Competitive ELISA method for accurately and quantitatively detecting feline calicivirus antigen

Abstract

The invention discloses a competitive ELISA (Enzyme-Linked Immunosorbent Assay) kit capable of accurately quantifying a feline calicivirus antigen. The kit comprises an elisa plate, feline calicivirus negative / positive serum, a feline calicivirus standard antigen and an elisa secondary antibody. Wherein the elisa plate is coated with a feline calicivirus VP1 recombinant protein, and the sequence of the feline calicivirus VP1 recombinant protein is as shown in SEQ ID No. 2. A competitive ELISA method is adopted, the feline calicivirus VP1 gene recombinant protein expressed by an escherichia coli expression system is small in antigen dosage and high in sensitivity and specificity, and whether feline calicivirus antigen exists or not can be efficiently detected; meanwhile, the protein content of the feline calicivirus VP1 gene or the virus content of the feline calicivirus in the antigen or the vaccine can be accurately quantified by using different samples. 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] The present invention belongs to the field of biotechnology. More specifically, the present invention relates to a competitive ELISA method for accurately quantifying feline calicivirus antigen. Background Art

[0002] FCV belongs to the genus Vesivirus of the family Caliciviridae, and is a spherical non-enveloped virus with icosahedral symmetry, with a diameter of 35-39 nm. The virus capsid is composed of 180 protein molecules arranged in dimers, forming 90 capsomers. The capsid contains only one kind of protein (VP1) in its biological composition. The FCV genome is a single-stranded positive-strand linear RNA with a full length of 7,683 bp.

[0003] FCV infection usually mainly affects the upper respiratory tract and oral cavity of cats. The frequent appearance of blisters and ulcers on the tongue is a typical infection feature. In addition, it can also cause limb ulcers, lameness, abortion, pneumonia and feline chronic gingivostomatitis, and highly virulent FCV strains can even cause death. Feline calicivirus (FCV) is a highly contagious pathogen widely distributed in the cat population. Almost all felids are susceptible to FCV, and there are also reports of its infection in dogs. Given its highly transmissible characteristics, the research on FCV vaccines is particularly important.

[0004] At present, there is only one FCV vaccine, Nobivac TriCat, produced by Zoetis in China. With the rapid development of the domestic pet industry, the research and development of domestic feline triple vaccines have emerged one after another. Therefore, the evaluation of vaccine quality, antigen content, intermediate products, etc. is particularly important. At present, there are colloidal gold test strips for detecting FCV virus produced by multiple different manufacturers on the market, but their disadvantages are low sensitivity and inability to quantify; while the commonly used cell detection methods for detecting virus titers, one is that it takes several days to obtain results, and in addition, the samples detected are limited to live viruses. Currently, inactivated vaccines, attenuated live vaccines, and subunit vaccines each have their own advantages, and different animal health manufacturers will naturally choose different vaccine processes. In this context, the present invention aims to provide a competitive ELISA method that can accurately quantify FCV antigen, which is used to quantitatively detect the content of FCV antigen in vaccines, intermediate products, protein concentrations of vector expression products or cell cultures, so as to provide assistance for the research of FCV-related biological products. Summary of the Invention

[0005] The purpose of the present invention is to provide a competitive ELISA method that can accurately quantify feline calicivirus (FCV) antigen. This method uses the FCV VP1 recombinant protein as the coating antigen to establish a competitive ELISA method with good specificity, sensitivity and repeatability, which is used to quantitatively detect the content of FCV antigen in vaccines, intermediate products, protein concentrations of vector expression products or cell cultures.

[0006] To achieve the above object, the present invention first analyzes and predicts to screen out the immunogenic dominant region in the excellent performance feline calicivirus VP1, and then obtains the antigen protein by tandem connection through a linker. The recombinant feline calicivirus VP1 protein provided by the present invention is a recombinant feline calicivirus VP1 protein expressed by an Escherichia coli expression system, and its amino acid sequence is SEQ ID No.2. The nucleotide sequence of the recombinant feline calicivirus VP1 protein is SEQ ID No.1.

[0007] The present invention provides a competitive ELISA kit for accurately quantifying FCV antigen. The kit includes an enzyme-linked reaction plate coated with the recombinant feline calicivirus VP1 protein as an antigen, FCV positive serum, FCV negative serum, FCV purified standard antigen, and an enzyme-labeled secondary antibody; the amino acid sequence of the recombinant feline calicivirus VP1 protein is SEQ ID No.2.

[0008] For the competitive ELISA kit for accurately quantifying FCV antigen, the enzyme-linked reaction plate is a detachable 96-well microtiter plate; the recombinant feline calicivirus VP1 protein is a protein expressed by a prokaryotic system.

[0009] The best preparation method and conditions of the enzyme-linked reaction plate are to dissolve the recombinant feline calicivirus VP1 protein in a carbonate solution with a pH of 9.6, and then add it to a 96-well polystyrene enzyme-linked reaction plate, 100 μl per well, 1 μg / mL recombinant feline calicivirus VP1 gene recombinant protein, composition, and place it at 2-8 °C for 8-12 hours to allow the composition antigen to fully bind to the enzyme-linked reaction plate. Then, add a PBS buffer solution containing 1% (g / ml) bovine serum albumin (BSA) with a pH of 7.4 at 300 μl / well, and perform a blocking treatment at 37 °C for 2-3 hours. After centrifuging to dryness, store it sealed at 4 °C after the enzyme-linked reaction plate is dried.

[0010] The FCV positive serum is rabbit serum or guinea pig serum collected after immunization with inactivated FCV virus; the FCV negative serum is SPF guinea pig or rabbit serum.

[0011] The FCV purified standard antigen is a recombinant protein expressed by Escherichia coli, purified by a protein purification column and with its concentration determined, or an antigen purified by sucrose density gradient centrifugation, with its virus content determined and inactivated.

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

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

[0014] The method further includes a sample diluent and a concentrated washing solution (20 times); the sample diluent is a 0.01 M phosphate buffer with a pH value of 7.4 containing 0.5% casein; the concentrated washing solution is a 0.01 M phosphate buffer with a pH value of 7.4 containing 0.8% - 1.2% (ml / ml) of Tween-20.

[0015] The detection procedure of the method of the present invention is as follows:

[0016] 1. Equilibration: Take out all samples related to the detection from the refrigerated environment, place them at room temperature for 30 min for standby; mix the liquid reagents before use.

[0017] 2. Solution preparation: Dilute the concentrated washing solution 20 times with distilled water or deionized water to obtain a washing buffer solution; 3. Setting: Set 2 negative control wells and 2 positive control wells, and the rest are wells for samples to be tested.

[0018] 4. Pre-dilution of samples to be tested: Use the sample diluent to dilute the samples to be tested, such as the recombinant protein expressing feline calicivirus VP1 gene, inactivated FCV antigen, processed vaccine, etc., in an appropriate ratio, and then perform serial dilutions at a two-fold ratio.

[0019] 5. Dilution of standards: According to different sources of antigens, select appropriate standards (i.e., select protein expression standards for expressed proteins and inactivated antigen standards for inactivated antigens) for serial dilutions at a two-fold ratio.

[0020] 6. Sample addition: Add 50 μl of positive serum to each well as pre-set, and then add 50 μl of the sample to be tested diluted in proportion. The time span during the sample addition process should be as short as possible.

[0021] 7. Incubation: Mix well by shaking, place in an incubator at 37 °C, and react for 30 min.

[0022] 8. Plate washing: Discard the reaction solution, add 300 μl of the diluted washing buffer solution to each well, soak for 15 s, discard the washing solution by centrifugation, wash the plate continuously 4 times, and then pat dry.

[0023] 9. Enzyme addition: 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 to guinea pig serum).

[0024] 10. Incubation: Place in an incubator at 37 °C and react for 30 min.

[0025] 11. Plate washing: Discard the reaction solution, add 300 μl of diluted washing buffer to each well, soak for 15 s, discard the washing solution by shaking off, and pat dry after washing the plate 4 times continuously.

[0026] 12. Color development: Add 100 μl of substrate working solution to each well (mix equal amounts of substrate solution A and substrate solution B to obtain the substrate working solution, prepare it freshly before use), mix well by shaking, place it in an incubator at 37 °C, and react for 15 min in the dark.

[0027] 13. Add 50 μl of color development termination solution to each well, mix well by shaking to terminate the reaction.

[0028] 14. Measure the OD 450nm value of each well (the reaction plate with the termination solution added should be read for the OD 450nm value within 15 min).

[0029] Judgment of test results:

[0030] 1. The average OD 450nm value of the negative control should be < 0.3, otherwise it is invalid.

[0031] 2. Each test value of the positive control should be between 0.7 and 2.0, otherwise it is invalid.

[0032] 3. Establishment of the standard curve: Use the OD 450nm value of the standard product as the abscissa (X-axis), and the protein concentration of the standard product as the ordinate (Y-axis) to establish the standard curve. The R 2 value should be ≥ 0.98.

[0033] 4. Determination of the concentration of the sample to be tested: Substitute the two groups of data with the OD 450nm value of the sample to be tested close to 1 into the standard curve, calculate the original concentration, and take the average value as the concentration of the sample to be tested.

[0034] The above detection method of the present invention can be used to detect feline calicivirus antigen proteins, feline calicivirus antigens or vaccines from different sources.

[0035] The positive effects of the present invention are as follows: The recombinant protein of the feline calicivirus VP1 gene expressed by Escherichia coli is used in the present invention, making the method have the advantages of high sensitivity and strong specificity.

[0036] This method has good specificity and has no cross-reaction with common feline pathogenic viruses such as feline rhinotracheitis virus, feline infectious peritonitis virus, feline panleukopenia virus, rabies virus, etc.;

[0037] This method can detect FCV antigens from different sources, such as FCV live virus, inactivated virus, and the protein of the VP1 gene expressed by Escherichia coli. The lowest detection limit of the method can reach 0.2 μg / ml or 105.5 TCID 50 / mL; It can be applied to the whole process of the preparation of the corresponding vaccine;

[0038] In summary, this method uses the recombinant protein of feline calicivirus VP1 gene expressed by Escherichia coli to coat the enzyme-linked reaction plate, with less antigen consumption, high sensitivity and strong specificity, and can effectively detect FCV antigens or vaccines from different sources. The experimental results show that the detection method of the present invention has good repeatability, strong specificity and high sensitivity. It can meet the needs of people at different levels and has broad market prospects and good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the SDS-PAGE picture of the purified VP1 recombinant protein;

[0040] Figure 2 It is the SDS-PAGE result of the purified rabbit anti-FCV positive serum;

[0041] Figure 3 It is the Western-blot result of the purified VP1 protein and the rabbit anti-FCV positive serum;

[0042] Figure 4 It is the Western-blot result of the purified VP1 protein and the rabbit negative serum;

[0043] Figure 5 It is the P / N value of different blocking solutions;

[0044] Figure 6 It is the P / N value of different blocking times;

[0045] Figure 7 It is the P / N value of different dilution degrees and reaction times of the anti-rabbit enzyme-labeled secondary antibody;

[0046] Figure 8 It is the OD 450nm value distribution diagram of the rabbit anti-FCV positive serum;

[0047] Figure 9 It is the OD 450nm value distribution diagram of the rabbit negative serum;

[0048] Figure 10 It is the competitive ELISA standard curve. DETAILED DESCRIPTION OF THE INVENTION

[0049] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of this invention are for the purpose of describing specific embodiments only and are not intended to limit the invention.

[0051] It should be clear that the experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0052] As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover non-exclusive inclusion. For example, a composition, step, method or article containing the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method or article.

[0053] The methods in the following examples are all conventional methods unless otherwise specified.

[0054] Example 1 Expression and Identification of Feline Calicivirus VP1 Protein

[0055] 1. Construction of Recombinant VP1 Protein Expression Vector

[0056] First, the present invention screened the immunogenic dominant regions in feline calicivirus VP1 with excellent performance through analysis and prediction, obtained the antigen protein by tandem connection through linker, optimized the codons of the constructed antigen protein sequence for Escherichia coli, and obtained the recombinant protein sequence of feline calicivirus VP1, and its sequence is SEQ ID No.2. The nucleotide sequence of the recombinant protein of feline calicivirus VP1 is SEQ ID No.1.

[0057] The recombinant protein sequence of feline calicivirus VP1 was synthesized into the pET-32a(+) vector, and the restriction enzyme sites were EcorⅠ and SacⅠ. The constructed pET-32a-VP1 was transformed into BL21 competent cells, and the expression of the recombinant plasmid in Escherichia coli was accurately induced by IPTG. After identification and purification, the recombinant VP1 protein of feline calicivirus was obtained.

[0058] 2. Identification of Expression Results

[0059] The SDS-PAGE expression identification results of the VP1 recombinant protein showed that, compared with the control before induction, after IPTG induction, specific proteins with the expected size could be expressed in the whole bacteria, the supernatant and precipitate of the disrupted bacteria, and the expression products were mainly expressed in the form of inclusion bodies. The optimal induction expression conditions for the VP1 recombinant protein were: inducing at 37 °C for 4 hours and harvesting the centrifugal precipitate after disrupting the bacteria. It was measured that the expression level of the VP1 recombinant protein reached 0.6 μg / ml, which was better than that of the unmodified VP1 protein control (0.3 μg / ml). Estimated by SDS-PAGE gel scanning analysis with Coomassie brilliant blue staining, the size of this recombinant protein was about 70 kDa, which was consistent with the expected result. The results are shown in Figure 1 。

[0060] 3. Purification results and specific identification

[0061] The SDS-PAGE and Western-blot results of the purified VP1 recombinant protein showed that the expressed VP1 protein was purified, and the collected samples were concentrated by ultrafiltration (ultrafiltration tube), with a purity > 90%.

[0062] Example 2 Preparation of rabbit anti-FCV positive serum

[0063] 1. Initial preparation of rabbit anti-FCV positive serum

[0064] Two healthy and unimmunized male New Zealand white rabbits were selected and immunized with inactivated feline calicivirus and recombinant VP1 protein of feline calicivirus expressed by Escherichia coli expression system as antigens. The immunization protocol was as follows:

[0065] (1) First immunization: The inactivated FCV virus and recombinant VP1 protein of feline calicivirus expressed by Escherichia coli expression system were fully mixed with an equal volume of Freund's complete adjuvant and emulsified completely, and 2 ml of the above emulsion was injected subcutaneously at multiple points on the back of the rabbit;

[0066] (2) Second immunization: 14 days after the first immunization, the above composition was mixed with an equal volume of Freund's incomplete adjuvant and emulsified completely, and 2 ml of the above emulsion was injected subcutaneously at multiple points on the back of the rabbit;

[0067] (3) Third immunization: 14 days after the second immunization, repeat the operation in (2);

[0068] (4) 7 days after the third immunization, blood was collected and indirect ELISA was performed. If the antibody titer in the measured serum met the conditions, no booster immunization was required, and blood was directly collected to collect serum for standby; if the conditions were not met, booster immunization was continued until the qualified titer; it was measured that the antibody titer was good after the third immunization in the VP1 recombinant protein immunized group and was better than that of the unmodified VP1 protein control group.

[0069] (5) The negative serum is the SPF rabbit serum collected and centrifuged.

[0070] 2. Purification and identification of polyclonal antibody

[0071] The rabbit serum with qualified titer above was purified using a Protein G Sepharose affinity purification chromatography column. The serum prepared after immunization was slowly loaded onto the column after being mixed equally with PBS buffer. After the antigen-antibody binding was complete, elution was carried out with glycine elution buffer, and the purified antibody was collected. An appropriate amount of PBS buffer was added to the collected purified antibody, and dialysis was carried out overnight at 4 °C. The purified antibody was taken out the next day, which was the polyclonal antibody. After aliquoting, it was stored at -80 °C for standby.

[0072] Through SDS-PAGE analysis, the purity of the purified antibody was observed (the results are shown in Figure 5 ), and the specificity of negative and positive sera was detected by WB ( Figure 3 , Figure 4 ).

[0073] Example 3 Establishment of competitive ELISA method (based on rabbit anti-FCV positive serum)

[0074] 1. Determination of the optimal coating concentration of antigen and the optimal working concentrations of negative and positive sera

[0075] Square titration tests were carried out according to the orthogonal matrix method. The VP1 recombinant protein was diluted to 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 μg / ml with coating buffer; the positive serum was diluted to 1:100, 1:200, 1:400, 1:800-fold in 4 dilutions with sample diluent; the negative serum was diluted to 1:100, 1:200, and 1:400-fold in 3 dilutions with sample diluent, and at the same time, the sample diluent was set as the blank control. According to the ELISA operation procedure, the OD 450nm value was measured. Taking the OD 450nm value of the positive control well around 1.0 and the optimal antigen coating concentration and the optimal working concentrations of negative and positive sera when the P / N value (P: OD 450nm value of the positive control well, N: OD 450nm value of the negative control well) was the largest as the optimal antigen coating concentration and the optimal working concentrations of negative and positive sera.

[0076] The results showed that when the antigen concentration was 1 μg / mL, the OD 450nm values of positive sera at each dilution reached a plateau; when the coating concentration was 2 μg / mL and the dilution of positive serum was 1:400, the OD 450nm value was close to 1.0; when the dilution of negative serum was 1:200, the P / N value was the largest at 11.47, and the OD 450nm value of negative serum was higher than the OD of the blank control450nm The value is high. Therefore, the optimal coating concentration of the antigen was determined to be 2 μg / mL, the optimal dilution of the positive serum was 1:400, and the optimal dilution of the negative serum was 1:200. The results are shown in Tables 1 to 4.

[0077] Table 1 Results of ELISA checkerboard titration of coated antigen and negative and positive sera (OD 450nm )

[0078]

[0079]

[0080] Table 2 Ratio of positive serum to negative serum diluted 1:100 (P / N value)

[0081]

[0082] Table 3 Ratio of positive serum to negative serum diluted 1:200 (P / N value)

[0083]

[0084] Table 4 Ratio of positive serum to negative serum diluted 1:400 (P / N value)

[0085]

[0086]

[0087] 2. Determination of antigen coating time

[0088] The antigen was diluted to the optimal coating concentration with 0.1 mol / L carbonate buffer at pH 9.6 and used to coat the ELISA plates at 100 μl / well. After incubation at 37°C for 3 hours and overnight at 4°C respectively, the ELISA procedure was followed for the test, and the OD 450nm value was measured and the P / N value was calculated to determine the optimal antigen coating conditions.

[0089] When the antigen-coated reaction plates were incubated overnight at 4°C, the P / N value was the largest, which was 12.03. Therefore, the optimal coating condition was determined to be overnight incubation at 4°C. The results are shown in Table 5.

[0090] Table 5 Results of OD450nm value determination under different coating times and conditions

[0091] Coating conditions 37℃3h Overnight at 4°C P value 1.012 1.131 N value 0.091 0.094 P / N value 11.12 12.03

[0092] 3. Determination of blocking solution and blocking time

[0093] Dilute the antigen to the optimal coating concentration with 0.1 mol / L carbonate buffer solution at pH 9.6, coat the reaction plate, and block it with three kinds of blocking solutions containing 1.0% gelatin, 1.0% BSA, and 5.0% skim milk powder, 200 μl / well, incubate at 37 °C for 1, 2, and 3 hours, with the same other conditions. Conduct the experiment according to the ELISA operation procedure, read the OD 450nm value, calculate the P / N value, and determine the optimal blocking solution and blocking time.

[0094] The blocking effects of the three blocking solutions are in the order of 1.0% BSA > 1.0% gelatin > 5.0% skim milk powder. When the blocking time is 2 hours, the P / N value is the largest, which is 13.34. Therefore, select PBS containing 1.0% BSA as the blocking solution and 2 hours as the blocking time as the optimal blocking conditions. The results are shown in Figure 5 , Figure 6 .

[0095] 4. Determination of the working conditions of the enzyme-labeled secondary antibody against rabbit

[0096] Dilute the HRP-labeled goat anti-rabbit IgG with PBS (0.01 mol / L, pH 7.2) buffer solution at dilutions of 1:5000, 1:10000, 1:20000, and 1:40000 respectively, 100 μl / well, and the reaction conditions are to incubate at 37 °C for 0.5, 1, and 2 hours respectively, with the same other conditions. Conduct the experiment according to the ELISA operation procedure, read the OD450nm value, calculate the P / N value, and determine the dilution ratio and action time of the enzyme-labeled secondary antibody.

[0097] When the dilution ratio of the enzyme-labeled secondary antibody is 1:10000 and the action time is 1 h, the OD 450nm value is closest to 1.0, and the P / N value at this time is 11.69. Therefore, determine the dilution ratio of the enzyme-labeled secondary antibody to be 1:10000 and the action time to be 1 hour. The results are shown in Figure 7 .

[0098] 5. Determination of the substrate color development time

[0099] Let the substrate color development solution develop color at room temperature for 10, 15, and 20 minutes respectively, terminate the reaction with 2 mol / L sulfuric acid, with the same other conditions. Conduct the experiment according to the ELISA operation procedure, read the OD 450nm value, calculate the P / N value, and determine the substrate color development time.

[0100] When the substrate solution develops color at room temperature for 15 minutes, the OD 450nm value is close to 1.0, and the P / N value at this time is 12.84. Therefore, determine the color development time to be 15 minutes at room temperature. The results are shown in Table 6.

[0101] Table 6 OD 450nm value measurement results

[0102]

[0103] 6. Determination of the standards for positive and negative controls

[0104] Dilute the positive serum at 1:400 and the negative serum at 1:200. According to the indirect ELISA test, perform 50 detections on the positive and negative sera. Through the statistical analysis of the OD 450nm values of 50 positive and negative sera, determine the OD 450nm range of the positive and negative sera.

[0105] Perform 50 tests on the same positive serum. The average OD value (X) is 1.361, and the standard deviation (S) is 0.218. For the data of 50 tests, the probability that the OD value is within the range of X ± 3S (0.706 - 2.015) is 100%, which conforms to the normal distribution and has good stability. Therefore, set the OD 450nm absorbance value of the positive serum to 0.7 - 2.0. The results are shown in Figure 8 .

[0106] Perform 50 tests on the same negative serum. The average OD value (X) is 0.129, and the standard deviation (S) is 0.038. For the data of 50 tests, the probability that the OD value is within the range of X ± 3S (0.014 - 0.243) is 100.0%, which conforms to the normal distribution and has good stability. Therefore, set the OD 450nm absorbance value of the negative serum to less than 0.3. The results are shown in Figure 9 .

[0107] Example 4. Determination of the within - batch repeatability of the competitive ELISA method using rabbit - derived or guinea - pig - derived positive sera

[0108] Take 6 enzyme - labeled plates coated with the same batch, and use rabbit - derived or guinea - pig - derived positive sera as competitive antibodies to detect the calibrated 50 μg / ml FCV - VP1 protein. Each positive serum is used to detect 3 plates, and each plate has 5 replicates. According to the operating procedure of the competitive ELISA test, examine the final OD 450nm value and calculate the within - batch coefficient of variation.

[0109] Using the competitive ELISA method, repeat the detection of the OD 450nm value of the calibrated 50 μg / ml FCV - VP1 protein 5 times with the reaction plates coated with the same batch. When using rabbit - derived positive serum as the competitive antibody, the within - batch coefficient of variation is 2.54%; when using guinea - pig - derived positive serum as the competitive antibody, the within - batch coefficient of variation is 1.84%. It shows that the within - batch repeatability of this method is good. The results are shown in Tables 7 and 8.

[0110] Table 7 OD Results of Intra - batch Repeatability Detection Based on Rabbit Anti - FCV Positive Serum 450nm Value Results

[0111]

[0112] Table 8 OD Results of Intra - batch Repeatability Detection Based on Guinea Pig Anti - FCV Positive Serum 450nm Value Results

[0113]

[0114]

[0115] Example 5. Establishment of Competitive ELISA Method

[0116] The detection procedure of the competitive ELISA invention method is as follows:

[0117] 1. Equilibration: Take out all detection - related samples from the refrigerated environment, place them at room temperature for 30 min for equilibration and reserve; mix the liquid reagents before use.

[0118] 2. Solution preparation: Dilute the concentrated washing solution 20 - fold with distilled water or deionized water to obtain the washing buffer;

[0119] 3. Setting: Set up 2 negative control wells and 2 positive control wells, and the rest are wells for samples to be tested.

[0120] 4. Pre - dilution of samples to be tested: After inactivating the FCV antigen, recombinant expressed feline calicivirus VP1 protein, etc. in the samples to be tested with the sample diluent and diluting them in an appropriate ratio, perform serial two - fold dilutions.

[0121] 5. Dilution of standards: According to different sources of antigens, select appropriate standards (i.e., inactivated antigen standards for inactivated antigens and expressed protein standards for expressed proteins) and perform serial two - fold dilutions.

[0122] 6. Sample addition: Add 50 μl of positive serum to each well as pre - set, and then add 50 μl of the proportionally diluted sample to be tested. The time span during the sample addition process should be as short as possible.

[0123] 7. Incubation: Mix well by shaking, place in an incubator at 37 °C for 30 min.

[0124] 8. Plate washing: Discard the reaction solution, add 300 μl of the diluted washing buffer to each well, soak for 15 s, discard the washing solution, wash the plate continuously 4 times and then pat dry.

[0125] 9. Enzyme addition: Add 100 μl of the corresponding horseradish peroxidase - labeled IgG antibody to each well (add goat anti - rabbit IgG antibody to rabbit serum and goat anti - guinea pig IgG antibody to guinea pig serum).

[0126] 10. Incubation: Place in an incubator at 37°C and react for 30 min.

[0127] 11. Plate washing: Discard the reaction solution, add 300 μl of diluted washing buffer to each well, soak for 15 s, discard the washing solution, and pat dry after washing the plate 4 times continuously.

[0128] 12. Color development: Add 100 μl of substrate working solution to each well (mix equal amounts of substrate solution A and substrate solution B to obtain the substrate working solution, prepare it freshly before use), mix well by shaking, place in an incubator at 37°C, and react in the dark for 15 min.

[0129] 13. Add 50 μl of color development stop solution to each well and mix well by shaking to terminate the reaction.

[0130] 14. Measure the OD of each well 450nm value (the reaction plate with the addition of the stop solution should have the OD 450nm value read within 15 min).

[0131] Judgment of test results:

[0132] 1. The average OD of the negative control 450nm should be < 0.3, otherwise it is invalid.

[0133] 2. Each test value of the positive control should be between 0.7 and 2.0, otherwise it is invalid.

[0134] 3. Establishment of the standard curve: Use the OD of the standard product 450nm value as the abscissa (X-axis), and the protein concentration of the standard product as the ordinate (Y-axis) to establish the standard curve. The R 2 value should be ≥ 0.98.

[0135] 4. Determination of the titer or protein concentration of the sample to be tested: Substitute the two sets of data with the OD 450nm value of the sample to be tested close to 1 into the standard curve, calculate the original titer or protein concentration, and take the average value as the titer or protein concentration of the sample to be tested.

[0136] Example 6. Establishment of the standard curve

[0137] Dilute the feline calicivirus standard antigen with the sample diluent to obtain a standard series of virus contents, namely 10 9.5 TCID 50 / ml, 10 9.0 TCID 50 / ml, 10 8.5 TCID 50 / ml, 10 8.0 TCID 50 / ml, 10 7.5 TCID 50 / ml, 107.0 TCID 50 / ml, 10 6.5 TCID 50 / ml, 10 6.0 TCID 50 / ml, 10 5.5 TCID 50 / ml, 0,, the detection was carried out according to the operation of Example 5. Taking the OD value with the competitive antigen concentration of 0 as B 450nm value, and the OD value of each corresponding antigen titer was B. Using the logarithm of the competitive antigen concentration as the abscissa and the binding rate (B / B 0 %) as the ordinate, a standard curve was plotted. 450nm value, and the OD value of each corresponding antigen titer was B. Using the logarithm of the competitive antigen concentration as the abscissa and the binding rate (B / B 0 %) as the ordinate, a standard curve was plotted.

[0138] Applying competitive antigens with different virus titers, the antigen-antibody competitive reaction established with the coated antigen has a good linear relationship (R 2 = 0.9932), and the linear regression equation is Y = -18.803x + 190.28. The detection range is 10 5.5 TCID 50 / mL to 10 9.5 TCID 50 / mL, and the minimum detection limit is 10 5.5 TCID 50 / mL. The results are shown in Figure 10 .

[0139] Example 7 Specificity Test

[0140] The established ELISA method was used to detect the liquid of eight strains of pathogens, such as feline parvovirus (FPV), feline herpesvirus (FHV), feline infectious peritonitis virus type I (FIP-1), feline infectious peritonitis virus type II (FIP-2), Mycoplasma felis (MF), Chlamydia felis (CF), Bordetella bronchiseptica (Bb), and feline calicivirus (FCV), respectively. At the same time, negative controls and blank controls were set up. To identify whether the established ELISA detection method has non-specific cross-reactions with other common respiratory viruses of cats except feline calicivirus. The results showed that except for FCV, the OD 450nm values of other pathogen detections were all less than 0.3, and the results were all negative. As shown in Table 9, it was proved that the established method has no cross-reaction with the above viruses and has good specificity.

[0141] Table 9 Results of OD 450nm values in the specificity test

[0142]

[0143] Example 8 Sensitivity Test

[0144] The feline calicivirus was diluted at 10 different concentrations, namely 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, 1:6561, 1:19683, and 1:59049, and measured using the established ELISA method. The results showed that the lowest detection dilution of the method for FCV reached the critical value at 1:19683, indicating that the detection method established in this experiment had high sensitivity, as shown in Table 10.

[0145] Table 10 Sensitivity Test

[0146]

[0147] The foregoing examples are merely illustrative and are used to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as can be contemplated, and the embodiments presented herein are merely illustrative of selected implementations from all possible combinations of embodiments. Therefore, the applicant's intention is that the appended claims not be limited by the selection of examples that illustrate the features of the present invention. Some of the 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 whenever possible.

Claims

1. A recombinant protein of feline calicivirus VP1, the sequence of which is shown in SEQ ID No.

2.

2. The coding gene of the feline calicivirus VP1 recombinant protein according to claim 1, whose sequence is shown in SEQ ID No.

1.

3. A competitive ELISA kit for accurate quantification of feline calicivirus antigen, characterized in that: The kit comprises an enzyme-linked reaction plate coated with a feline calicivirus VP1 recombinant protein as an antigen, a feline calicivirus positive serum, a feline calicivirus negative serum, a feline calicivirus standard antigen and an enzyme-labeled secondary antibody; the feline calicivirus VP1 recombinant protein is the feline calicivirus VP1 recombinant protein according to claim 1.

4. The kit according to claim 3, characterized in that The preparation method of the enzyme-linked reaction plate is as follows: the feline calicivirus VP1 recombinant protein according to claim 1 is dissolved in 100 μl of a carbonate solution with a pH of 9.6, and then added to a 96-well polystyrene enzyme-linked reaction plate, with 100 μl of a feline calicivirus VP1 recombinant protein with a concentration of 1 μg / mL in each well, and placed at 2 to 8° C. for 8 to 12 hours to allow the feline calicivirus VP1 recombinant protein to fully combine with the enzyme-linked reaction plate, and then 300 μl / well of a PBS buffer with a pH of 7.4 is added, and the plate is sealed at 37° C. for 2 to 3 hours, and after being dried, the enzyme-linked reaction plate is sealed and stored at 4° C. after drying.

5. The kit according to claim 3, characterized in that The feline calicivirus-positive serum is rabbit serum or guinea pig serum collected after immunization with feline calicivirus; the feline calicivirus-negative serum is SPF guinea pig or rabbit serum.

6. The kit according to claim 3, characterized in that The feline calicivirus standard antigen is an antigen that has been purified by sucrose density gradient centrifugation, tested for viral content, and inactivated.

7. The kit according to claim 3, characterized in that The enzyme-labeled secondary antibody is a goat anti-rabbit IgG antibody or a goat anti-guinea pig IgG antibody labeled with horseradish peroxidase.

8. The kit according to claim 3, characterized in that The kit also includes substrate solution A, substrate solution B and stop solution, wherein the substrate solution A is a citric acid phosphate buffer solution containing 0.6 mg / ml hydrogen peroxide urea, the substrate solution B is a 0.2 mg / ml tetramethylbenzidine solution; and the stop solution is a 2 mol / L sulfuric acid solution.

9. The kit according to claim 3, characterized in that The kit also includes a sample diluent and a concentrated washing solution; the sample diluent is a phosphate buffer solution of 0.01 mol / L and pH 7.4 containing 0.005 g / ml casein; the concentrated washing solution is a phosphate buffer solution of 0.01 mol / L and pH 7.4 containing 0.8% to 1.2% Tween-20 by volume.

10. Use of a recombinant protein of the feline calicivirus VP1 gene in the preparation of a kit for detecting feline calicivirus antigens; preferably, the kit is a competitive ELISA kit for quantitatively detecting feline calicivirus antigens.

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