ELISA kit, method and application for detecting antibodies to feline infectious peritonitis virus

Abstract

The present invention discloses an ELISA kit, method and application for detecting antibodies to feline infectious peritonitis virus, and belongs to the technical field of feline infectious peritonitis virus. The ELISA kit uses the recombinant protein S1121-1176 of feline infectious peritonitis virus as a coating antigen; the amino acid sequence of the recombinant protein S1121-1176 is shown in SEQ ID NO: 1. The present invention also discloses the application of the ELISA kit in the preparation of a diagnostic product for feline infectious peritonitis. The present invention prepares the S1121-1176 recombinant protein by prokaryotic expression, uses the recombinant protein as a coating antigen, optimizes and establishes an indirect ELISA detection method for diagnosing FIP, and the method has high specificity and sensitivity, is simple to operate, short time consumption, low cost, can be detected in large quantities, and provides a simple and effective diagnostic tool for clinical diagnosis of FIP in veterinary medicine.

Description

Technical Field

[0001] The invention relates to the technical field of feline infectious peritonitis virus, and in particular to an ELISA kit, method and application for detecting feline infectious peritonitis virus antibodies. Background Art

[0002] Feline Infectious Peritonitis (FIP) is a systemic disease that endangers the health of cats and is caused by the Feline Infectious Peritonitis Virus (FIPV). Once clinical symptoms appear in cats, they usually develop high inflammation and systemic infection. Cats infected with FIPV can die within 1 to 3 weeks without effective treatment. It is almost a 100% fatal disease.

[0003] FIPV is a non-segmented single-stranded RNA virus with a genome of about 29kb. It contains 11 open reading frames (ORFs), encoding four structural proteins: spike protein (S), nucleocapsid protein (N), envelope protein (E), and membrane protein (M). Among them, the S protein plays an important role in the pathogenesis of FIPV because it participates in the process of receptor binding and virus-cell membrane fusion. There are cell receptor binding sites, action sites for fusion with cell membranes, and major antigenic sites on the S protein. Studies have reported that there are at least 5 neutralization sites on the S protein.

[0004] At present, the main detection methods for FIP are clinical examination and laboratory examination. The clinical examination process is cumbersome, the symptoms are not specific, and it is easy to miss or misdiagnose. Laboratory examination can be divided into antigen detection and antibody detection. Although immunohistochemistry (IHC) is the gold standard for diagnosing FIP, this method is mostly used for postmortem examination. Pre-mortem diagnosis will cause invasive damage, and the long cycle and high cost may be difficult for pet owners to accept. RT-PCR cannot distinguish between feline coronavirus (FECV) and FIPV, and there is interference from false positives. Serological diagnostic methods (such as ELISA) are simple to operate, sensitive, rapid, and low-cost, but the biggest difficulty in the serological diagnosis of FIP lies in the limitations in sensitivity and specificity. Therefore, there is currently a lack of an effective serological detection method for FIP. Summary of the invention

[0005] The purpose of the present invention is to provide an ELISA kit, method and application for detecting antibodies to feline infectious peritonitis virus, so as to solve the problems existing in the above-mentioned prior art. The present invention uses S1121-1176 recombinant protein as a coating antigen, optimizes and establishes an indirect ELISA detection method for diagnosing FIP. The method has high specificity and sensitivity, is simple to operate, short in time, low in cost, can be used for large-scale detection, and provides a simple and effective diagnostic tool for veterinary clinical diagnosis of FIP.

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

[0007] The present invention provides an ELISA kit for detecting antibodies to feline infectious peritonitis virus. The ELISA kit uses recombinant protein S1121-1176 of feline infectious peritonitis virus as a coating antigen.

[0008] The amino acid sequence of the recombinant protein S1121-1176 is shown in SEQ ID NO:1.

[0009] Furthermore, the method for preparing the coated antigen comprises the following steps:

[0010] The coding gene of the recombinant protein S1121-1176 of feline infectious peritonitis virus is connected to a prokaryotic expression vector and transformed into a host bacterium to induce expression, and a bacterial liquid is obtained, which is then broken and purified to harvest the coated antigen.

[0011] Furthermore, the prokaryotic expression vector is pEGX-6P-1; and the host bacteria is Escherichia coli.

[0012] Furthermore, it also includes enzyme-labeled secondary antibody, blocking solution, washing solution, diluent, positive control sample, negative control sample, color development solution and stop solution.

[0013] Furthermore, the enzyme-labeled secondary antibody is HRP-sheep anti-cat IgG antibody; and the blocking solution is PBST blocking solution containing 2% BSA.

[0014] The present invention also provides an ELISA detection method for detecting antibodies to feline infectious peritonitis virus for non-diagnostic purposes, comprising the following steps:

[0015] The recombinant protein S1121-1176 was coated onto an ELISA plate, and a blocking solution was added to block the plate for 1-2 hours;

[0016] Dilute the serum with diluent and incubate the diluted serum for 15-90 minutes;

[0017] Add diluted HRP-sheep anti-cat IgG antibody and incubate for 15-60 minutes;

[0018] Add color developing solution and develop color at room temperature in the dark for 5-20 minutes;

[0019] Add stop solution to stop color development;

[0020] Finally, the OD values ​​at wavelengths of 450nm and 630nm were measured using an enzyme-labeled instrument.

[0021] Furthermore, if the OD450 nm-OD630 nm of the serum to be tested is ≥0.59, it is positive; if the OD450 nm-OD630 nm of the serum to be tested is ≤0.42, it is negative; if the OD450 nm-OD630 nm of the serum to be tested is greater than 0.42 and less than 0.59, it is suspicious.

[0022] Furthermore, the coating concentration of the recombinant protein S1121-1176 is 26.4 μg / mL; the dilution multiple of the serum is 1:1600, and the incubation time is 60 min; the dilution multiple of the HRP-sheep anti-cat IgG antibody is 1:15000, and the incubation time is 60 min; and the blocking solution is PBST blocking solution containing 5% skimmed milk powder.

[0023] The present invention also provides application of the ELISA kit in preparing a diagnostic product for feline infectious peritonitis.

[0024] The present invention discloses the following technical effects:

[0025] The ELISA detection method for detecting antibodies to feline infectious peritonitis virus established by the present invention has a sensitivity of up to 1:6400; there is no cross reaction with FPV, FHV, and Toxoplasmosis positive sera; repeatability tests show that the coefficient of variation of the samples detected within the same batch is between 1.890% and 17.318%, all less than 20%; and the coefficient of variation within different batches is between 2.306% and 18.466%, all less than 20%, indicating that the method has good intra-batch and inter-batch repeatability; 6 FIPV antibody-positive samples were detected from clinical samples.

[0026] In summary, the recombinant S1121-1176 protein prepared by the present invention has good antigenicity, and the established ELISA detection method for detecting feline infectious peritonitis virus antibodies is sensitive, specific, and efficient, simple to operate, short in time, and low in cost. It can detect cats infected with FIPV in large quantities, providing a simple and effective diagnostic tool for veterinary clinical diagnosis of FIP. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 The results of bacterial liquid PCR identification; M: 215bp; 1-4: S1121-1176 target bands;

[0029] Figure 2 The results of SDS-PAGE determination of recombinant protein; M: 33KDa; S1121-1176: concentrated recombinant protein S1121-1176;

[0030] Figure 3 The results of Western-bloting immunogenicity test of the recombinant protein; M: 33KDa; S1121-1176: purified recombinant protein S1121-1176. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] This test uses Escherichia coli (E.coli) BL21 (DE3) competent cells prepared by our laboratory; standard feline panleukopenia virus (FPV), feline herpes virus (FHV), feline toxoplasmosis antibody positive serum, standard feline infectious peritonitis virus (FIPV) antibody positive serum and negative serum are all stored in our laboratory.

[0037] The main reagents used in the experiment are as follows:

[0038] Glutathione Sepharose TM 4Fast Flow was purchased from cytiva; protein marker was purchased from Thermo; SDS and IPTG were purchased from Biosharp; HRP-sheep anti-cat IgG (H+L) was purchased from Dakoway Biotechnology Co., Ltd.; TMB colorimetric solution was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Beyotime); PBS powder was purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0039] Example 1 Construction of prokaryotic expression vector of recombinant plasmid pEGX-6P-1-S1121-1176:

[0040] 1.1 The amino acid sequence 1121-1176 of the FIPV S protein with good antigenicity and strong hydrophilicity (as shown in SEQ ID NO: 1) was selected as the target antigen protein, and a pair of amplification primers were designed. The primer sequence S1121-1176-F was shown in SEQ ID NO: 2, and S1121-1176-R was shown in SEQ ID NO: 3.

[0041] PCR amplification was performed using the cDNA of type I FIPV strain stored in the laboratory as a template;

[0042] The PCR reaction system is as follows:

[0043] 2×Phanta Max Master Mix 25μL, S1121-1176-F 2μL, S1121-1176-R 2μL, cDNA 1μL and H2O 20μL, the total reaction system is 50μL.

[0044] The PCR reaction procedure is as follows:

[0045]

[0046] 1.2 The pEGX-6P-1 plasmid stored in the laboratory was used for inverse PCR to obtain a linearized vector. The sequence of the upstream primer pEGX-F is shown in SEQ ID NO: 4, and the sequence of the downstream primer pEGX-R is shown in SEQ ID NO: 5;

[0047] The PCR reaction system is as follows:

[0048] 2×Phanta Max Master Mix 25μL, pEGX-F 2μL, pEGX-R 2μL, pEGX-6P-1 plasmid 1μL and H2O 20μL, the total reaction system is 50μL.

[0049] The PCR reaction procedure is as follows:

[0050]

[0051]

[0052] The obtained amplified product and linearized vector were recovered using the OMEGA gel recovery kit. The recovered linearized vector and the target gene fragment were connected by single fragment homologous recombination. The connection reaction system is as follows:

[0053] 2×Uniclone SeamLess Cloning Mix 5μL, linearized vector 90ng, target gene fragment 10ng and H2O2.9μL, the total reaction system is 10μL.

[0054] React at 50℃ for 30min. Take 10μL of the above ligation product to transform E.coli competent cells DH5α, and spread it on LB solid plate (tryptone 10g, agar powder 15g, yeast extract 5g, NaCl 10g, deionized water to 1L) containing ampicillin resistance (final concentration is 100mg / L) for overnight culture; the next day, pick the white clone strain on the plate and perform gene sequencing identification (commissioned by Wuhan Qingke Innovation Biotechnology Co., Ltd.).

[0055] The amino acid sequence of the S1121-1176 protein in this example is shown in SEQ ID NO: 1:

[0056] TKIQSVVNQQGEALSQLTSQLQKNFQAISSSIAEIYNRLEKVEADAQVDRLITGRL(SEQ ID NO: 1);

[0057] S1121-1176-F: ACCAAGATTCAGAGTGTG (SEQ ID NO: 2);

[0058] S1121-1176-R: TAATCTGCCAGTAATGAGACG (SEQ ID NO: 3);

[0059] pEGX-F: CTCGAGCGGCCGCATCGTGA (SEQ ID NO: 4);

[0060] pEGX-R: GGATCCCAGGGGCCCCTGGA (SEQ ID NO: 5).

[0061] Example 2 Induction and expression of recombinant protein S1121-1176

[0062] 2.1 Transformation and identification of recombinant plasmid pEGX-6P-1-S1121-1176

[0063] The correctly sequenced recombinant plasmid pEGX-6P-1-S1121-1176 was transferred into E. coli BL21 (DE3) competent cells, spread on the plate, picked a single positive colony and inoculated it in Amp-LB liquid medium for culture, and then the PCR-positive samples of the bacterial liquid were sent to Wuhan Qingke Innovation Biotechnology Co., Ltd. for sequencing.

[0064] The PCR system is as follows:

[0065] 2×Tap Mix 5μL, S1121-1176-F 1μL, S1121-1176-R 1μL, template 1μL and H2O 2μL.

[0066] The PCR reaction procedure is as follows:

[0067]

[0068] The test results showed that the target band size was 168 bp after PCR identification of the bacterial solution and electrophoresis on 1.5% agarose gel, which was consistent with the expected size ( Figure 1 ).

[0069] 2.2 Induced expression and purification of recombinant protein S1121-1176

[0070] 2.2.1 Inducible expression of recombinant protein

[0071] The positive bacterial liquid identified by PCR and sequenced correctly was inoculated into Amp-LB liquid culture medium, placed in an incubator at 26°C and cultured with shaking at 200r / min until OD450nm reached 0.6, 1% IPTG inducer was added to continue inducing expression for 6h, and the negative control strain was cultured simultaneously and the bacterial liquid was collected.

[0072] 2.2.2 Recombinant protein purification

[0073] (1) The induced bacterial solution was centrifuged at 10,000 r / min for 10 min, resuspended in GST Binding Buffer, and then crushed under high pressure and centrifuged at 10,000 r / min for 50 min. The supernatant was collected and filtered through a 0.45 μm filter.

[0074] (2) Glutathione Sepharose TM 4. Mix the Fast Flow filler and add it to the chromatography column. Let it stand at room temperature for 10 minutes. After the gel and solution are separated, open the liquid outlet at the bottom to allow the ethanol to flow out slowly by gravity.

[0075] (3) Add 5 volumes of deionized water to the loaded column to rinse away the ethanol, and then equilibrate the column with 5 column volumes of GST Binding Buffer.

[0076] (4) Load the supernatant onto the column at a flow rate of 0.5 ml / min and collect the flow-through.

[0077] (5) Rinse the column with 10 column volumes of GST Binding Buffer to remove contaminating proteins.

[0078] (6) Elute the target protein using a Bio-Rad NGC protein chromatography system purifier, using an appropriate amount of Elution Buffer, and collect the elution peaks in separate tubes, with 1 tube containing 2 ml of each peak.

[0079] (7) After elution, wash the column with 5 column volumes of GST Binding Buffer and 5 column volumes of deionized water, and then equilibrate with 3 column volumes of 20% ethanol (the ethanol should be enough to submerge the filler). Seal the column and store at 2-8°C.

[0080] (8) Concentrate the eluted peaks collected in the sub-tubes using a 10 kDa protein concentrator at 4500 r / min at 4°C for 1 h. Store the concentrated protein at -80°C.

[0081] 2.2.2 Identification of recombinant protein by SDS-PAGE

[0082] Take 40 μL of the above concentrated protein, add 10 μL of 5×SDS loading buffer, denature at 105°C for 10 min, perform SDS-PAGE, and after SDS-PAGE, use Coomassie Brilliant Blue staining to identify the expression and solubility of the recombinant protein S1121-1176.

[0083] The separation gel and stacking gel for SDS-PAGE were prepared as follows:

[0084] (1) 12% separation gel

[0085] H2O.3 mL, 30% acrylamide 4.0 mL, Tris-HCl (pH=8.8) 2.5 mL, 10% SDS 0.1 mL, 10% AP 0.1 mL and TEMED 0.004 mL.

[0086] (2) 5% concentrated gel

[0087] H2O 2.1mL, 30% acrylamide 0.5mL, Tris-HCl (pH=6.8) 0.38mL, 10% SDS 0.03mL,

[0088] 10% AP 0.03 mL and TEMED 0.003 mL.

[0089] The test results showed that the S1121-1176 protein was expressed at 33 kDa ( Figure 2 ).

[0090] 2.2.3 Identification of recombinant protein by Western blotting

[0091] The concentrated protein was subjected to SDS-PAGE and transferred to a PVDF membrane at 120V for 45min. The membrane was blocked with TBST containing 5% skimmed milk powder overnight at 4°C, and FIPV antibody-positive serum was added and incubated at 37°C for 2h. The membrane was washed three times with TBST and then incubated with 1:10000 diluted HRP-sheep anti-cat IgG (H+L) for 2h. ECL colorimetric solution was used for color development to verify the immunogenicity of the recombinant protein S1121-1176.

[0092] The test results showed that the purified recombinant protein S1121-1176 had a single band at 33 kDa ( Figure 3 ).

[0093] Example 3 Establishment of an indirect ELISA antibody detection method based on S1121-1176 protein

[0094] 3.1 Determination of the optimal coating concentration of recombinant protein and the optimal dilution factor of serum

[0095] The purified recombinant protein S1121-1176 was diluted with coating buffer at a ratio of 1:100 (26.4 μg / mL), 1:200 (13.2 μg / mL), 1:400 (6.6 μg / mL), 1:800 (3.3 μg / mL), 1:1600 (1.65 μg / mL), 1:3200 (0.825 μg / mL), 1:6400 (0.4125 μg / mL). L), 1:12800 (0.20625μg / mL), and added to the 1st to 8th rows of a 96-well ELISA plate respectively. Each dilution was added to a row of 12 wells, 100μL per well, and coated at 4℃ overnight. After coating, wash three times with PBST, adding 200μL each time, shaking and washing for 5min, and then patting dry; add 100μL of PBST blocking solution containing 2% BSA to each well, incubate at 37℃ for 1h, discard the blocking solution and wash the plate;

[0096] The standard negative and positive sera were diluted with PBST in a serial ratio starting from 1:100 and then respectively formed into square arrays with the recombinant protein S1121-1176, 100 μL per well, incubated at 37°C for 1 hour, and the plate was washed again; 100 μL of HRP-sheep anti-cat IgG (H+L) diluted 1:5000 with PBST was added to each well, incubated at 37°C for 1 hour, the plate was washed three times and patted dry; 50 μL of TMB colorimetric solution was added to each well, and after 10 minutes at room temperature and away from light, 50 μL of 2M H2SO4 was added to each well to terminate the color development reaction, and the OD450nm and OD630nm values, the optimal protein concentration and the optimal serum dilution were measured with an enzyme marker. The judgment criteria were: positive OD450nm-OD630nm / negative OD450nm-OD630nm (P / N) was the maximum.

[0097] 3.2 Determination of dilution concentration of enzyme-labeled secondary antibody

[0098] With HRP-sheep anti-cat IgG (H+L) diluted at 1:5000, 1:10000, and 1:15000 as the only variable, the recombinant protein S1121-1176 was coated with the above-determined protein concentration, and the primary antibody was incubated with the above-determined serum concentration. The other steps were the same as 3.1. The optimal dilution multiple was determined by the OD450nm-OD630nm values ​​of the positive and negative serum and the P / N value at different enzyme-labeled secondary antibody dilutions.

[0099] 3.3 Determination of the optimal blocking solution

[0100] Under the condition that only the blocking solution is used as a variable, four groups of blocking solutions are set, including 1% BSA, 2% BSA, 5% BSA, and 5% skim milk powder. The recombinant protein S1121-1176 is coated with the above determined protein concentration, and the primary antibody is incubated with the above determined serum concentration. The other steps are the same as 3.1. The criterion for determining the optimal blocking solution is: the maximum P / N value.

[0101] 3.4 Determination of the optimal serum reaction time

[0102] When only serum reaction time was used as a variable, five groups of reaction times were set: 15min, 30min, 45min, 60min, and 90min. The recombinant protein S1121-1176 was coated with the above-determined protein concentration, and the primary antibody was incubated with the above-determined serum concentration. The other steps were the same as 3.1. The criterion for determining the optimal serum reaction time was: the maximum P / N value.

[0103] 3.5 Determination of the optimal enzyme-labeled secondary antibody reaction time

[0104] Under the condition that only the enzyme-labeled secondary antibody reaction time is used as a variable, four groups of reaction times are set: 15min, 30min, 45min, and 60min. The recombinant protein S1121-1176 is coated with the above-determined protein concentration, and the primary antibody is incubated with the above-determined serum concentration. The other steps are the same as 3.1. The criterion for determining the optimal enzyme-labeled secondary antibody reaction time is: the maximum P / N value.

[0105] 3.6 Determination of critical value

[0106] The optimized indirect ELISA method was used to detect 30 FIPV antibody negative sera, and the OD450nm-OD630nm values ​​were determined and statistically analyzed. When the OD450nm-OD630nm value of the test sample ≥ the average value of the negative control When the negative control standard deviation (SD) is greater than the negative control standard deviation, it can be judged as positive; when the OD450nm-OD630nm value of the test sample is less than the negative control average value When the sample is 0.0447 W / m3 / s, it can be judged as negative; if it is between the two, it is judged as suspicious.

[0107] Preliminary conditions for the indirect ELISA antibody detection method based on S1121-1176 protein:

[0108] The optimal coating concentration of recombinant protein S1121-1176 is 26.4μg / mL, and the optimal dilution of serum is 1:1600 (Table 1); the optimal dilution of enzyme-labeled secondary antibody is 1:15000 (Table 2); the optimal blocking solution is PBST containing 5% skimmed milk powder (Table 3); the optimal reaction time of enzyme-labeled secondary antibody is 1h (Table 4); the optimal reaction time of serum is 1h (Table 5); the critical values ​​of S1121-1176 are 0.59 and 0.42, the tested serum OD450nm-OD630nm ≥ 0.59 is positive, the tested serum OD450nm-OD630nm ≤ 0.42 is negative, and those between the two are suspicious (Table 6).

[0109] Table 1 Determination of the optimal coating concentration of S1121-1176 protein and the optimal dilution multiple of serum

[0110]

[0111]

[0112] Table 2 Determination of the optimal enzyme-labeled secondary antibody dilution

[0113]

[0114] Table 3 Determination of the best blocking solution

[0115]

[0116] Table 4 Determination of enzyme-labeled secondary antibody reaction time

[0117]

[0118]

[0119] Table 5 Determination of the optimal serum reaction time

[0120]

[0121] Table 6 Determination of critical values ​​of S1121-1176

[0122]

[0123] Example 4 Performance measurement of indirect ELISA antibody detection method based on S1121-1176 protein

[0124] 4.1 Specificity test

[0125] According to the above-mentioned conditions, the positive sera for FPV, FHV and Toxoplasmosis antibodies were tested, and positive and negative controls were set up to test the specificity of the ELISA detection method established in this experiment.

[0126] The results showed that the ELISA detection method established in this experiment detected FIPV standard positive serum as positive, and other positive sera were judged as negative, indicating that the indirect ELISA antibody detection method for detecting FIPV established in this experiment had no cross-reaction with other sera and had good specificity (Table 7).

[0127] 4.2 Sensitivity test

[0128] The established indirect ELISA method was used to test the standard positive serum after multiple dilution to verify the sensitivity of this method.

[0129] The results showed that the detection sensitivity of the ELISA detection method for S1121-1176 protein to the standard positive serum was 1:6400, indicating that the method had a high sensitivity (Table 8).

[0130] 4.3 Repeatability test

[0131] Using the established indirect ELISA detection method, 5 sera with different antibody levels were tested in parallel, with each sample repeated 3 times in three ELISA plates from the same batch. Then, the same 5 sera with different antibody levels were tested in parallel, with each sample repeated 3 times in three ELISA plates from different batches. The OD450nm-OD630 nm values ​​of the samples were counted, and the intra- and inter-batch coefficients of variation were calculated.

[0132] The test results showed that the coefficient of variation of the FIPV indirect ELISA antibody detection method within the same batch was between 1.890% and 17.318%, all less than 20%; and the coefficient of variation within different batches was between 2.306% and 18.466%, all less than 20%, indicating that the method had good intra-batch and inter-batch repeatability (Tables 9 and 10).

[0133] Table 7 Specificity test

[0134]

[0135] Table 8 Sensitivity test

[0136]

[0137]

[0138] Table 9S1121-1176 Protein Batch Repeat Test

[0139]

[0140] Table 10S1121-1176 protein intra-batch repeat test

[0141]

[0142] Example 5 Clinical application of indirect ELISA antibody detection method based on S1121-1176 protein

[0143] The FIPV indirect ELISA antibody detection method established in this study was used to detect 80 sera from the clinic (serum numbers 1-6 are known to be FIPV positive sera, and the rest are negative sera), and the positive rate was calculated. The results of detecting clinical serum FIPV antibodies using the method of the present invention are shown in Table 11. From the test results, the method established in this study is completely consistent with the actual results, which proves that this method is accurate and reliable and can be applied to clinical testing.

[0144] Table 11 Clinical serum FIPV antibody test results

[0145]

[0146] 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. An ELISA kit for detecting antibodies to feline infectious peritonitis virus, characterized in that: The ELISA kit uses the recombinant protein S1121-1176 of feline infectious peritonitis virus as the coating antigen; The amino acid sequence of the recombinant protein S1121-1176 is shown in SEQ ID NO:

1.

2. The ELISA kit according to claim 1, characterized in that The method for preparing the coated antigen comprises the following steps: The coding gene of the recombinant protein S1121-1176 of feline infectious peritonitis virus is connected to a prokaryotic expression vector and transformed into a host bacterium to induce expression, and a bacterial liquid is obtained, which is then broken and purified to harvest the coated antigen.

3. The ELISA kit according to claim 2, characterized in that The prokaryotic expression vector is pEGX-6P-1; the host bacteria is Escherichia coli.

4. The ELISA kit according to claim 1, characterized in that It also includes enzyme-labeled secondary antibody, blocking solution, washing solution, diluent, positive control sample, negative control sample, color development solution and stop solution.

5. The ELISA kit according to claim 4, characterized in that The enzyme-labeled secondary antibody was HRP-sheep anti-cat IgG antibody; the blocking solution was PBST blocking solution containing 2% BSA.

6. An ELISA method for detecting antibodies to feline infectious peritonitis virus for non-diagnostic purposes, characterized in that: The following steps are involved: The recombinant protein S1121-1176 of feline infectious peritonitis virus was coated onto the ELISA plate and blocked with blocking solution for 1-2 hours; Dilute the serum with diluent and incubate the diluted serum for 15-90 minutes; Add diluted HRP-sheep anti-cat IgG antibody and incubate for 15-60 minutes; Add color developing solution and develop color at room temperature in the dark for 5-20 minutes; Add stop solution to stop color development; Finally, the OD values ​​at wavelengths of 450nm and 630nm were measured using an enzyme reader; The amino acid sequence of the recombinant protein S1121-1176 is shown in SEQ ID NO:

1.

7. The ELISA detection method according to claim 6, characterized in that If the OD450 nm-OD630 nm of the tested serum is ≥ 0.59, it is positive; if the OD450 nm-OD630 nm of the tested serum is ≤ 0.42, it is negative; if the OD450 nm-OD630 nm of the tested serum is greater than 0.42 and less than 0.59, it is suspicious.

8. The ELISA detection method according to claim 6, characterized in that The coating concentration of the recombinant protein S1121-1176 is 26.4 μg / mL; the dilution multiple of the serum is 1:1600, and the incubation time is 60 min; the dilution multiple of the HRP-sheep anti-cat IgG antibody is 1:15000, and the incubation time is 60 min; the blocking solution is PBST blocking solution containing 5% skimmed milk powder.

9. application of the ELISA kit as described in any one of claims 1-5 in the preparation of a feline infectious peritonitis diagnosis product.

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