L sulfuric acid solution (1 bottle, 12 ml / bottle).

[0057] (9) 20-fold concentrated washing solution: 0.01M phosphate buffer (50ml / bottle, 2 bottles) containing 0.8%~1.2% (ml / ml) Tween-20 at pH 7.4.

[0058] I. Purification and Identification of Rabbit Anti-FPV Positive Serum FPV-positive serum was purchased from Qianxun Biotechnology Co., Ltd. The qualified rabbit serum was purified using a Protein G Sepharose affinity chromatography column. The purified antibody, which is a polyclonal antibody, was aliquoted and stored at -80℃ for later use.

[0059] The purity of the purified antibody was observed by SDS-PAGE analysis (results are shown in [link to results]). Figure 6 Western blot (WB) was used to detect the specificity of positive and negative serum samples. Figure 7 , Figure 8 ).

[0060] II. Determination of the optimal antigen coating concentration and the optimal concentration of negative and positive sera. Square matrix titrations were performed using the orthogonal matrix method. VP2-1 protein was diluted to 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 μg / ml with coating buffer; positive serum was diluted to four dilutions (1:100, 1:200, 1:400, and 1:800) with sample dilution buffer; negative serum was diluted to three dilutions (1:100, 1:200, and 1:400) with sample dilution buffer. Sample dilution buffer was also used as a blank control. OD was measured according to the ELISA procedure. 450nm Value. OD of the positive control well. 450nm The value is around 1.0 and the P / N value (P: positive control well OD) is... 450nm Value, N: OD of negative control well 450nm The optimal antigen coating concentration and the optimal concentration of negative and positive sera for use when the value is maximized are the optimal antigen coating concentration and the optimal concentration of negative and positive sera for use.

[0061] The results showed that when the antigen concentration was 4 μg / mL, the OD of positive serum at each dilution was... 450nm The value reached a plateau; when the coating concentration was 8 μg / mL and the positive serum dilution was 1:800, OD... 450nm The value is close to 1.0; when the negative serum dilution is 1:200, the P / N value is the highest at 10.98, and the negative serum OD... 450nm Value compared to blank control OD 450nm The values ​​were high; therefore, the optimal antigen coating concentration was determined to be 8 μg / mL, the optimal dilution for positive serum was 1:800, and the optimal dilution for negative serum was 1:200. The results are shown in Tables 4-7.

[0062] Table 4 Results of ELISA square array titration assays using coated antigen and negative / positive sera (OD) 450nm )

[0063] Table 5. Ratio of positive serum to 1:100 diluted negative serum (P / N value)

[0064] Table 6. Ratio of positive serum to negative serum diluted 1:200 (P / N value)

[0065] Table 7. Ratio of positive serum to negative serum diluted 1:400 (P / N value)

[0066] III. Optimization of Enzyme-Linked Reaction Plate Preparation Conditions 1. Determination of antigen coating time Dilute the antigen to the optimal coating concentration using 0.1 mol / L pH 9.6 carbonate buffer and coat the ELISA plate with 100 μl / well. Incubate at 37°C for 3 hours and 4°C overnight, respectively. Perform the ELISA test according to the procedure and read the OD value. 450nm The values ​​are calculated, and the P / N ratio is determined to identify the optimal antigen coating conditions.

[0067] The P / N ratio was highest (12.04) when the coating plate was incubated overnight at 4°C. Therefore, the optimal coating condition was determined to be overnight at 4°C. The results are shown in Table 8.

[0068] Table 8 OD under different coating times and conditions 450nm Value measurement results Envelope conditions 37℃ 3h 4℃ overnight p-value 0.915 1.108 N value 0.083 0.092 P / N value 11.02 12.04 2. Determination of sealing solution and sealing time The antigen was diluted to the optimal coating concentration using 0.1 mol / L pH 9.6 carbonate buffer to coat the reaction plate. Blocking was performed using three different blocking solutions: 1.0% (g / ml) gelatin, 1.0% (g / ml) BSA, and 5.0% (g / ml) skim milk powder, 200 μl / well. Incubation was performed at 37°C for 1, 2, and 3 hours, respectively, under the same conditions. The experiment was conducted according to the ELISA procedure, and OD values ​​were measured. 450nm Calculate the P / N ratio to determine the optimal sealing solution and sealing time.

[0069] The blocking effects of the three blocking solutions were as follows: 1.0% (g / ml) BSA > 1.0% (g / ml) gelatin > 5.0% (g / ml) skim milk powder. The highest P / N value (12.45) was observed at a blocking time of 2 hours. Therefore, PBS containing 1.0% BSA and a blocking time of 2 hours were selected as the optimal blocking conditions. See the results below. Figure 9 , Figure 10 .

[0070] 3. Preparation method of enzyme-linked reaction plate: The feline panleukopenia virus VP2 recombinant protein VP2-1 described in claim 1 was dissolved in a carbonate solution at pH 9.6 and then added to a 96-well polystyrene ELISA plate. 100 μl of feline panleukopenia virus VP2 recombinant protein at a concentration of 8 μg / mL was added to each well. The plate was incubated overnight (8-12 hours) at 4°C to allow the feline panleukopenia virus VP2 recombinant protein to fully bind to the ELISA plate. Then, 300 μl of PBS buffer containing 0.01 g / mL bovine serum albumin at pH 7.4 was added to each well. The plate was blocked at 37°C for 2 hours, dried, and then sealed and stored at 4°C.

[0071] 4. Determination of working conditions for anti-rabbit enzyme-labeled secondary antibody HRP-labeled goat anti-rabbit IgG was diluted 1:5000, 1:10000, 1:20000, and 1:40000 with PBS (0.01 mol / L, pH 7.2), 100 μl / well. The reaction was carried out at 37°C for 0.5, 1, and 2 hours respectively, with all other conditions remaining the same. The experiment was performed according to the ELISA procedure, and OD values ​​were measured. 450nm The P / N ratio is calculated to determine the dilution and reaction time of the enzyme-labeled secondary antibody.

[0072] When the enzyme-labeled secondary antibody was diluted 1:10000 and incubated for 1 hour, the OD450nm value was closest to 1.0, and the P / N ratio was 11.12. Therefore, the optimal enzyme-labeled secondary antibody dilution was determined to be 1:10000, and the incubation time was determined to be 1 hour. Results are shown below. Figure 11 .

[0073] 5. Determining the substrate development time The substrate solution was incubated at room temperature for 10, 15, and 20 minutes, respectively. The reaction was terminated with 2 mol / L sulfuric acid, and all other conditions remained the same. The experiment was performed according to the ELISA procedure, and the OD values ​​were measured. 450nm Calculate the P / N value to determine the substrate development time.

[0074] OD of substrate solution after 15 minutes of color development at room temperature 450nm The value is close to 1.0, at which point the P / N ratio is 12.45, which is close to the value after 20 minutes of color development. Therefore, the color development time is determined to be 15 minutes at room temperature. The results are shown in Table 9.

[0075] Table 9 OD at different color development times 450nm Value measurement results Color development time (min) p-value N value P / N value 10 0.814 0.067 10.66 15 1.108 0.089 12.45 20 1.287 0.104 12.38 6. Determination of negative and positive control criteria Positive serum was diluted 1:800 and negative serum was diluted 1:200. Following an indirect ELISA assay, 50 tests were performed on both positive and negative sera. The OD values ​​of the 50 positive and negative sera were then analyzed. 450nm Statistical analysis of OD values ​​was performed to determine the OD values ​​of positive and negative serum. 450nm scope.

[0076] Fifty tests were performed on the same positive serum sample, with a mean OD value (X) of 1.476 and a standard deviation (S) of 0.157. The probability of OD values ​​falling within the range of X ± 3S (1.004–1.948) was 100% across the 50 tests, conforming to a normal distribution and demonstrating good stability. Therefore, the OD values ​​of positive serum... 450nm The absorbance was set at 1.0–2.0. Results are shown below. Figure 12 .

[0077] Fifty experiments were conducted on the same negative serum sample, with a mean OD value (X) of 0.119 and a standard deviation (S) of 0.032. The probability that the OD value fell within the range of X ± 3S (0.022–0.216) was 100.0% across the 50 experiments, conforming to a normal distribution and demonstrating good stability. Therefore, the OD values ​​of negative serum... 450nm The absorbance was set to be less than 0.3. Results are shown below. Figure 13 .

[0078] Example 3: Establishment of a Competitive ELISA Method The detection procedure for competing ELISA invention methods is as follows: 1. Equilibration: Remove all relevant samples from the refrigerated environment and allow them to equilibrate at room temperature for 30 minutes before use; mix liquid reagents thoroughly before use.

[0079] 2. Solution preparation: Dilute the concentrated washing solution 20 times with distilled water or deionized water to obtain the washing buffer solution; 3. Setup: 2 negative control wells and 2 positive control wells, with the rest being sample wells to be tested.

[0080] 4. Pre-dilution of the sample to be tested: Dilute the inactivated FPV antigen and recombinant feline panleukopenia virus VP2-1 protein in the sample to be tested in an appropriate ratio using sample diluent, and then perform serial dilution at a ratio of 2.

[0081] 5. Dilution of standards: Select appropriate standards (i.e., inactivated antigen standards for inactivated antigens and expressed protein standards for expressed proteins) according to the different sources of antigens and perform serial dilutions of 2-fold.

[0082] 6. Sample addition: Add 50µl of positive serum to each well according to the pre-set instructions, followed by 50µl of the proportionally diluted test sample. The sample addition process should be as short as possible.

[0083] 7. Incubation: Shake well and place in a 37°C incubator for 30 minutes.

[0084] 8. Washing the plate: Discard the reaction solution, add 300 μl of diluted washing buffer to each well, soak for 15 seconds, discard the washing solution, wash the plate 4 times and then pat dry.

[0085] 9. Add enzyme: Add 100 μl of the corresponding horseradish peroxidase-labeled IgG antibody to each well (add goat anti-rabbit IgG antibody to rabbit serum, and add goat anti-guinea pig IgG antibody to guinea pig serum).

[0086] 10. Incubation: Place in a 37℃ incubator and react for 30 minutes.

[0087] 11. Washing the plate: Discard the reaction solution, add 300µl of diluted washing buffer to each well, soak for 15s, discard the washing solution, wash the plate 4 times and then pat dry.

[0088] 12. Add 100µl of substrate working solution to each well for color development (the substrate working solution is obtained by mixing equal volumes of substrate solution A and substrate solution B, and should be prepared fresh before use), shake to mix well, and place in a 37℃ incubator to react for 15 min in the dark.

[0089] 13. Add 50µl of colorimetric stop solution to each well and shake to mix and terminate the reaction.

[0090] 14. Measure the OD of each well. 450nm The OD value (the reaction plate after adding the stop solution should be read within 15 minutes) 450nm value).

[0091] Interpretation of test results: 1. Negative control OD 450nm The average value should be less than 0.3; otherwise, it is invalid.

[0092] 2. The positive control should have a test value between 0.7 and 2.0; otherwise, it is invalid.

[0093] 3. Establishment of the standard curve: using the OD of the standard product... 450nm A standard curve is constructed using the values ​​on the x-axis (x-axis) and the concentration of standard protein on the y-axis (y-axis). R0 2 The value should be ≥0.98. 4. Determination of toxicity or protein concentration in the sample: The OD value of the sample is... 450nm Substitute two sets of data with values ​​close to 1 into the standard curve, calculate the original toxic value or protein concentration, and take the average value as the toxic value or protein concentration of the sample to be tested.

[0094] Example 4: Determination of the sensitivity of a competitive ELISA method using rabbit or guinea pig positive sera. The sensitivity of the competitive ELISA kit prepared according to the method in Example 2 was tested. FPV-positive serum was guinea pig or rabbit serum, and FPV-negative serum was SPF guinea pig or rabbit serum. The enzyme-labeled secondary antibody was horseradish peroxidase-labeled goat anti-rabbit IgG antibody or goat anti-guinea pig IgG antibody. The detection method is as shown in Example 3.

[0095] The sensitivity of two positive serum competitive ELISA methods was verified by purifying and inactivating FPV after sucrose density gradient centrifugation, purifying E. coli expressing feline panleukopenia virus VP2 protein (sequence 2 in the sequence listing), and purifying E. coli expressing recombinant feline panleukopenia virus VP2 protein (sequence 4 in the sequence listing). (The initial concentrations of the purified antigens were calibrated using BCA and SDS-PAGE, and the average value was taken.) The results are shown in Table 10. Table 10 Sensitivity Detection Results of Competitive ELISA Methods

[0096] Example 5: Specificity testing of two methods The competitive ELISA kit prepared according to the method in Example 2 was used for specific detection. FPV-positive serum was guinea pig or rabbit serum, and FPV-negative serum was SPF guinea pig or rabbit serum. The enzyme-labeled secondary antibody was horseradish peroxidase-labeled goat anti-rabbit IgG antibody or goat anti-guinea pig IgG antibody. The detection method is as shown in Example 3. The reactivity of positive control, feline calicivirus, feline rhinotracheitis, feline infectious peritonitis, feline panleukopenia, and rabies viruses were detected. The results showed that only the detection result with added FPV showed a specific reaction; the results for the other methods were all higher than 1.5, indicating good method specificity. The results are shown in Table 11. Table 11 Specific detection results Method 1 uses rabbit serum FCV FHV FIP FPV <![CDATA[Rab + ]]> <![CDATA[OD 450 Value 1.887 1.896 1.792 0.231 1.911 Method 2 uses guinea pig serum FCV FHV FIP FPV <![CDATA[Rab + ]]> <![CDATA[OD 450 Value 1.776 1.683 1.726 0.246 1.729 Example 7: Results of Competitive ELISA Standard Curve Plotting OD was performed using inactivated viruses of different viral titers. 450nm Value detection was performed, and a standard curve of viral titer - B / B0% was plotted. The OD value was set to 0 for the competing antigen. 450nm The value is B0, representing the OD values ​​of the corresponding antigen concentrations. 450nm The value is B. A standard curve is plotted with the logarithm of the competing antigen concentration on the x-axis and the binding rate (B / B0%) on the y-axis. The antigen-antibody competitive reactions established using different viral titers and the coated antigen show a good linear relationship (R0). 2 =0.992), the linear regression equation is Y = -18.463x + 145.77. The detection range is 103.5 TCID. 50 / mL~107.0 TCID50 / mL, limit of detection 103.5 TCID 50 / mL. Results are shown in Table 12 and... Figure 14 .

[0097] Table 12 Results of the determination of the competitive antigen standard curve <![CDATA[TCID 50 Logarithmic (X-axis) OD value <![CDATA[B / B0% (Y-axis)]]> 7.0 0.282 15.95 6.5 0.486 27.49 6.0 0.633 35.80 5.5 0.731 41.35 5.0 0.911 51.53 4.5 1.152 65.16 4 1.307 73.93 3.5 1.406 79.52 0 (virus-free) 1.768 / Example 8: Application of this competitive ELISA method in laboratory samples The established competitive ELISA method was used to detect samples from each process of the existing feline triple inactivated virus vaccine production and each sample from the FPV production process. The results are shown in Table 13, indicating that the two methods have good monitoring effects in the above antigen production (the VP2-1 protein expressed in E. coli is soluble).

[0098] Table 13 Results of the application of the competitive ELISA method in laboratory samples Method 1 uses rabbit serum live virus Inactivated virus After inactivated virus concentration and purification E. coli expression lysate supernatant 1 (10-fold dilution) Escherichia coli expression inclusion body 1 (10-fold dilution) Concentrated and purified protein (10-fold dilution) <![CDATA[OD 450 Value 0.487 0.482 0.282 0.572 1.618 0.446 Detecting antigen content <![CDATA[10 6.3 TCID 50 / ml]]> <![CDATA[10 6.3 TCID 50 / ml]]> <![CDATA[10 7.0 TCID 50 / ml]]> 43 ug / ml - 51 ug / ml Method 2 uses guinea pig serum Live virus antigen Inactivated antigen After inactivated antigen concentration and purification E. coli expression lysate supernatant 1 (10-fold dilution) Escherichia coli expression inclusion body 1 (10-fold dilution) Concentrated and purified protein (50-fold dilution) <![CDATA[OD 450 Value 0.492 0.494 0.314 0.596 1.677 0.482 Detecting antigen content <![CDATA[10 6.3 TCID 50 / ml]]> <![CDATA[10 6.3 TCID 50 / ml]]> <![CDATA[10 7.0 TCID 50 / ml]]> 43 ug / ml - 51 ug / ml The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are merely illustrative of selected implementations based on combinations of all possible embodiments. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A feline panleukopenia virus VP2 recombinant protein, the amino acid sequence of which is shown in sequence 4 in the sequence list.

2. The gene encoding the feline paneleukopenia virus VP2 recombinant protein according to claim 1, characterized in that, The nucleotide sequence of the coding gene is shown in sequence 3 in the sequence list.

3. A competitive ELISA kit for the accurate quantification of feline paneleukopenia virus antigen, characterized in that, The kit comprises a feline panleukopenia virus VP2 recombinant protein as an antigen coated enzyme-linked reaction plate, feline panleukopenia virus positive serum, feline panleukopenia virus negative serum, feline panleukopenia virus purified standard antigen and enzyme-labeled secondary antibody; the feline panleukopenia virus VP2 recombinant protein is the feline panleukopenia virus VP2 recombinant protein of claim 1.

4. The method of claim 3, wherein, The preparation method of the enzyme-linked reaction plate is to dissolve the feline panleukopenia virus VP2 recombinant protein of claim 1 in a carbonate solution with pH 9.6, then add to a 96-hole polystyrene enzyme-linked reaction plate, 100 μl of the feline panleukopenia virus VP2 recombinant protein with a concentration of 8 μg / mL in each hole, and place at 2-8℃ for 8-12 hours to make the feline panleukopenia virus VP2 recombinant protein fully combined with the enzyme-linked reaction plate, then add 300 μl / hole of PBS buffer containing 0.01 g / ml bovine serum albumin with pH 7.4, and seal and store at 4℃ after drying after blocking treatment at 37℃ for 2-3 hours.

5. The method of claim 3, wherein, The feline panleukopenia virus positive serum is rabbit serum or guinea pig serum collected after immunization with one or both of feline panleukopenia virus inactivated virus and E. coli expression system expressed feline panleukopenia virus VP2 protein; the feline panleukopenia virus negative serum is SPF guinea pig or rabbit serum.

6. The method of claim 3, wherein, The feline panleukopenia virus purified standard antigen is an antigen inactivated after sucrose density gradient centrifugation purification and virus content determination.

7. The method of claim 3, wherein, The enzyme-labeled secondary antibody is horseradish peroxidase labeled goat anti-rabbit IgG antibody or goat anti-guinea pig IgG antibody.

8. The kit of claim 3, wherein The kit further comprises substrate solution A, substrate solution B and termination solution, the substrate solution A is citric acid phosphate buffer containing 0.6 mg / ml hydrogen peroxide urea, the substrate solution B is 0.2 mg / ml tetramethyl benzidine solution, and the termination solution is 2 mol / L sulfuric acid solution.

9. The kit of claim 3, wherein The kit further comprises sample diluent and concentrated washing solution; the sample diluent is 0.01 mol / L phosphate buffer with pH value of 7.4 containing 0.005 g / ml casein, and the concentrated washing solution is 0.01 mol / L phosphate buffer with pH value of 7.4 containing 0.8%-1.2% volume percentage Tween-20.

10. Use of the feline panleukopenia virus VP2 recombinant protein of claim 1 in the preparation of a kit for detecting feline panleukopenia virus.

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