Indirect ELISA kit for detecting cat hepatophilic virus core antibody and application of indirect ELISA kit

Abstract

The invention discloses an indirect ELISA (enzyme-linked immuno sorbent assay) kit for detecting a cat hepatophilic virus core antibody and application of the indirect ELISA kit, and belongs to the technical field of immunodetection. The invention firstly discloses application of the recombinant protein core1-149 of the feline hepatotrophy virus in preparation of the kit for detecting the core antibody of the feline hepatotrophy virus, and secondly discloses an indirect ELISA kit for detecting the core antibody of the feline hepatotrophy virus and a detection method. The recombinant protein core1-149 of the feline hepatotrophy virus is constructed, an ELISA method for detecting the antibody of the feline hepatotrophy virus (DCH) is established, and the method has the advantages of high sensitivity, specificity and efficiency, simplicity, convenience and rapidness in operation and lower cost, and provides a new detection means for clinical diagnosis of the feline hepatotrophy virus.

Description

Technical Field

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

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

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

[0004] At present, the detection method of DCH is not mature, and there is no commercially available detection kit. The detection method mainly adopts conventional laboratory detection techniques such as polymerase chain reaction (PCR), real-time fluorescence quantitative polynucleotide chain reaction (qPCR), immunohistochemistry (IHC) and serum enzyme-linked immunosorbent assay (ELISA). The most commonly used detection method is qPCR, but it has the disadvantages of complex operation and high cost. In addition, the latent infection of hepatotropic virus makes its DNA difficult to detect. Therefore, it is urgent to establish a simple, sensitive, rapid and low-cost antibody detection method. Summary of the invention

[0005] The purpose of the present invention is to provide an indirect ELISA kit for detecting feline hepatotropic virus core antibodies and its application to solve the problems existing in the above-mentioned prior art. The present invention constructs a recombinant protein core1-149 of feline hepatotropic virus and establishes an ELISA method for detecting feline hepatotropic virus (DCH) antibodies. The method has high sensitivity, specificity and high efficiency, is simple and fast to operate, and has low cost, providing a new detection method for 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 a feline hepatotropic virus core antibody. 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 the core protein core1-149 of the feline hepadnavirus 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 application of the indirect ELISA kit in detecting feline hepatotropic virus core antibody.

[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 hepadnavirus was coated, and after washing, it was 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, add diluted enzyme-labeled secondary antibody, and incubate with secondary antibody;

[0020] After the incubation is completed, 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); the serum incubation time is 1-2h.

[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 hour.

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

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

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

[0031] The invention obtains the feline hepatotropic virus recombinant protein core1-149 by prokaryotic induction expression, and the recombinant protein has good binding ability with feline hepatotropic virus positive serum. The recombinant protein is used to construct an indirect ELISA detection method for detecting feline hepatotropic virus antibodies, and the sensitivity can reach 1:1600, with high sensitivity; the intra-batch coefficient of variation and the inter-batch coefficient of variation are 0.68-9.21% and 0.82-4.83% respectively, both less than 10%, with good repeatability; there is no cross reaction with feline infectious peritonitis virus, calicivirus, herpes virus, parvovirus and toxoplasma, and the specificity is strong; the feline hepatotropic virus antibodies can be accurately and efficiently detected, the operation is simple and fast, the cost is low, and a simple and effective diagnostic method is provided for clinical diagnosis of feline hepatotropic virus. 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

[0034] Figure 2 The double restriction enzyme electrophoresis results of the recombinant plasmid pET-28a-core1-149; wherein, M: 5000 Marker, lane 1: double restriction enzyme 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 bacterial 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; wherein, M: Marker, lane 1: 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

[0042] The biological materials involved in the embodiments of the present invention can be purchased through conventional channels or obtained in other ways besides the indicated obtaining methods.

[0043] Example 1

[0044] 1. Experimental Materials

[0045] Escherichia coli competent DH5α and BL21 (DE3) were stored 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, and positive and negative sera for feline hepatotropic virus were stored by the Veterinary Pathology Laboratory of Huazhong Agricultural University. HRP-goat anti-cat IgG was purchased from Dakoway Biotechnology Co., Ltd.; TMB colorimetric solution was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; His-tag protein purification prepacked column (1 mL) was purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0046] 2. Experimental methods

[0047] 2.1 Construction of 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 coding gene (SEQ ID NO.1). EcoRⅠ and XhoⅠ restriction sites were introduced at both ends of the upstream primer and the downstream primer, and protective bases were added at the same time. The upstream primer: 5'-CCGGAATTCATGGACATTGACCCTTATAAAGAATTTGG-3' (SEQ ID NO.2); the downstream primer: 5'-CCGCTCGAGGATAACCGTATGCTCCGGAAGA-3' (SEQ ID NO.3). DCH (GenBank: MK117078.1) DNA preserved in the Veterinary Pathology Laboratory of Huazhong Agricultural University was used as a template and the above primers were used for PCR amplification. The PCR system was 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; PCR reaction program: pre-denaturation at 94°C for 1 min; denaturation at 98°C for 10 sec, annealing at 60°C for 15 sec, extension at 68°C for 30 sec, and 35 cycles; the reaction was terminated at 12°C.

[0049] SEQ ID NO.1:

[0050] ATGGACATTGACCCTTATAAAAGAATTTGGAACTACATCTCAGCTTATCTCTTTTTTGCCGTCTGACTTTTTTCCTGCTCTCAATGATCTGGTCGATACGATTCAAGCGCTCTATGAAGAGGAACTTACAGGTAGGGAACACTGTTCCCCTCACCATACGGCCCTTTCGTGTGCTCTTGAACTGTTGGGAGGAGTCAGCTAGAATGGCTACATGGGTTAGAGCCAA CGTGGAAGGAGCCCCATTGCAGGATGCCATTGTGGCTTATGTTAATTCTACTGTCAGTTTGAAATTGAGGCAGCAAATGTGGTTCCATCTATCTTGCCTCACTTTTGGACAACATACAGTATTGGAGTTTTTAGTATCTTTTGGTACCTGGATTCGCACACCTGCACCGTATAGACCCCCCTATGCACCCATTCTCTCCACTCTTCCGGAGCATACGGTTATC.

[0051] The amplified product was tested by nucleic acid electrophoresis. Figure 1As shown, there is a clear band at 465bp, indicating that the amplification was successful. 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 by gel. The recovered linearized vector and the target fragment were connected 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, and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing after the bacterial solution became turbid.

[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 product was detected by electrophoresis. The detection results were as follows: Figure 2 As shown, the results showed that the recombinant plasmid was successfully constructed, and the plasmid was named pET-28a-core1-149. The recombinant plasmid pET-28a-core1-149 was transformed into BL21 (DE3) competent cells, spread on a plate containing kanamycin and chloramphenicol, and cultured overnight at 37°C. Single colonies of uniform size were picked and inoculated into LB liquid culture medium containing kanamycin and chloramphenicol for culture, and the bacterial solution was sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing.

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

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

[0056] a. Resuspend the bacteria with 1 / 10 volume of Binding Buffer, and use a high-pressure disruptor to disrupt the E. coli cells at low temperature, with an operating temperature of 4°C and a working pressure of 650 bar;

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

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

[0059] d. Before loading, wash with filtered ultrapure water and fill the constant flow pump pipeline, install the binding column on the flow tube, add the connector, first use about 10mL ultrapure water to rinse the His-Trap column, and then use about 5mL Binding Buffer to balance the His-Trap column;

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

[0061] f. After loading, use about 30mL Washing Buffer to remove the impurities in the His-Trap column;

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

[0063] h. Use 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 in sequence, and seal it with 20% ethanol;

[0065] j. Use a 10 kDa ultrafiltration tube to concentrate the collected protein by ultrafiltration at 4000 r / min for 15 min at 4°C, and replace the imidazole in the Elution Buffer with GE Buffer 4-5 times. The final concentrated protein is packaged and stored at -80°C for later use.

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

[0067] The uninduced samples, whole bacteria samples after induction, supernatant samples after induction, precipitate samples after induction, flow-through and elution samples were mixed with loading buffer in a volume ratio of 4:1, heated in a boiling water bath, centrifuged (4°C, 12000r / min, 2min), and the supernatant was taken for loading; electrophoresis buffer was added, the glass plate was installed, and the sample comb was slowly and vertically pulled out in the buffer solution, and 10μL protein sample and 3μL Marker were added to the hole; the positive and negative poles of the power cord were correctly connected, and the initial voltage of the electrophoresis instrument was set to 80V. When the Marker ran to the separation gel, the voltage was adjusted to 120V, and the electrophoresis was continued for 1-1.5h, and the length of time depended on the molecular weight of the protein to be tested; the gel was peeled off from the gel plate, placed in Coomassie Brilliant Blue staining solution, and stained at room temperature for 2h. Then the staining solution was discarded, and an appropriate amount of decolorizing solution was added for decolorization several times until the background color was weak and the target protein band was 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 size of the gel. Activate the PVDF membrane with methanol for 3 minutes. Immerse the gel, PVDF membrane and filter paper in the transfer buffer to wet them. Place them in the order of positive electrode-filter paper-PVDF membrane-gel-filter paper-negative electrode, with 5 filter papers on each side. Avoid bubbles and drying of the gel and membrane, cover the lid and put it in ice. Transfer the membrane for 18 minutes at a constant voltage of 65V and 1kDa / 1min. Use TBST containing 5% skim milk to block at room temperature on a shaker for 2 hours; after blocking, wash the membrane three times with TBST, and then use TBST to dilute DCH positive serum 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 at a ratio of 1:10000 with TBST, 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 them 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, and 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 chessboard titration method. The specific process is as follows:

[0076] Use coating solution (0.1mol / L pH9.6 carbonate buffer) to dilute the purified recombinant protein core1-149 according to seven gradients of 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL, and 0.125μg / mL. Each concentration is coated in a vertical row of 6 wells, 100μL per well, and coated overnight at 4°C. Add 250μL PBST to each well for washing, 5 minutes each time, repeat 5 times, and pat the liquid in the plate vertically several times after washing. Use 5% BSA as the blocking solution (prepared with PBST), 200μL per well, and block 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 an enzyme 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 largest 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 samples to be tested

[0078]

[0079] 2. Determination of the best 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, 200 μL 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 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 1h.

[0085] Table 3 Determination of the optimal sealing time

[0086]

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

[0088] The optimized coating concentration, serum dilution ratio, blocking solution and blocking time were used for indirect ELISA. The other conditions were the same as in step 1. Four different serum incubation 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. 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, and the optimal serum incubation time is 37°C for 1.5h.

[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 above optimized optimal coating concentration, optimal serum dilution ratio, optimal blocking solution, optimal blocking time and optimal serum incubation time were used for indirect ELISA determination. The other conditions were the same as 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. OD 450 Value, select P / N (positive serum OD 450 Value / negative serum OD 450 The dilution ratio of the secondary antibody with the largest value is the optimal dilution ratio of the secondary antibody. The test results are shown in Table 5, and the optimal dilution ratio of the secondary antibody is 1:10000.

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

[0095]

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

[0097] The above optimized optimal coating concentration, optimal serum dilution ratio, optimal blocking solution, optimal blocking time, optimal serum incubation time and optimal secondary antibody dilution ratio were used for indirect ELISA determination. The other conditions were the same as 3.1. Four different secondary antibody incubation times were set, namely 37℃ 0.5h, 37℃ 1h, 37℃ 1.5h, and 37℃ 2h. Three parallel wells were set in each group for indirect ELISA detection. 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, and the optimal incubation time of the secondary antibody is 1 h at 37°C. 450 value / OD value of negative serum 450 value) value as the optimal incubation time of the secondary antibody. The test results are shown in Table 6, and the optimal incubation time of the secondary antibody is 1 h 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, which are 5 min, 10 min, 15 min, and 20 min in sequence. Set 3 parallel wells in each group, perform indirect ELISA detection, and 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, and the optimal TMB color development time is 10 min at 37°C. 450 value / OD value of negative serum 450 value) value as the optimal color development time. The test results are shown in Table 7, and the optimal TMB color development time is 10 min 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 h, serum dilution ratio is 1:400, serum incubation time is 1.5 h, secondary antibody dilution ratio is 1:10000, secondary antibody incubation time is 1 h, TMB color development time is 10 min). Calculate the average value X and standard deviation SD of its OD 450 value. Taking 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] Take the same batch of coated ELISA plates, and use the optimized indirect ELISA method to test 4 DCH positive sera and 3 DCH negative sera, with 3 parallel wells for each serum, to test the intra-batch repeatability of this indirect ELISA method. In addition, take 3 batches of coated ELISA plates and use the optimized indirect ELISA method to test the inter-batch repeatability of this indirect ELISA method. The calculation formula of the coefficient of variation is standard deviation (SD) / mean value (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 repeatability.

[0112] Table 9 Intra-batch coefficient of variation

[0113]

[0114] Table 10 Inter-batch coefficient of variation

[0115]

[0116] 10. Specificity test

[0117] The above optimized conditions were used to perform ELISA tests on the 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 of positive serum against 5 other common cat pathogens is 450 All of them were below the critical value of 0.465, which fully proved that the established indirect ELISA method had good specificity.

[0119] Table 11 core1-149 specificity test readings

[0120] Serum samples <![CDATA[OD 450 ]]> Judgment result Positive samples 1.449 + Negative samples 0.063 - FWf 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 to perform indirect ELISA detection 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 result + + + + + - - -

[0126] 12. Compliance rate detection

[0127] Thirty-four cat sera were selected and tested by Western Blot using core1-149 protein loading. ELISA was also used to test the 34 sera and compare the consistency of the two methods.

[0128] The test results are shown in Table 13. The results show that the indirect ELISA test method detected 22 positive samples, Western Blot detected 21 positive samples, and the positive coincidence rate was 95.5% (21 / 22); the indirect ELISA test method detected 12 negative samples, Western Blot detected 13 negative samples, and the negative coincidence rate was 92.3% (12 / 13); there was 1 serum that was judged as positive in ELISA, but negative in Western Blot; the total coincidence rate of the indirect ELISA test method and Western Blot test 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 detection 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 antibody in Wuhan was 84.88%.

[0133] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of feline hepatotropic virus recombinant protein core1-149 in the preparation of 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.

4.

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

4. The indirect ELISA kit according to claim 3, characterized in that The blocking solution was 5% BSA.

5. Use of the indirect ELISA kit described in any one of claims 2 to 4 in detecting feline hepadnavirus core antibodies.

6. A method for detecting feline hepadnavirus core antibodies, characterized in that: Detection using the indirect ELISA kit according to any one of claims 2 to 4 comprises the following steps: The recombinant protein core1-149 of feline hepatotropic virus was coated, washed and blocked; After blocking is completed, the diluted serum to be tested is added and the serum is incubated; After incubation, wash, add diluted enzyme-labeled secondary antibody, and incubate with secondary antibody; After the incubation is completed, wash, add color development solution, and incubate for color development; After adding the stop solution, read the OD 450 Numeric value.

7. The method according to claim 6, characterized in that The coating concentration is 0.5-2 μg / mL; the blocking time is 1-2 hours.

8. The method according to claim 6, characterized in that The dilution ratio of the serum to be tested is 1:(200-400); the serum incubation time is 1-2h.

9. The method according to claim 6, characterized in that The dilution ratio of the enzyme-labeled secondary antibody is 1:(10000-20000); the incubation time of the secondary antibody is 1.5-2h.

10. The method according to claim 6, characterized in that The color development incubation time is 10-15 min.

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