A competitive elisa kit for accurate quantification of feline rhinotracheitis virus antigen

Abstract

The application discloses a competitive ELISA kit for accurately quantifying feline rhinotracheitis virus antigen. The kit comprises an enzyme-labeled plate, feline rhinotracheitis virus negative / positive serum, feline rhinotracheitis virus standard antigen and enzyme-labeled secondary antibody. The enzyme-labeled plate is coated with feline rhinotracheitis virus gD recombinant protein, and the sequence of the protein is shown in sequence 5 in the sequence list. The application adopts the competitive ELISA method, uses the feline rhinotracheitis virus gD gene recombinant protein expressed by an E. coli expression system, has less antigen consumption, high sensitivity and high specificity, can efficiently detect whether the feline rhinotracheitis virus antigen exists, and can accurately quantify the feline rhinotracheitis gD gene protein content or the feline rhinotracheitis virus titer in different samples. The kit has high sensitivity, good specificity and convenient operation, 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 kit for the accurate quantitative detection of feline rhinotracheitis virus antigen. Technical Background

[0002] The causative agent of feline infectious rhinobronchitis (FHV) is feline herpesvirus (FHV), belonging to the Herpesviridae family, Alphaherpesvirinae subfamily. It is a linear double-stranded DNA virus characterized by a short replication cycle, rapid spread, induction of cell lysis, and latent infection. It is a large virus; the viral particle is round or oval, icosahedral, and consists of a core, capsid, and envelope. The core is a uniform, round, electron-dense region. The outer capsid consists of 162 capsomeres, and the envelope contains the viral membrane and spikes. Typical clinical symptoms of FHV infection include fever, sneezing, coughing, keratoconjunctivitis, serous or purulent nasal and ocular discharge, and periorbital skin ulcers. It can occur in both kittens and adult cats, primarily infecting cats aged 2-4 months, with a morbidity rate as high as 100%. While it does not cause death in adult cats, the mortality rate in kittens can reach 50%, making it one of the most serious known feline respiratory diseases. Recovered cats can remain carriers, causing persistent or latent infections.

[0003] The gD protein of FHV is a major component of the viral envelope, present on the cell membrane of infected cells. It is highly conserved and antigenic, capable of specifically binding to molecules on the cell surface. It induces cellular and humoral immunity, plays a crucial role in viral penetration of susceptible cells, and is one of the main targets of host cellular and humoral immune responses. The gD protein is a highly conserved immunogenic protein, often chosen as a target protein for research on herpesvirus subunit vaccines, and possesses good immunogenicity.

[0004] Currently, with the improvement of people's living standards, more and more people are choosing cats as pets, and cat health has gradually become a hot topic. Studies have shown that the most effective measure against FHV remains vaccination; therefore, more and more animal health companies are beginning to develop commercial vaccines against FHV. The testing of intermediate products and the efficacy testing of finished products for related vaccines mainly involve methods such as cell experiments, animal experiments, quantitative PCR, and agar amplification. These methods either require enormous financial and material resources or are time-consuming and cumbersome. Therefore, this invention aims to provide a competitive ELISA method that can accurately quantify FHV antigens, used for the quantitative detection of FHV antigen content in vaccines, intermediate products, vector expression products, or cell cultures, thus providing support for FHV-related biopharmaceutical research. Summary of the Invention

[0005] The purpose of this invention is to provide a competitive ELISA method that can accurately quantify feline rhinotracheitis virus (FHV) antigen. This method uses feline rhinotracheitis virus gD recombinant protein as the coating antigen and establishes a competitive ELISA method with good specificity, sensitivity and reproducibility. It can be used to quantitatively detect the protein concentration of vaccines, intermediate products, vector expression products or the content of FHV antigen in cell cultures.

[0006] To achieve the above objectives, this invention first analyzes and predicts the immunogenic dominant regions in feline rhinotracheitis virus (FRV) gD with superior screening performance, and then obtains the antigen protein through tandem linker. The recombinant FRV gD protein provided by this invention is a recombinant FRV gD protein expressed by an Escherichia coli expression system. The amino acid sequence of the recombinant FRV gD protein is SEQ ID No. 5, and its encoding nucleotide sequence is SEQ ID No. 1.

[0007] This invention provides a competitive ELISA kit for accurate quantification of FHV antigen. The kit comprises an enzyme-linked reaction plate coated with feline rhinotracheitis virus gD recombinant protein as antigen, FHV positive serum, FHV negative serum, purified FHV standard antigen, and enzyme-labeled secondary antibody; the amino acid sequence of feline rhinotracheitis virus gD recombinant protein is SEQ ID No. 5.

[0008] The accurate quantitative FHV antigen competitive ELISA kit includes a removable 96-well ELISA plate; the feline rhinotracheitis virus gD recombinant 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 rhinotracheitis virus gD recombinant protein in a carbonate solution at pH 9.6, and then add 100 μl of 1 μg / mL feline rhinotracheitis virus gD gene recombinant protein to each well of a 96-well polystyrene enzyme-linked reaction plate. Incubate the plate at 2–8°C for 8–12 hours to allow the antigen in the composition 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 the plate at 37°C for 2–3 hours. After drying, seal and store the plate at 4°C.

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

[0011] The FHV purified standard antigen is a recombinant protein expressed by Escherichia coli that has been purified by a protein purification column and had its concentration determined, or an antigen that has been purified by sucrose density gradient centrifugation, and then inactivated after virus content determination.

[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 diluent and a concentrated wash buffer (20-fold); the sample diluent is a 0.01M phosphate buffer containing 0.5% 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:

[0016] 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.

[0017] 2. Solution preparation: Dilute the concentrated washing solution 20 times with distilled water or deionized water to obtain the washing buffer solution;

[0018] 3. Setup: 2 negative control wells and 2 positive control wells, with the rest being the sample wells to be tested.

[0019] 4. Pre-dilution of the sample to be tested: The recombinant protein of feline rhinotracheitis virus gD gene, inactivated FHV antigen, and treated vaccine expressed in the sample to be tested are diluted in appropriate proportions using sample diluent, and then serially diluted in a 2-fold ratio.

[0020] 5. Dilution of standards: Select appropriate standards (i.e., express protein standards for expressed proteins and inactivated antigen standards for inactivated antigens) according to the different sources of antigens and perform serial dilutions of 2-fold.

[0021] 6. Sample addition: Add 50 μl of positive serum to each well according to the pre-set procedure, followed by 50 μl of the proportionally diluted test sample. The sample addition process should be as short as possible.

[0022] 7. Incubation: Shake well and place in a 37°C incubator for 30 minutes.

[0023] 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.

[0024] 9. Add enzyme: Add 100 μl of the corresponding horseradish peroxidase-labeled IgG antibody to each well (rabbit serum with goat anti-rabbit IgG antibody, guinea pig serum with goat anti-guinea pig IgG antibody).

[0025] 10. Incubation: Place in a 37℃ incubator and react for 30 minutes.

[0026] 11. 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.

[0027] 12. Add 100 μl 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°C incubator to react in the dark for 15 min.

[0028] 13. Add 50 μl of colorimetric stop solution to each well and shake to mix and terminate the reaction.

[0029] 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).

[0030] Interpretation of test results:

[0031] 1. Negative control OD 450nm The average value should be less than 0.3; otherwise, it is invalid.

[0032] 2. The positive control should have a test value between 0.7 and 2.0; otherwise, it is invalid.

[0033] 3. Establishment of the standard curve: using the OD of the standard product... 450nm A standard curve is constructed using the x-axis (values) as the x-axis and the standard protein concentration as the y-axis (values). R0 2 The value should be ≥0.98.

[0034] 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.

[0035] The detection method described above in this invention can be used to detect feline rhinotracheitis virus antigen protein, feline rhinotracheitis virus antigen, or vaccines from different sources.

[0036] The positive effects of this invention are as follows: This invention uses recombinant protein of feline rhinotracheitis virus gD gene expressed by Escherichia coli, which makes the method have the advantages of high sensitivity and strong specificity.

[0037] 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.

[0038] This method can detect FHV antigens from different sources, such as live FHV, inactivated FHV, and E. coli expressing the gD gene protein. The limit of detection for this method is as low as 0.781 μg / ml or 10 μg / ml. 5.5 TCID 50 / mL; can be applied to the entire process of corresponding vaccine preparation;

[0039] In summary, this method uses recombinant protein of the feline rhinotracheitis virus (FHV) gD gene expressed in *E. coli* to coat an enzyme-linked reaction (ELISA) plate. It requires a small amount of antigen, exhibits high sensitivity and specificity, and can effectively detect FHV 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

[0040] Figure 1 SDS-PAGE image of purified gD-1-3 recombinant protein;

[0041] Figure 2 Results of Western blot (anti-His antibody) for the purification of recombinant gD-1-3 protein;

[0042] Figure 3 Serological antigenicity analysis of recombinant proteins

[0043] Figure 4 The results of SDS-PAGE analysis of purified rabbit anti-FHV positive serum;

[0044] Figure 5 The purified gD-1-3 recombinant protein was reacted with rabbit anti-FHV positive serum ( Figure 5 (A) or rabbit negative serum ( Figure 5 Western blot results for B);

[0045] Figure 6 OD of rabbit anti-FHV positive serum 450nm Value distribution chart;

[0046] Figure 7 Rabbit negative serum OD 450nm Value distribution chart;

[0047] Figure 8 To compete with the ELISA standard curve. Detailed Implementation

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0053] Example 1: Expression and identification of recombinant protein containing the gD gene antigenic epitope of feline rhinotracheitis virus.

[0054] 1. Expression and preparation of recombinant gD gene antigenic epitope tandem recombinant protein

[0055] The FHV viral gD protein was analyzed using the DNAstar software Protean to obtain information related to antigenic epitopes, including antigenicity, accessibility, hydrophilicity, flexibility, and epitope localization. Preliminary predictions of the dominant antigenic region were made. The predicted nucleotide fragments were tandemly linked using a linker, and the tandem nucleotide sequences were optimized and artificially synthesized according to codons preferred by *E. coli*. The recombinant protein nucleotide sequences are SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, and the recombinant protein amino acid sequences are SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively, denoted as gD-1-3, gD-1, gD-2, and gD-3. The protein gene fragment synthesized in vitro was inserted into the pET-30a(+) expression vector using NdeⅠ and HindⅢ to obtain a 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, and positive single colonies containing the specific protein expression plasmid were picked and inoculated into LB medium containing kanamycin. After incubation at 37°C with shaking overnight, the positive recombinant bacteria were named pET30-gD-1-3, pET30-gD-1, pET30-gD-2, and pET30-gD-3.

[0056] 2. Identification of Expression Results

[0057] SDS-PAGE expression identification of the recombinant protein showed that, compared with the pre-induction control, the recombinant bacterial strain expressed mainly insoluble protein after IPTG induction. The optimal induction conditions for the recombinant protein were: induction at 37℃ for 4 hours, followed by harvesting of bacterial cells and supernatant. SDS-PAGE gel scanning analysis stained with Coomassie Brilliant Blue estimated the size of the recombinant protein to be approximately 39–43 kDa, consistent with expectations. See results below. Figure 1 .

[0058] 3. Purification results and specificity identification

[0059] Protein purification was performed according to the protein purification method of the BeyoGold™ His-tag Purification Resin kit.

[0060] SDS-PAGE of purified gD gene recombinant protein gD-1-3 ( Figure 1The recombinant protein gD-1-3 expressed by the gD gene was purified using Western blot results. The collected samples were concentrated by ultrafiltration (ultrafiltration tube), and the purity was >90% (see results). Figure 1 ); specific Western blot analysis of the recombinant protein gD-1-3 of the gD gene is shown in [reference needed]. Figure 2 .

[0061] 4. Serological antigenicity analysis of recombinant gD gene protein

[0062] Recombinant proteins gD-1-3, gD-1, gD-2, and gD-3 were used as coating antigens, each at a concentration of 4 μg / ml, 100 μl / well to coat the ELISA plate. FHV-positive control serum diluted 1:400 was used as the primary antibody, and HRP-labeled goat anti-rabbit IgG diluted 1:10000 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. 450 The P / N ratio is used to determine the antigenicity of recombinant proteins gD-1-3, gD-1, gD-2, and gD-3. Serological antigenic analysis results show that the P / N ratio (OD) of recombinant protein gD-1-3 is relatively high. 450 The highest value was obtained from the FHV gD-1-3 antigen fragment. Based on the results of antigenicity analysis using the bioinformatics software DNAstar and Western blotting, the dominant antigen fragment was finally identified. The results are shown in Table 1. Figure 3 .

[0063] Table 1. Antigenicity analysis of recombinant proteins

[0064] P / N value 11.8 1.3 4.45 5.85

[0065] Example 2: Preparation of a competitive ELISA kit for accurate quantification of feline rhinotracheitis virus (FHV) antigen

[0066] The competitive ELISA kit for accurate quantification of feline rhinotracheitis virus (FHV) antigen of this invention comprises:

[0067] (1) A 96-well removable polystyrene enzyme-linked reaction plate coated with recombinant protein gD-1-3 of feline rhinotracheitis virus gD gene; 2 × 96 wells.

[0068] (2) Positive control serum: FHV positive serum, purchased from Qianxun Biotechnology Co., Ltd. (1 tube, 1.0 ml / tube).

[0069] (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).

[0070] (4) Enzyme-labeled secondary antibody: Horseradish peroxidase-labeled goat anti-rabbit IgG antibody or goat anti-guinea pig IgG antibody. Purchased from EarthOx.

[0071] (5) Sample dilution solution: 0.01M phosphate buffer containing 0.5% casein, pH 7.4, 1 bottle (24ml / bottle).

[0072] (6) Substrate solution A: citrate phosphate buffer containing 0.6 mg / ml hydrogen peroxide urea (1 bottle, 12 ml / bottle)

[0073] (7) Substrate solution B: 0.2 mg / ml tetramethylbenzidine (TMB) solution (1 bottle, 12 ml / bottle).

[0074] (8) Termination solution: 2 mol / L sulfuric acid solution (1 bottle, 12 ml / bottle).

[0075] (9) 20x concentrated washing solution: 0.01M phosphate buffer (50ml / bottle, 2 bottles) containing 0.8% to 1.2% (ml / ml) Tween-20 at pH 7.4.

[0076] The preparation method of the above-mentioned positive serum and enzyme-labeled plate is illustrated below, using rabbit serum as the positive and negative sera and horseradish peroxidase-labeled goat anti-rabbit IgG antibody as the enzyme-labeled secondary antibody:

[0077] I. Purification and Identification of Positive Serum

[0078] FHV positive serum was purchased from Qianxun Biotechnology Co., Ltd., and the rabbit serum with the above-mentioned qualified titer was purified using a Protein G Sepharose affinity chromatography column. The purified polyclonal antibody was aliquoted and stored at -80℃ for later use.

[0079] The purity of the purified antibody was observed by SDS-PAGE analysis (results are shown in [link to results]). Figure 4 Western blot (WB) was used to detect the specificity of positive and negative serum samples. Figure 5 China A Figure 5 (B)

[0080] II. Determination of Optimal Antigen Coating Concentration and Optimal Concentration for Use of Negative and Positive Serum

[0081] Square matrix titrations were performed using the orthogonal matrix method. The gD-1-3 recombinant protein was diluted to 0.06, 0.13, 0.25, 0.5, 1.0, 2.0, and 4.0 μg / ml with coating buffer; positive serum was diluted to four dilutions (1:100, 1:200, 1:400, and 1:800) with sample diluent; negative serum was diluted to three dilutions (1:100, 1:200, and 1:400) with sample diluent, with the sample diluent serving 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.

[0082] The results showed that when the antigen concentration was 1 μg / mL, the OD of positive serum at each dilution was... 450nm The value reached a plateau; when the positive serum dilution was 1:400, 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 11.17, 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 1 μg / mL, the optimal dilution for positive serum was 1:400, and the optimal dilution for negative serum was 1:200. The results are shown in Tables 2-5.

[0083] Table 2. Results of ELISA square array titration assays using coated antigen and negative / positive serum (OD) 450nm )

[0084]

[0085]

[0086] Table 3. Ratio of positive serum to negative serum diluted 1:100 (P / N value)

[0087]

[0088] Table 4. Ratio of positive serum to negative serum diluted 1:200 (P / N value)

[0089]

[0090] Table 5. Ratio of positive serum to negative serum diluted 1:400 (P / N value)

[0091]

[0092]

[0093] III. The preparation method of a 96-well detachable polystyrene ELISA plate coated with feline rhinotracheitis virus gD recombinant protein is as follows: Dissolve the gD gene recombinant protein gD-1-3 antigen prepared in Example 1 in 100 μl of pH 9.6 carbonate solution, and then add it to a 96-well polystyrene ELISA plate, with 100 μl of feline rhinotracheitis virus gD recombinant protein at a concentration of 1 μg / mL per well. Incubate at 2-8°C for 8-12 hours to allow the feline rhinotracheitis virus gD recombinant protein to fully bind to the ELISA plate. Then add 300 μl of pH 7.4 PBS buffer per well, block at 37°C for 2-3 hours, spin dry, and store at 4°C after the ELISA plate has dried.

[0094] IV. Determination of negative and positive control criteria

[0095] Positive serum was diluted 1:400, 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.

[0096] Fifty tests were performed on the same positive serum sample, with a mean OD value (X) of 1.321 and a standard deviation (S) of 0.231. The data from these 50 tests showed that the probability of OD values ​​falling within the range of X ± 3S (0.691–1.979) was 100%, conforming to a normal distribution and demonstrating good stability. Therefore, the OD values ​​of positive serum... 450nm The absorbance was set at 0.7–2.0. Results are shown below. Figure 6 .

[0097] Fifty experiments were conducted on the same negative serum sample, with a mean OD value (X) of 0.122 and a standard deviation (S) of 0.031. The probability that the OD value fell within the range of X ± 3S (0.028–0.215) 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 7 .

[0098] The competitive ELISA kit for accurately quantifying feline rhinotracheitis virus (FHV) antigen of this invention was prepared according to the optimized serum and antibody concentrations described above. Example 3: Detection procedure of the competitive ELISA method of this invention.

[0099] The detection procedure for the competing ELISA method of this invention is as follows:

[0100] 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.

[0101] 2. Solution preparation: Dilute the concentrated washing solution 20 times with distilled water or deionized water to obtain the washing buffer solution;

[0102] 3. Setup: 2 negative control wells and 2 positive control wells, with the rest being the sample wells to be tested.

[0103] 4. Pre-dilution of the sample to be tested: Dilute the FHV antigen to be tested with the sample diluent at an appropriate ratio, and then perform serial dilution at a ratio of 2.

[0104] 5. Dilution of feline rhinotracheitis virus antigen: Select appropriate standards (i.e., express protein standards for expressed proteins and inactivated antigen standards for inactivated antigens) for serial dilution in two-fold serial proportions according to the different sources of antigens.

[0105] 6. Sample addition: Add 50 μl of positive serum to each well according to the pre-set procedure, followed by 50 μl of the proportionally diluted test sample. The sample addition process should be as short as possible.

[0106] 7. Incubation: Shake well and place in a 37°C incubator for 30 minutes.

[0107] 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.

[0108] 9. Add enzyme: Add 100 μl of the corresponding horseradish peroxidase-labeled IgG antibody to each well (rabbit serum with goat anti-rabbit IgG antibody, guinea pig serum with goat anti-guinea pig IgG antibody).

[0109] 10. Incubation: Place in a 37℃ incubator and react for 30 minutes.

[0110] 11. 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.

[0111] 12. Add 100 μl 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°C incubator to react in the dark for 15 min.

[0112] 13. Add 50 μl of colorimetric stop solution to each well and shake to mix and terminate the reaction.

[0113] 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).

[0114] Interpretation of test results:

[0115] 1. Negative control OD 450nm The average value should be less than 0.3; otherwise, it is invalid.

[0116] 2. The positive control should have a test value between 0.7 and 2.0; otherwise, it is invalid.

[0117] 3. Establishment of the standard curve: using the OD of the standard product... 450nm A standard curve is constructed using the x-axis (values) as the x-axis and the standard protein concentration as the y-axis (values). R0 2 The value should be ≥0.98.

[0118] 4. Determination of protein concentration or toxicity value 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 protein concentration or toxicity value, and take the average value to obtain the protein concentration or toxicity value of the sample to be tested.

[0119] Example 4: Determination of the sensitivity of a competitive ELISA method using rabbit or guinea pig positive sera.

[0120] The sensitivity of the competitive ELISA kit prepared according to the method in Example 2 was tested. FHV positive serum was guinea pig or rabbit serum, and FHV 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.

[0121] The sensitivity of the two positive serum competitive ELISA methods was verified by purifying and inactivating FHV after sucrose density gradient centrifugation and expressing the feline rhinotracheitis virus gD gene protein in purified E. coli. (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 8.

[0122] Table 8. Sensitivity test results of the competitive ELISA method

[0123]

[0124] Example 5: Establishment of Standard Curve

[0125] The purified *E. coli* expressing the feline rhinotracheitis virus gD gene protein (KT963466) was diluted with sample diluent to obtain a standard series of virus concentrations: 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 1.563 μg / ml, 0.781 μg / ml, and 0. The concentrations were then measured according to the procedure in Example 5. The OD concentration was set to 0 (the concentration of the competing antigen was 0). 450nmThe value is B0, representing the OD of each corresponding antigen titer. 450nm With the value being B, a standard curve is plotted using the logarithm of the competitive antigen concentration as the x-axis and the binding rate (B / B0%) as the y-axis.

[0126] Using competitive antigens with different viral titers, the antigen-antibody competitive reaction established with the coated antigen showed a good linear relationship (R0). 2 =0.9927), and the linear regression equation was Y = -24.908x + 57.066. The detection range was 50 μg / ml to 0.781 μg / ml, and the limit of detection was 0.781 μg / ml. Results are shown in […]. Figure 8 See Table 9.

[0127] Table 9. Establishment of antigen standard curve for feline rhinotracheitis virus gD gene protein standard.

[0128] 0.3905 -0.40838 66.0535 0.781 -0.10735 59.822 1.563 0.193959 51.5905 3.125 0.49485 47.359 6.25 0.79588 38.21275 12.5 1.09691 29.896 25 1.39794 19.5645 50 1.69897 15.433

[0129] Example 7: Application of the competitive ELISA kit of the present invention in laboratory samples.

[0130] The established competitive ELISA method was used to detect samples from each process of the existing feline triple inactivated virus vaccine production and FHV production process. The results are shown in Table 10, indicating that the two methods have good monitoring effects in the above antigen production (E. coli expressing gD gene protein is expressed as inclusion body).

[0131] Table 10 Results of the application of the competitive ELISA method in laboratory samples.

[0132]

[0133]

[0134] 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. Recombinant gD protein of feline rhinotracheitis virus, the sequence of which is shown in SEQ ID No.

5.

2. A competitive ELISA kit for accurately quantifying feline rhinotracheitis virus antigen, characterized in that, The kit comprises an enzyme-linked reaction plate coated with feline rhinotracheitis virus gD recombinant protein as an antigen, feline rhinotracheitis virus positive serum, feline rhinotracheitis virus negative serum, feline rhinotracheitis virus standard antigen, and enzyme-labeled secondary antibody; the feline rhinotracheitis virus gD recombinant protein is the feline rhinotracheitis virus gD recombinant protein as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The method for preparing the enzyme-linked reaction plate is as follows: dissolve the feline rhinotracheitis virus gD recombinant protein as described in claim 1 in 100 μl of pH 9.6 carbonate solution, and then add it to a 96-well polystyrene enzyme-linked reaction plate, with 100 μl of feline rhinotracheitis virus gD recombinant protein at a concentration of 1 μg / mL in each well. Incubate at 2-8°C for 8-12 hours to allow the feline rhinotracheitis virus gD recombinant protein to fully bind with the enzyme-linked reaction plate. Then, add 300 μl of pH 7.4 PBS buffer at 37°C per well, block at 37°C for 2-3 hours, spin dry, and store the enzyme-linked reaction plate at 4°C after drying.

4. The reagent kit according to claim 2, characterized in that, The positive serum for feline rhinotracheitis virus (FRV) is rabbit serum or guinea pig serum collected after immunization with FRV; the negative serum for FRV is SPF guinea pig or rabbit serum.

5. The reagent kit according to claim 2, characterized in that, The standard antigen for feline rhinotracheitis virus is an antigen purified by sucrose density gradient centrifugation, followed by virus content determination and inactivation.

6. The reagent kit according to claim 2, characterized in that, The enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-rabbit IgG antibody or goat anti-guinea pig IgG antibody.

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

8. The reagent kit according to claim 2, characterized in that, The kit also includes a sample diluent and a concentrated wash buffer; the sample diluent is a 0.01 mol / L phosphate buffer containing 0.005 g / ml casein at pH 7.4; the concentrated wash buffer is a 0.01 mol / L phosphate buffer containing 0.8%–1.2% Tween-20 at pH 7.

4.

9. The use of the feline rhinotracheitis virus gD recombinant protein according to claim 1 in the preparation of a kit for detecting feline rhinotracheitis virus antigen.

10. The application according to claim 9, wherein the kit is a competitive ELISA kit for quantitative detection of feline rhinotracheitis virus antigen.

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