An indirect ELISA kit for detecting feline hepatotropic virus core antibodies and its application

Abstract

The present invention discloses an indirect ELISA kit for detecting feline hepatotropic virus core antibodies and its application, which belongs to the field of immunoassay technology. The present invention first discloses the application of feline hepatotropic virus recombinant protein core1-149 in the preparation of a kit for detecting feline hepatotropic virus core antibodies, and secondly discloses an indirect ELISA kit and detection method for detecting feline hepatotropic virus core antibodies. The present invention constructs feline hepatotropic virus recombinant protein core1-149 and establishes an ELISA method for detecting feline hepatotropic virus (DCH) antibodies. The method has high sensitivity, specificity and efficiency, is simple and quick to operate, and has low cost, providing a new detection means for clinical diagnosis of feline hepatotropic virus.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, in particular to an indirect ELISA kit for detecting feline hepatotropic virus core antibodies and application thereof. Background Art

[0002] Domestic cat hepadnavirus (DCH) was first reported in Australia in 2018. Studies have shown that DCH is closely associated with chronic hepatitis and hepatocellular carcinoma in cats, causing symptoms such as chronic liver inflammation and liver damage in infected cats.

[0003] DCH belongs to the Hepadnaviridae family, genus Orthohepadnavirus, and is a small, partially double-stranded circular DNA virus with only 3.2 kb of bases. DCH has four overlapping open reading frames (ORFs), encoding four proteins: core protein (C), surface protein (S), polymerase protein (P), and X protein. The core protein is highly conserved across genotypes and is a component of the viral nucleocapsid. It also plays an important role in nucleic acid transport and reverse transcription. The nucleocapsid formed by the core antigen is also highly immunogenic.

[0004] Currently, DCH detection methods are immature, with no commercially available test kits. Detection primarily relies on conventional laboratory techniques, including polymerase chain reaction (PCR), real-time quantitative polynucleotide chain reaction (qPCR), immunohistochemistry (IHC), and serum enzyme-linked immunosorbent assay (ELISA). qPCR is currently the most widely used method, but it is complex and expensive. Furthermore, the occult nature of hepatotropic virus infection makes its DNA difficult to detect. Therefore, there is an urgent need for a simple, sensitive, rapid, and low-cost antibody detection method. Summary of the Invention

[0005] The present invention aims to provide an indirect ELISA kit for detecting antibodies to feline hepatotropic virus core and its use to address the problems of the prior art. The present invention constructs a recombinant protein of feline hepatotropic virus core1-149 and establishes an ELISA method for detecting antibodies to feline hepatotropic virus (DCH). This method is highly sensitive, specific, and efficient, simple to operate, and low in cost, providing a new detection method for the clinical diagnosis of feline hepatotropic virus.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of a feline hepatotropic virus recombinant protein core1-149 in preparing a kit for detecting feline hepatotropic virus core antibodies. The amino acid sequence of the feline hepatotropic virus recombinant protein core1-149 is shown in SEQ ID NO.4.

[0008] The preparation method of the feline hepatotropic virus recombinant protein core1-149 of the present invention is:

[0009] The coding gene of feline hepadnavirus core protein core1-149 was connected to the pET-28a vector, transformed into Escherichia coli, induced for expression, and then extracted and purified.

[0010] The nucleotide sequence of the gene encoding the feline hepadnavirus core protein core1-149 is shown in SEQ ID NO.1.

[0011] The present invention also provides an indirect ELISA kit for detecting feline hepadnavirus core antibodies, wherein the indirect ELISA kit uses the feline hepadnavirus recombinant protein core1-149 as a coating antigen;

[0012] The amino acid sequence of the feline hepadnavirus recombinant protein core1-149 is shown in SEQ ID NO.4.

[0013] Furthermore, the indirect ELISA kit also includes an enzyme-labeled secondary antibody, a blocking solution, a washing solution, a diluent, a positive control sample, a negative control sample, a color developing solution, and a stop solution.

[0014] Optionally, the blocking solution is 5% BSA.

[0015] The present invention also provides use of the indirect ELISA kit in detecting feline hepadnavirus core antibodies.

[0016] The present invention also provides a method for detecting feline hepadnavirus core antibodies, which is detected using the above-mentioned indirect ELISA kit, comprising the following steps:

[0017] The recombinant protein core1-149 of feline hepatotropic virus was coated, washed and blocked with blocking solution;

[0018] After blocking is completed, the diluted serum to be tested is added and the serum is incubated;

[0019] After incubation, wash and add diluted enzyme-labeled secondary antibody for incubation;

[0020] After incubation, wash, add color development solution, and incubate for color development;

[0021] After adding the stop solution, read the OD 450 Numeric value.

[0022] Optionally, the coating concentration is 0.5-2 μg / mL; and the blocking time is 1-2 h.

[0023] Further optionally, the coating concentration is 1 μg / mL; and the blocking time is 1 h.

[0024] Optionally, the dilution ratio of the serum to be tested is 1:(200-400); and the serum incubation time is 1-2 hours.

[0025] Further optionally, the dilution ratio of the serum to be tested is 1:400; and the serum incubation time is 1.5 h.

[0026] Optionally, the dilution ratio of the enzyme-labeled secondary antibody is 1:(10000-20000); the incubation time of the secondary antibody is 1-1.5h.

[0027] Further optionally, the dilution ratio of the enzyme-labeled secondary antibody is 1:10000; and the incubation time of the secondary antibody is 1 h.

[0028] Optionally, the color development incubation time is 10-15 minutes.

[0029] Further optionally, the color development incubation time is 10 minutes.

[0030] The present invention discloses the following technical effects:

[0031] The present invention obtains a recombinant protein of feline hepatotropic virus (FHV) core1-149 through prokaryotic induction expression. The recombinant protein has good binding ability with FHV-positive serum. An indirect ELISA assay for detecting FHV antibodies using the recombinant protein has a high sensitivity of 1:1600, with intra-assay and inter-assay coefficients of variation of 0.68-9.21% and 0.82-4.83%, respectively, both less than 10%, demonstrating good reproducibility. The assay exhibits no cross-reactivity with feline infectious peritonitis virus, calicivirus, herpesvirus, parvovirus, and Toxoplasma gondii, demonstrating high specificity. The assay can accurately and efficiently detect FHV antibodies, is simple and quick to operate, and is low-cost, providing a simple and effective diagnostic method for the clinical diagnosis of FHV. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1The electrophoresis results of the amplified products are shown in Figure 2. M: 2000 Marker, lane 1: target gene;

[0034] Figure 2 The double enzyme digestion electrophoresis results of the recombinant plasmid pET-28a-core1-149 are shown in Figure 1. M: 5000 Marker, lane 1: double enzyme digestion fragment of the recombinant plasmid pET-28a-core149;

[0035] Figure 3 The SDS-PAGE results of the recombinant protein core1-149 are shown in Figure 1. M: Marker, lane 1: uninduced sample, lane 2: whole cell sample after induction, lane 3: supernatant sample after induction, lane 4: precipitate sample after induction, lane 5: flow-through, lanes 6-8: eluted samples.

[0036] Figure 4 The Western Blot results of the recombinant protein core1-149 are shown in FIG. 1 , where M is a marker and lane 1 is a recombinant protein core1-149. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] In addition to the indicated methods of obtaining, the biological materials involved in the embodiments of the present invention can also be purchased through conventional channels or obtained in other ways.

[0043] Example 1

[0044] 1. Experimental Materials

[0045] Competent Escherichia coli DH5α and BL21(DE3) strains were maintained by the Veterinary Pathology Laboratory of Huazhong Agricultural University. Positive sera for feline infectious peritonitis virus, feline calicivirus, feline herpesvirus, feline parvovirus, and Toxoplasma gondii, as well as positive and negative sera for feline hepatotropic virus, were also maintained by the Veterinary Pathology Laboratory of Huazhong Agricultural University. HRP-goat anti-feline IgG was purchased from Dakoway Biotechnology Co., Ltd.; TMB colorimetric solution was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; and prepacked columns (1 mL) for His-tagged protein purification were purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0046] 2. Experimental methods

[0047] 2.1 Construction of the prokaryotic expression vector pET-28a-core1-149

[0048] The amino acid sequence 1-149 of the DCH core protein with high stability and strong antigenicity was selected as the target protein. A pair of amplification primers was designed based on its encoding gene (SEQ ID NO.1). EcoRI and XhoⅠ restriction sites were introduced at both ends of the upstream primer and downstream primer, and protective bases were added at the same time. The upstream primer was: 5'-CCGGAATTCATGGACATTGACCCTTATAAAGAATTTGG-3' (SEQ ID NO.2); the downstream primer was: 5'-CCGCTCGAGGATAACCGTATGCTCCGGAAGA-3' (SEQ ID NO.3). DCH (GenBank: MK117078.1) DNA stored in the Veterinary Pathology Laboratory of Huazhong Agricultural University was used as a template and PCR amplification was performed using the above primers. The PCR system consisted of 10 μL of 2×ApexHF FS PCR Master Mix, 1 μL of upstream and downstream primers, 1 μL of template DNA, and 7 μL of ddH2O. The PCR reaction procedure was as follows: pre-denaturation at 94°C for 1 min; denaturation at 98°C for 10 sec, annealing at 60°C for 15 sec, and extension at 68°C for 30 sec, for 35 cycles; and termination at 12°C.

[0049] SEQ ID NO.1:

[0050] ATGGACATTGACCCTTATAAAAGAATTTGGAACTACATCTCAGCTTATCTCTTTTTTGCCGTCTGACTTTTTTCCTGCTCTCAATGATCTGGTCGATACGATTCAAGCGCTCTATGAAGAGGAACTTACAGGTAGGGAACACTGTTCCCCTCACCATACGGCCCTTTCGTGTGCTCTTGAACTGTTGGGAGGAGTCAGCTAGAATGGCTACATGGGTTAGAGCCAA CGTGGAAGGAGCCCCATTGCAGGATGCCATTGTGGCTTATGTTAATTCTACTGTCAGTTTGAAATTGAGGCAGCAAATGTGGTTCCATCTATCTTGCCTCACTTTTGGACAACATACAGTATTGGAGTTTTTAGTATCTTTTGGTACCTGGATTCGCACACCTGCACCGTATAGACCCCCCTATGCACCCATTCTCTCCACTCTTCCGGAGCATACGGTTATC.

[0051] The amplified products were subjected to nucleic acid electrophoresis detection, and the test results were as follows: Figure 1As shown, there is a clear band at 465bp, indicating successful amplification. The amplified product was recovered using the Omega gel recovery kit. The recovered and purified core1-149 target fragment and pET-28a vector were double-digested with EcoRⅠ and XhoⅠ endonucleases and then recovered from the gel. The recovered linearized vector and target fragment were ligated with T4 ligase at 16°C overnight to construct the recombinant plasmid pET-28a-core1-149. 10μL of the ligation product was transformed into 100μL of DH5α competent medium, spread on an LB plate containing kanamycin, and cultured at 37°C overnight. The next day, single colonies of uniform size on the plate were picked and cultured in LB liquid culture medium containing kanamycin. After the bacterial solution became turbid, it was sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing.

[0052] 2.2 Inducible expression of recombinant core1-149 protein

[0053] The plasmid was extracted from the bacterial solution with correct sequencing, and the plasmid was double-digested with EcoRⅠ and XhoⅠ endonucleases. The digestion products were detected by electrophoresis. The detection results were as follows: Figure 2 The results showed that the recombinant plasmid was successfully constructed and named pET-28a-core1-149. The recombinant plasmid pET-28a-core1-149 was transformed into BL21(DE3) competent cells and plated onto plates containing kanamycin and chloramphenicol. After overnight incubation at 37°C, single colonies of uniform size were selected and inoculated into LB liquid medium containing kanamycin and chloramphenicol. The bacterial culture was then sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing.

[0054] The positive bacterial solution with correct sequencing was inoculated into 1 L of LB liquid medium containing kanamycin and chloramphenicol and cultured at 37 ° C and 220 r / min for 3 h. 600 When the p-value reaches 0.6-0.8, 1 mL of the culture medium is taken as an uninduced sample. IPTG at a final concentration of 1 mmol / L is added to the remaining culture medium for induction. Induction is carried out at 16°C overnight. After induction, the E. coli pellet is collected by centrifugation at 5000 g for 10 minutes. The pellet is washed with an appropriate amount of PBS and centrifuged again. The pellet is collected and purified for recombinant core1-149 protein.

[0055] The purification process of recombinant core1-149 protein is as follows:

[0056] a. Resuspend the cells in 1 / 10 the volume of Binding Buffer and disrupt the E. coli cells using a high-pressure disruptor at a temperature of 4°C and a pressure of 650 bar.

[0057] b. Centrifuge the disrupted bacterial suspension at 30,000 g for 20 min at 4°C, collect the supernatant and precipitate, and place on ice until ready for use.

[0058] c. Filter the supernatant with a 0.22 μm filter;

[0059] d. Before loading, wash the His-Trap column with filtered ultrapure water and fill the constant flow pump tubing. Install the binding column onto the flow tube and attach a connector. Rinse the His-Trap column with approximately 10 mL of ultrapure water, then equilibrate the His-Trap column with approximately 5 mL of Binding Buffer.

[0060] e. The filtered supernatant containing the recombinant protein is loaded onto the His-Trap column via a peristaltic pump;

[0061] f. After loading, use approximately 30 mL of Washing Buffer to remove contaminants from the His-Trap column.

[0062] g. After washing, elute the target protein with Elution buffer containing 400 mM imidazole;

[0063] h. Use the Broadford protein concentration detection kit to detect the concentration of the collected protein in the centrifuge tube and collect the high-concentration protein;

[0064] i. Wash the His-Trap column with Elution buffer, Binding buffer and ultrapure water, and seal it with 20% ethanol;

[0065] j. Use a 10 kDa ultrafiltration tube to concentrate the collected protein by ultrafiltration at 4000 rpm for 15 min at 4°C. Replace the imidazole in the Elution Buffer with GE Buffer 4-5 times. Aliquot the final concentrated protein and store at -80°C until use.

[0066] 2.3 SDS-PAGE identification of recombinant protein core1-149

[0067] The uninduced sample, the whole bacterial sample after induction, the supernatant sample after induction, the precipitate sample after induction, the flow-through and the elution sample were mixed with the loading buffer in a volume ratio of 4:1, heated in a boiling water bath, and centrifuged (4°C, 12000r / min, 2min) to prepare the supernatant for loading; add electrophoresis buffer, install the glass plate, slowly pull out the sample comb vertically in the buffer solution, add 10μL protein sample and 3μL Marker to the hole; correctly connect the positive and negative poles of the power cord, set the initial voltage of the electrophoresis instrument to 80V, adjust the voltage to 120V when the Marker runs to the separation gel, and continue electrophoresis for 1-1.5h. The length of time depends on the molecular weight of the protein to be tested; peel the gel from the gel plate, place it in Coomassie brilliant blue staining solution, and stain it at room temperature for 2h. Then discard the staining solution and add an appropriate amount of decolorizing solution for several times until the background color is weak and the target protein band is clear. The SDS-PAGE results are as follows Figure 3 As shown, the results showed that the core1-149 protein was successfully expressed at 18 kDa.

[0068] 2.4 Western Blot Identification of Recombinant Protein Core1-149

[0069] After SDS-PAGE electrophoresis of the recombinant protein core1-149, cut a PVDF membrane according to the gel size. Activate the PVDF membrane with methanol for 3 minutes. Wet the gel, PVDF membrane, and filter paper in transfer buffer. Arrange the membrane in the order of positive electrode, filter paper, PVDF membrane, gel, filter paper, and negative electrode, with five filter papers on each side. Avoid air bubbles and drying of the gel and membrane, cover the membrane, and place on ice. Transfer the membrane at a constant voltage of 65V and 1kDa / 1min for 18 minutes. Use TBST containing 5% skim milk to block on a shaker at room temperature for 2 hours; after blocking, wash the membrane three times with TBST, then dilute DCH positive serum with TBST at a ratio of 1:2000 and incubate at 4°C overnight; after washing the membrane with TBST, add HRP-goat anti-cat IgG diluted with TBST at a ratio of 1:10000 and incubate at room temperature for 1 hour; after washing the membrane with TBST, mix ECL chemiluminescent substrate A solution and B solution in a volume ratio of 1:1, add it dropwise to the membrane for color development, and take pictures with a chemiluminescent imaging system.

[0070] The results are as follows Figure 4 As shown, the results showed that there was a specific band at about 18 kDa on the PVDF membrane, which was consistent with the expected band size. The recombinant protein core1-149 could specifically bind to the DCH positive serum.

[0071] The amino acid sequence of the recombinant protein core1-149 is shown in SEQ ID NO.4:

[0072] MDIDPYKEFGTTSQLISFLPSDFFPALNDLVDTIQALYEEELTGREHCSPHHTALRVLLN CWEESARMATWVRANVEGAPLQDAIVAYVNSTVSLKLRQQMWFHLSCLTFGQHTVLEFLVSFGTWIRTPAPYRPPYAPILSTLPEHTVI.

[0073] Example 2 Establishment of an indirect ELISA detection method based on recombinant protein core1-149

[0074] 1. Determination of the optimal coating concentration of recombinant protein and the optimal serum dilution ratio

[0075] The optimal coating concentration of the recombinant protein and the optimal serum dilution ratio were determined using the checkerboard titration method. The specific process is as follows:

[0076] The purified recombinant protein core1-149 was diluted with coating solution (0.1mol / L pH 9.6 carbonate buffer) in seven gradients of 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL, and 0.125μg / mL. Each concentration was coated in a vertical row of 6 wells, 100μL per well, and coated overnight at 4°C. 250μL PBST was added to each well for washing, 5 minutes each time, repeated 5 times, and the liquid in the plate was patted dry vertically several times after washing. 5% BSA was used as a blocking solution (prepared in PBST), 200μL per well, and blocked at 37°C for 2h. Use diluent (PBST containing 5% BSA) to dilute the positive serum and negative serum at a ratio of 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200, respectively. Add each dilution to 6 wells in a row, 100 μL per well, and incubate at 37°C for 1 hour. After incubation, wash 5 times with PBST. Use PBST containing 5% BSA to dilute HRP-goat anti-cat IgG at a ratio of 1:10000, mix well, add 100 μL to each well, and incubate at 37°C for 1 hour. After incubation, wash 5 times with PBST. Add TMB colorimetric solution in the dark, 100 μL per well, and react at 37°C in the dark for 10 minutes. After adding 100 μL of sulfuric acid stop solution to each well, immediately read the OD using a microplate reader. 450 Value, based on positive serum OD 450 The value is greater than and close to 1, negative serum OD 450 If the value is less than or equal to 0.2, select P / N (positive serum OD 450 Value / negative serum OD 450The combination with the highest value (value) is the optimal antigen coating concentration and the optimal serum dilution ratio. The test results are shown in Table 1. The optimal antigen coating concentration is 1 μg / mL and the optimal serum dilution ratio is 1:400.

[0077] Table 1 Optimal coating concentration of recombinant protein core1-149 and optimal dilution of test samples

[0078]

[0079] 2. Determination of the optimal blocking solution for recombinant protein

[0080] The indirect ELISA was performed using the optimized coating concentration and serum dilution ratio. The other conditions were the same as in step 1. 3% BSA, 5% BSA, 5% skim milk, and 10% skim milk were used as blocking solutions, respectively. 200 μL was used per well. Three replicate wells were set up for indirect ELISA. The OD values ​​were read. 450 Value, select P / N (positive serum OD 450 Value / negative serum OD 450 The best blocking solution is the one with the largest value. The test results are shown in Table 2. The best blocking solution is 5% BSA.

[0081] Table 2 Best blocking solution types

[0082]

[0083] 3. Determination of the optimal blocking time for recombinant protein

[0084] The indirect ELISA test was performed using the optimized coating concentration, serum dilution ratio, and blocking solution. The other conditions were the same as in step 1. Four different blocking times were set, namely, 37°C 0.5h, 37°C 1h, 37°C 1.5h, and 37°C 2h. Three parallel wells were set in each group for indirect ELISA test. The OD values ​​were read. 450 Value, select P / N (positive serum OD 450 Value / negative serum OD 450 The best sealing time is the sealing time with the largest value. The test results are shown in Table 3, and the best sealing time is 1 hour.

[0085] Table 3 Determination of optimal sealing time

[0086]

[0087] 4. Determination of the optimal serum incubation time

[0088] The indirect ELISA test was performed using the optimized coating concentration, serum dilution ratio, blocking solution, and blocking time. The other conditions were the same as in step 1. Four different serum incubation times were set, namely, 37°C for 0.5 h, 37°C for 1 h, 37°C for 1.5 h, and 37°C for 2 h. Three parallel wells were set in each group for indirect ELISA detection. The OD values ​​were read. 450 Value, select P / N (positive serum OD 450 Value / negative serum OD 450 The serum incubation time with the largest value is the optimal serum incubation time. The test results are shown in Table 4. The optimal serum incubation time is 1.5 hours at 37°C.

[0089] Table 4 Optimal serum incubation time

[0090]

[0091]

[0092] 5. Determination of the optimal dilution ratio of HRP-goat anti-cat IgG

[0093] The indirect ELISA test was performed using the optimized optimal coating concentration, optimal serum dilution ratio, optimal blocking solution, optimal blocking time, and optimal serum incubation time. The other conditions were the same as in step 1. Four different secondary antibody dilution ratios were set, namely 1:5000, 1:10000, 1:15000, and 1:20000. Three parallel wells were set in each group for indirect ELISA detection. The OD values ​​were read. 450 Value, select P / N (positive serum OD 450 Value / negative serum OD 450 The optimal dilution ratio of the secondary antibody was 1:10000.

[0094] Table 5 Optimal HRP-goat anti-cat antibody dilution ratio

[0095]

[0096] 6. Determination of the optimal incubation time for HRP-goat anti-cat IgG (secondary antibody)

[0097] The indirect ELISA test was performed using the optimized coating concentration, serum dilution ratio, blocking solution, blocking time, serum incubation time, and secondary antibody dilution ratio. The other conditions were the same as in 3.1. Four different secondary antibody incubation times were set, namely 37°C for 0.5h, 37°C for 1h, 37°C for 1.5h, and 37°C for 2h. Three parallel wells were set in each group for indirect ELISA test. The OD values ​​were read. 450Value, select the incubation time of the secondary antibody with the largest P / N (OD value of positive serum / OD value of negative serum) value as the optimal incubation time of the secondary antibody. The test results are shown in Table 6. The optimal incubation time of the secondary antibody is 1 hour at 37°C. 450 value / OD value of negative serum 450 value), and select the incubation time of the secondary antibody with the largest P / N value as the optimal incubation time of the secondary antibody. The test results are shown in Table 6. The optimal incubation time of the secondary antibody is 1 hour at 37°C.

[0098] Table 6 Optimal incubation time of HRP-goat anti-cat IgG

[0099]

[0100] 7. Determination of the optimal color development time

[0101] Perform indirect ELISA using the above-optimized optimal coating concentration, optimal serum dilution ratio, optimal blocking solution, optimal blocking time, optimal serum incubation time, optimal secondary antibody dilution ratio, and optimal secondary antibody incubation time. The other conditions are the same as in 3.1. Set 4 different color development times, namely 5 minutes, 10 minutes, 15 minutes, and 20 minutes. Set 3 parallel wells in each group and perform indirect ELISA detection. Read the OD 450 value, select the color development time with the largest P / N (OD value of positive serum / OD value of negative serum) value as the optimal color development time. The test results are shown in Table 7. The optimal TMB color development time is 10 minutes at 37°C. 450 value / OD value of negative serum 450 value) as the optimal color development time. The test results are shown in Table 7. The optimal TMB color development time is 10 minutes at 37°C.

[0102] Table 7 Optimal TMB color development time

[0103]

[0104] 8. Determination of the critical value

[0105] Perform indirect ELISA on 40 negative sera using the above-optimized conditions (antigen coating concentration is 1 μg / mL, blocking solution is 5% BSA, blocking time is 1 hour, serum dilution ratio is 1:400, serum incubation time is 1.5 hours, secondary antibody dilution ratio is 1:10000, secondary antibody incubation time is 1 hour, TMB color development time is 10 minutes). Calculate the average value X and standard deviation SD of its OD 450 , and take the positive and negative critical value = OD 450 average value + 3 × standard deviation as the standard. That is, when OD 450 ≥ X + 3SD, it is determined as antibody positive; when OD 450 < X + 3SD, it is determined as antibody negative.

[0106] The test results are shown in Table 8. The results show that the average OD 450 value X of 40 negative sera is 0.197, the standard deviation SD is 0.089, and the positive and negative critical value = average OD 450+3×standard deviation=0.197+3×0.089=0.465. 450 When ≥0.465, it is determined to be antibody positive; when the serum OD 450 <0.465, determined as antibody negative.

[0107] Table 8 Negative serum OD 450 reading

[0108]

[0109] 9. Repeatability test

[0110] Four DCH-positive sera and three DCH-negative sera were tested using the optimized indirect ELISA method using the same batch of coated ELISA plates. Three parallel wells were set up for each serum sample to test the intra-batch reproducibility of this indirect ELISA method. Three additional batches of coated ELISA plates were also tested using the optimized indirect ELISA method to test the inter-batch reproducibility of this indirect ELISA method. The coefficient of variation was calculated as standard deviation (SD) / mean (X) × 100% = coefficient of variation (CV).

[0111] The test results are shown in Tables 9 and 10. The results show that the intra-batch coefficient of variation is 0.68-9.21%, and the inter-batch coefficient of variation is 0.82-4.83%, indicating that the method has good intra-batch and inter-batch reproducibility.

[0112] Table 9 Intra-batch coefficient of variation

[0113]

[0114] Table 10 Inter-batch coefficient of variation

[0115]

[0116] 10. Specificity test

[0117] The optimized conditions were used to perform ELISA on positive sera of feline infectious peritonitis virus (FIPV), feline herpesvirus (FHV), feline calicivirus (FCV), feline parvovirus (FPV) and Toxoplasma gondii (TOXO) stored in the laboratory. DCH positive and negative sera were used as controls to evaluate the specificity of the established indirect ELISA method.

[0118] The test results are shown in Table 11. The OD values ​​of positive serum against five other common cat pathogens are 450 All of them were below the critical value of 0.465, which fully demonstrated that the established indirect ELISA method had good specificity.

[0119] Table 11 core1-149 specificity test readouts

[0120] serum samples <![CDATA[OD 450 ]]> Judgment results Positive samples 1.449 + Negative samples 0.063 - FHV 0.378 - TOXO 0.237 - FIPV 0.203 - FCV 0.101 - FPV 0.332 -

[0121] 11. Sensitivity test

[0122] The DCH-positive serum was diluted at a ratio of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, and each gradient was repeated three times. Indirect ELISA detection was performed under optimized conditions.

[0123] The test results are shown in Table 12. When the positive serum was diluted to 1:1600, its OD 450 It is still greater than the critical value and can still be detected as positive, which proves that the method has good sensitivity.

[0124] Table 12 core1-149-ELISA sensitivity test readings

[0125] Serum dilution 1:100 1:200 1:400 1:800 1:1600 1:3200 1:6400 1:12800 <![CDATA[OD 450 ]]> 2.849 2.280 2.038 1.146 0.731 0.443 0.289 0.196 Judgment results + + + + + - - -

[0126] 12. Compliance rate detection

[0127] 34 cat sera were selected and tested by Western Blot using core1-149 protein loading. ELISA was also used to detect these 34 sera, and the coincidence rates of the two methods were compared.

[0128] The test results are shown in Table 13. The results showed that the indirect ELISA detection method detected 22 positive samples, the Western Blot detected 21 positive samples, and the positive coincidence rate was 95.5% (21 / 22); the indirect ELISA detection method detected 12 negative samples, the Western Blot detected 13 negative samples, and the negative coincidence rate was 92.3% (12 / 13); there was 1 serum that was positive in ELISA but negative in Western Blot; the total coincidence rate of the indirect ELISA detection method and Western Blot detection results was 97.1% (33 / 34), indicating that this method has good clinical application value.

[0129] Table 13 Core1-149-ELISA and WB consistency test

[0130]

[0131] 13. Clinical sample testing

[0132] The indirect ELISA method established by the present invention was used to detect 377 cat sera collected in Wuhan to analyze the prevalence of serum antibodies against DCH in Wuhan. The results showed that 320 of the 377 sera were positive for DCH antibodies (OD 450 The positive rate of DCH core antibodies in Wuhan was 84.88%.

[0133] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of feline hepatotropic virus recombinant protein core1-149 in preparing a kit for detecting feline hepatotropic virus core antibodies, characterized in that: The amino acid sequence of the feline hepadnavirus recombinant protein core1-149 is shown in SEQ ID NO.

4.

2. An indirect ELISA kit for detecting feline hepadnavirus core antibodies, characterized in that: The indirect ELISA kit uses the feline hepatotropic virus recombinant protein core1-149 as the coating antigen; The amino acid sequence of the feline hepadnavirus recombinant protein core1-149 is shown in SEQ ID NO.

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3. The indirect ELISA kit according to claim 2, wherein The indirect ELISA kit also includes an enzyme-labeled secondary antibody, a blocking solution, a washing solution, a diluent, a positive control sample, a negative control sample, a color developing solution, and a stop solution.

4. The indirect ELISA kit according to claim 3, wherein The blocking solution is 5% BSA.

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