Antigen for detecting I-type and II-type feline coronavirus antibodies and kit and application thereof

Abstract

The invention belongs to the technical field of antibody detection, and particularly relates to an antigen for detecting I-type and II-type feline coronavirus antibodies and a kit and application thereof. Antigens for detecting the I-type and II-type feline coronavirus antibodies are purified recombinant N protein, IRBD protein (I-type) and IIRBD (II-type) protein of feline coronavirus, and an indirect ELISA detection kit is formed by using an ELISA plate of the purified recombinant N protein and RBD protein of feline coronavirus. Results of the embodiment of the invention show that the ELISA kit for the feline coronavirus antibody has the advantages of low cost, simplicity and convenience in use, high sensitivity and strong specificity, does not generate cross reaction with other common pathogens in a cat group, and can effectively distinguish type I and type II feline coronaviruses.

Description

Technical Field

[0001] The invention belongs to the technical field of antibody detection, and in particular relates to an antigen for detecting type I and type II feline coronavirus antibodies, a kit and an application thereof. Background Art

[0002] Feline coronavirus (FCOV) is an enveloped, single-stranded, positive-sense RNA virus with a relatively large genome of approximately 29,000 nucleotides. The genome structure of feline coronavirus has typical characteristics of coronaviruses and consists of 11 open reading frames (ORFs) that can encode 11 different proteins, including structural proteins, non-structural proteins, and accessory proteins. Structural proteins include spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). In the field of coronavirus research, the receptor binding domain (RBD) at the C-terminus of the spike protein S1 not only contains the main neutralizing epitope, but also has strong immunogenicity. It is a key target for the design of vaccines and antiviral drugs and is of great value for the development of vaccines and diagnostic reagents.

[0003] Feline coronavirus is prone to mutation during replication, which may cause it to transform from feline enteric coronavirus (FECV) to feline infectious peritonitis virus (FIPV), causing fatal feline infectious peritonitis (FIP), which has become one of the main causes of death in domestic and wild cats. There are two serotypes of FCOV: type I and type II. Type I can persist in cats for a long time, while type II may be more closely related to the lethality of feline infectious peritonitis. Therefore, distinguishing these two serotypes is of great significance for the treatment of FCOV-infected cats in clinical practice.

[0004] The early symptoms of feline infectious peritonitis are non-specific, with symptoms such as decreased appetite and mental depression, which are difficult to distinguish from enteritis or other diseases. Therefore, laboratory tests are required for diagnosis, which increases the difficulty of on-site diagnosis. At present, there is no good treatment and prevention method for FIP, resulting in a very high infection rate of FCOV in cats.

[0005] The in vitro isolation and culture of FCOV are extremely sensitive to the environment. The detection rate is usually low due to multiple factors such as sample quality, experimental operation and culture conditions. Polymerase chain reaction (PCR) and immunohistochemistry are the main methods for detecting FCOV. PCR technology has strict requirements on experimental conditions. Improper operation may lead to false positive results, while mutations in viral sequences may lead to mismatches with primers, resulting in false negative results. Real-time quantitative PCR (RT-PCR) technology monitors the starting amount of DNA through fluorescent probes. Compared with traditional PCR, it shows higher sensitivity in clinical practice, but the requirements for operation and instrument facilities are relatively high, and the detection time is long, which limits its application in the differential diagnosis of early FCOV infection. Immunohistochemistry detection usually requires invasive operations, which causes certain physical damage to cats and is mainly used for the detection of postmortem organ samples.

[0006] In contrast, ELISA technology is widely used in clinical diagnosis because of its advantages such as simple and rapid operation, no need for special equipment, and high stability, specificity and sensitivity of reagents. Indirect ELISA can provide results within 4-5 hours and is suitable for the detection of a variety of antigens or antibodies. However, there is currently no effective commercial feline coronavirus antibody ELISA detection kit on the market. The biggest difficulty in the serological diagnosis of FCOV lies in the limitations of sensitivity and specificity. The indirect ELISA method for detecting feline coronavirus antibodies is usually designed based on a single protein of feline coronavirus (S protein, M protein or N protein). Its detection range is narrow, and it cannot detect two serotypes of feline coronavirus at the same time, and cannot distinguish serotypes.

[0007] Therefore, there is an urgent need for a substance that can effectively distinguish FCOV serotypes. Summary of the invention

[0008] The purpose of the present invention is to provide an antigen for detecting type I and type II feline coronavirus antibodies. The antigen provided by the present invention can not only distinguish cat serotypes, but also detect quickly and accurately.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] The invention provides an antigen for detecting type I and type II feline coronavirus antibodies. The antigen is one or more groups of purified recombinant type I N protein, type I IRBD protein and type II IIRBD protein of feline coronavirus; the amino acid sequences of the recombinant type I N protein, type I IRBD protein and type II IIRBD protein are shown in SEQ ID NO.1-3.

[0011] The nucleotide sequences encoding the above-mentioned recombinant type I N protein, type I IRBD protein and type II IIRBD protein are shown in SEQ ID NOs. 4-6.

[0012] The present invention also provides an indirect ELISA detection kit for detecting type I and type II feline coronavirus antibodies, wherein the ELISA plate of the kit is coated with the above antigen.

[0013] Preferably, the kit further comprises a positive control sample, a negative control sample, a sample diluent, a washing solution, an HRG-labeled rabbit anti-cat IgG antibody, a color developing solution and a stop solution.

[0014] More preferably, the positive control sample is FCOV positive cat serum; the negative control sample is FCOV negative cat serum; the sample diluent is PBS; the washing solution is PBST containing 0.05% Tween-20; the color developing solution is TMB solution; and the stop solution is 2M sulfuric acid solution.

[0015] The present invention also provides an indirect ELISA detection method for type I and type II feline coronavirus antibodies, which specifically comprises the following steps:

[0016] S1. Incubation of serum to be tested: dilute the sample serum and add it to the ELISA plate coated with recombinant N protein, IRBD protein (type I) and IIRBD protein (type II). Incubate at 37°C for 1 hour, then wash 5 times with PBST and pat dry.

[0017] S2, enzyme-labeled antibody incubation: add 4000-fold diluted HRP-labeled rabbit anti-cat IgG antibody to the ELISA plate obtained in step S1, incubate at 37°C for 1 hour, discard the solution, wash with PBST 5 times and pat dry;

[0018] S3, color development: add TMB single-component color development solution to each well and color development for 4 minutes in the dark;

[0019] S4. Stop: Add stop solution to each well to stop the reaction and measure the OD450 nm value of the sample to be tested.

[0020] Preferably, the method further comprises determining whether the sample to be tested contains feline coronavirus antibodies.

[0021] Preferably, the standard for determining whether the sample to be tested contains feline coronavirus antibodies is: the critical value of recombinant N protein is 0.264, that is, the tested serum OD450 nm ≥ 0.264 is positive; the critical value of recombinant IRBD protein is 0.254, that is, the tested serum OD450 nm ≥ 0.254 is positive; the critical value of recombinant IIRBD protein is 0.222, that is, the tested serum OD450 nm ≥ 0.222 is positive.

[0022] The present invention also provides the use of the above antigen or the above kit in detecting type I and type II feline coronavirus antibodies.

[0023] Beneficial effects of the present invention:

[0024] (1) The present invention utilizes N recombinant protein as coating antigen. The N gene is a highly conserved gene, so the kit containing the protein can detect different strains of FCOV and can be used for preventive detection of FCOV in pet cats and experimental cats.

[0025] (2) In the kit of the present invention, the RBD protein on the S protein of type I feline coronavirus and the RBD protein on the S protein of type II feline coronavirus are selected as antigens. There is no cross-reaction between the RBD proteins of type I and type II viruses. Therefore, the indirect ELISA method established by the present invention can identify type I FCOV and type II FCOV.

[0026] (3) The combined detection of N antigen and RBD antigen in the present invention can improve the sensitivity of serological detection, especially in the early stage of the disease, and the combined diagnosis of N protein and RBD protein antibodies can improve the accuracy of FCoV detection.

[0027] (4) The indirect ELISA detection method established in the present invention has good sensitivity, specificity and repeatability. Preliminary clinical applications have also demonstrated that the kit can detect cats infected with FCOV in large quantities and has a good effect in identifying serotypes.

[0028] (5) The kit provided by the present invention provides an ELISA plate coated with recombinant N protein, IRBD protein and IIRBD protein, which can be tested on site without the need for specialized testing personnel. The test is fast and accurate, and good application results are obtained in preliminary applications, providing an effective tool for the early diagnosis of FCoV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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 labor.

[0030] Figure 1 The electrophoresis diagram of the bacterial solution PCR verification of the recombinant plasmid in Example 1, wherein lane M, 5000 DNA Marker; lane 1, recombinant plasmid pET-28a-N; lane 2-recombinant plasmid pET-28a-IRBD; lane 3, recombinant plasmid pET-32a-IIRBD;

[0031] Figure 2The induced expression and purification results of the recombinant protein in Example 1, wherein lane M, protein Marker; lane 1, recombinant protein pET-28a-N supernatant; lane 2, recombinant protein pET-28a-N precipitate; 3, recombinant protein pET-28a-IRBD precipitate; lane 4, recombinant protein pET-28a-IRBD supernatant; 5, recombinant protein pET-32a-IIRBD supernatant; lane 6, recombinant protein pET-32a-IIRBD precipitate;

[0032] Figure 3 This is a detection diagram of the product during the purification of the recombinant protein pET-28a-N in Example 1, wherein lane M is a protein marker; lane 1, flow-through; lanes 2-4, washing solution; lane 5, 200 mM imidazole eluent, and lanes 6-9, 500 mM imidazole eluent;

[0033] Figure 4 The detection diagram of the product during the purification process of the recombinant proteins pET-28a-IRBD and pET-32a-IIRBD in Example 1, wherein lane M is a protein marker; lane 1, 1M urea-dissolved inclusion body solution of pET-28a-IRBD cleavage precipitation; lane 2, 3M urea-dissolved inclusion body solution of pET-28a-IRBD cleavage precipitation; lane 3, 6M urea-dissolved inclusion body solution of pET-28a-IRBD cleavage precipitation; lane 4 Lane 5, 8M urea-dissolved inclusion body solution of pET-28a-IRBD cleavage precipitation; Lane 5, 8M urea-dissolved inclusion body solution of pET-32a-IIRBD cleavage precipitation; Lane 6, 6M urea-dissolved inclusion body solution of pET-32a-IIRBD cleavage precipitation; Lane 7, 3M urea-dissolved inclusion body solution of pET-32a-IIRBD cleavage precipitation; Lane 8, 1M urea-dissolved inclusion body solution of pET-32a-IIRBD cleavage precipitation;

[0034] Figure 5 The results of the identification of the reactivity of the recombinant protein after purification by ELISA in Example 1, wherein A is the ELISA result of detecting FCOV positive and negative sera using the recombinant N protein as the coating antigen; B is the ELISA result of detecting FCOV positive and negative sera using the recombinant IRBD protein as the coating antigen; C is the ELISA result of detecting FCOV positive and negative sera using the recombinant IIRBD protein as the coating antigen;

[0035] Figure 6 The optimization result of the antigen coating amount in Example 2;

[0036] Figure 7 This is the optimization result of the antigen coating conditions in Example 2;

[0037] Figure 8 The optimization results of the blocking solution screening in Example 2;

[0038] Fig. 9 This is the optimization result of the closing conditions in Example 2;

[0039] Fig.10 This is the optimization result of serum dilution in Example 2;

[0040] Fig.11 This is the optimization result of the serum incubation conditions in Example 2;

[0041] Fig.12 This is the optimization result of the enzyme-labeled antibody dilution in Example 2;

[0042] Fig.13 The optimized results of the enzyme-labeled antibody incubation conditions in Example 2;

[0043] Fig.14 The optimized result of TMB color development time in Example 2

[0044] Fig.15 is the calculation result of the critical value in Example 2;

[0045] Fig.16 It is the specificity test result figure in Example 3;

[0046] Fig.17 This is a graph showing the results of the intra-batch repeatability test in Example 3;

[0047] Fig.18 This is a diagram of the batch reproducibility test results in Example 3;

[0048] Fig.19 It is the sensitivity test result figure in Example 3;

[0049] Fig. 20 The statistical results of the positive antibody detection rate of clinical samples in Example 4;

[0050] Fig.21 The statistical analysis results of the antibody combined detection in Example 4;

[0051] Fig. 22 This is a correlation analysis diagram between antibodies and the occurrence of FCOV symptoms in Example 4;

[0052] Fig.23 This is a correlation analysis diagram between the antibody level and the occurrence of FCOV symptoms in Example 4. DETAILED DESCRIPTION

[0053] The invention provides an antigen for detecting antibodies to a feline coronavirus. The antigen is a purified recombinant N protein, an IRBD protein (type I) and an IIRBD protein (type II) of a feline coronavirus; the amino acid sequence of the recombinant N protein is shown in SEQ ID NO.1, the amino acid sequence of the recombinant IRBD protein (type I) is shown in SEQ ID NO.2, and the amino acid sequence of the recombinant IIRBD protein (type II) is shown in SEQ ID NO.3; genes encoding the proteins are shown in nucleotide sequence table SEQ ID NOs.4-6.

[0054] The above-mentioned recombinant N protein, IRBD protein (type I) and IIRBD protein (type II) are prepared by connecting the coding gene of the feline coronavirus N gene as shown in SEQ ID NO.4 to the prokaryotic expression vector pet-28a, connecting the coding gene of the feline coronavirus IRBD gene (type I) as shown in SEQ ID NO.5 to the prokaryotic expression vector pet-28a, and connecting the coding gene of the feline coronavirus IIRBD gene (type II) as shown in SEQ ID NO.6 to the prokaryotic expression vector pet-32a, respectively, and transforming them into BL21 Escherichia coli competent cells to induce expression, obtaining bacterial liquid, and performing ultrasonic fragmentation and purification to obtain the above-mentioned antigen.

[0055] The amino acid sequences of the N protein, IRBD protein (type I) and IIRBD protein (type II) in this example are shown in SEQ ID NO. 1-3.

[0056] N-AA:MGSSHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFELRRQAY MATQGQRVNWGDEPSKRRGRSNSRGRKNNIIPLSFYNPLTLEQGSKFWNVCPRDFVPKGIGNKDQQIGYWNRQERYRIVKGQRKELPERWFFYFLGTGPHADAKFKDKIDGVFWVARDGAMNKPTTLGTRGTNNESKPLKFDGKIPPQFQLEVNRSRNNSRSGSQSRSVSRNRSQSRGRQQSNNQNNNVEDTIVAVLQKLGVTDKQRSRSKSKDRSESKPRDTTPKNANKHTWKKTAGKGDVTNFYGARSASANFGDSDLVANGNAAKCYPQIAECVPSVSSMLFGSQWSAEDDGDQVKVTLTHTYYLPKDDAKTSQFLEQIDAYKRPSQVAKDQRQRKSRSKSADKKPEELSVTLVEAYTDVFDDTQVEMIDEVTNAAALEHHHHHHHH(SEQ IDNO.1)

[0057] IRBD-AA:MGSSHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFELRQ AYYCDSPNLRIKCQQLKHELPDGFYSAIMLVKKDLPKTFVTMPQFYNLMNVTLHVVLNDTEKGADIILAKAPELASLADIHFEIVQANGSVTNVTSLCVQARQLALFYKYTSLQGLYTYSNLVELQNYDCPFSPQQFNNHLQFETLCFYVSPAVAGCKWSLVHYNRWRTQFATITVSYKDGTMITAAAALEHHHHHH(SEQ ID NO.2)

[0058] IIRBD-AA:MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPIL DEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSYCNSHINNIKCSQLTANLNNGFYPVVSSEVGFV NKSVVLLPSFFTHTAVNITIDLGMKLSGYGQPIASTLSNITLPMQDNNTDVYCIRSDQFSVYVHSTCKSSLWDNIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVAARTRTNEQVVRSLYVIYEEGDNIV(SEQ ID NO.3)

[0059] The sequences encoding the target gene N fragment, IRBD (type I) fragment and IIRBD (type II) fragment are as follows:

[0060] N:ATTCGAGCTCCGTCGACAAGCTTATTATTGTGACTCACCCCTCAATAGAATTAAGTGTCAGCAATTGAAGCATGAGTTACCGGACGGTTTTTATTCTGCTATCATGCTCGTCAAAAAGGATTTACCTAAGACATTTGTAACTATGCCACAATTTTACAATTTGATGAATGTCACACTACACGTCGTGTTGAACGATACTGAAAAGGGGGCTGACATCATCTTGGCTAAGGCACCAGAGCTAGCATCACTTGCTGACATACATTTTGAAATAGTTCAGGCGAATGGAAGTGTAACTAATGTCACCAGCCTGTGTGTGCAAGCAAGACAATTGGCTTTATTCTACAAGTACACTAGCCTACAAGGCTTGTACACTTATTCAAATTTGGTTGAGTTACAAAATTACGACTGCCCTTTTTCACCACAGCAGTTTAACAATCATCTGCAGTTTGAAACTTTGTGTTTTTATGTGAGCCCAGCTGTGGCAGGTTGTAAGTGGTCGTTAGTTCATTACAACAGGTGGCGTACCCAGTTTGCCACTATCACTGTTTCTTACAAGGATGGTACTATGATCACTGCGGCCGCACTCGAGCA(SEQ IDNO.4)

[0061] IRBD:(SEQ ID NO.5)

[0062]

[0063] The present invention also provides an indirect ELISA kit for detecting antibodies against feline coronavirus and distinguishing serotypes. The highly conservative N protein is selected as an antigen, so that the kit can simultaneously detect different strains of FCOV. In addition, the kit also includes an RBD protein (IRBD) on the type I feline coronavirus S protein and an RBD (IIRBD) protein on the type II feline coronavirus S protein as antigens. There is no cross-reaction between the RBD proteins of type I and type II viruses. Therefore, the indirect ELISA method established by the present invention can distinguish type I FCOV from type II FCOV.

[0064] The present invention also provides an application of an indirect ELISA kit for detecting antibodies to feline coronavirus. The kit adopts indirect ELISA to detect antibodies to infected cat coronavirus and distinguish serotypes, and can be used for preventive detection of FCOV.

[0065] The results of the embodiments of the present invention show that the indirect ELISA detection method for detecting FCOV antibodies established by the present invention has a sensitivity of up to 1:64000; there is no cross reaction with common pathogens (FHV, FCV, FPV) in cats; the repeatability test shows that the coefficient of variation of the samples detected in the same batch is between 3.220% and 8.413%, and the coefficient of variation in different batches is between 1.802% and 8.948%, both of which are less than 10%, indicating that the method has good intra-batch and inter-batch repeatability; 123 cat clinical samples were tested for clinical samples, and the positive rate of feline coronavirus antibodies was detected to be 73.17%. Among these positive samples, type I FCOV infection accounted for 91.11%, type II FCOV infection accounted for 1.11%, and mixed infection accounted for 2.22%. In addition, 5.56% of the samples were detected to be positive for feline coronavirus N antibodies but RBD antibodies were not detected.

[0066] The recombinant N protein, IRBD protein (type I) and IIRBD protein (type II) prepared by the present invention have good reactivity, and the established ELISA detection method for detecting feline coronavirus antibodies has the characteristics of sensitivity, specificity and high efficiency, simple operation, short time consumption and low cost, can detect cats infected with FCOV in large quantities, and has a good effect on distinguishing serotypes, thereby providing a simple and effective diagnostic tool for veterinary clinical diagnosis.

[0067] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0068] The positive sera of feline panleukopenia virus (FPV), feline herpesvirus (FHV), feline calicivirus (FCV) antibody, and feline coronavirus antibody positive and negative sera were stored in our laboratory. 123 feline clinical serum samples were collected from several pet hospitals in Guangzhou.

[0069] HIS protein purification kit was purchased from Shanghai Biotime Biotechnology Co., Ltd.; Phanta Max Super-Fidelity DNA Polymerase, DNAmaker, and BL21 Escherichia coli competent cells were purchased from Nanjing Novozymes Biotechnology Co., Ltd.; HRP-labeled rabbit anti-cat IgG enzyme-labeled antibody was purchased from Beijing Bioson Biotechnology Co., Ltd.; TMB color development solution and ELISA stop solution were purchased from Beijing Solebow Technology Co., Ltd.

[0070] The production process, experimental method or detection method involved in the embodiments of the present invention, unless otherwise specified, are conventional methods in the prior art, and their names and / or abbreviations are conventional names in the art, and are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the name, and implement them according to conventional conditions or conditions recommended by the manufacturer. The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention have no special restrictions on the source, and are all conventional products that can be purchased through regular commercial channels, and can also be prepared according to conventional methods well known to those skilled in the art.

[0071] Example 1 Preparation of recombinant protein

[0072] 1.1 Construction of recombinant plasmid

[0073] (1) Primer design: Feline coronavirus N protein, IRBD protein (type I) and IIRBD protein (type II) were selected as target antigen proteins, and specific primers were designed. The upstream and downstream primers of the N gene and IRBD gene (type I) respectively contained HindIII and NotI restriction sites (underlined), and the homologous sequence of the PET-28a vector was added after the restriction sites. The upstream and downstream primers of the IIRBD gene (type II) respectively contained BamHI and EcoRI restriction sites (underlined), and the homologous sequence of the PET-32a vector was added after the restriction sites. The primer sequences were submitted to Shanghai Sangon Biotech Co., Ltd. for synthesis. The detailed information of the sequences is as follows:

[0074] Table 1 Primer sequence list

[0075]

[0076] (2) Amplification of target gene: PCR amplification was performed using the cDNA of the FCOV strain stored in the laboratory as a template. The PCR reaction system is shown in Table 2. The reaction conditions are: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 sec, 55°C annealing for 15 sec, 72°C extension for 15 sec, for a total of 35 cycles; 72°C extension for 5 min. After the reaction, the target fragment was recovered by gel.

[0077] Table 2 PCR reaction system

[0078]

[0079] (3) Enzyme digestion of the vector: pET-28a plasmid was double-digested with HindIII and NotI restriction endonucleases, and pET-32a plasmid was double-digested with BamHI and EcoRI restriction endonucleases.

[0080] (4) Plasmid construction and identification: The amplified N gene and IRBD gene (type I) were connected to the pET-28a restriction vector by homologous recombination, and the amplified IIRBD gene (type II) was connected to the pET-32a restriction vector by homologous recombination. Then, the plasmids were transformed into BL21 Escherichia coli competent cells for bacterial liquid PCR identification and sequencing identification.

[0081] The results of bacterial liquid PCR test showed that Figure 1 As shown, after PCR identification of N, IRBD (type I), and IIRBD (type II) bacterial liquid, the target band sizes obtained by agarose gel electrophoresis were 1173 bp, 591 bp, and 601 bp, respectively, which were consistent with the expected sizes.

[0082] 1.2 Inducible expression of recombinant protein

[0083] The positive bacterial liquid identified by PCR and sequencing was inoculated into the corresponding resistance liquid culture medium, and cultured on a shaker at 37°C and 180rpm for 3-4h. The absorbance of the bacterial liquid was measured using a spectrophotometer. When the OD600 nm value was 0.6-0.8, the bacterial liquid was taken out, and 1mmoL isopropyl-β-D-thiogalactoside (IPTG) was added. After cultured at 16°C and 120rpm for 24h, ultrasonic lysis was performed (the parameters were set as power 300W, working 5s, pause 5s, and time 30min). After lysis, the supernatant and precipitate were separated and collected.

[0084] 1.3 Expression and identification of recombinant protein

[0085] Use SDS-PAGE to analyze the expression of the target protein in the supernatant and precipitate. The specific steps are as follows: place the prepared PAGE gel in the electrophoresis tank, pour in 1×SDS-PAGE electrophoresis solution, and dot the supernatant and precipitate samples collected in 1.2 into the wells, and set the protein marker wells at the same time. Assemble the electrophoresis tank device, 80V voltage for 30min, and 120V voltage for 60min after the sample enters the separation gel layer from the concentrated gel layer. Stop electrophoresis when the sample approaches the bottom of the gel. After the electrophoresis separation is completed, cut the gel to a suitable size, soak it in pure water for several minutes to wash away the electrophoresis solution and discard it, add Coomassie brilliant blue dye solution and soak it on a shaker for 30 minutes until the dye has bands, discard the Coomassie brilliant blue dye solution, and add an appropriate amount of pure water to wash away the dye solution.

[0086] The experimental results show that Figure 2 As shown, N, IRBD, and IIRBD proteins were successfully expressed. Among them, the recombinant N protein was expressed in both the inclusion body and the supernatant, with a molecular weight of approximately 49 kDa, which was in line with the expected size, and had good soluble expression. The supernatant was subsequently used for protein purification; the recombinant proteins IRBD (type I) and IIRBD (type II) were expressed in the inclusion body, with molecular weights of approximately 27 kDa and 38 kDa, which were in line with the expected size, and were subsequently purified by precipitation.

[0087] 1.4 Purification of soluble protein pet-28a-N

[0088] After ultrasound, a large amount of soluble protein N expressed in the supernatant was obtained. The target protein was purified by affinity chromatography using the Bio-Time HIS protein purification kit. The specific steps are as follows:

[0089] (1) First, add about 4 mL of 50% BeyoGold mixed evenly into the nickel column. TM His-tag Purification Resin (reduction-resistant chelating type), let it stand for 10 minutes to allow the filler and the protective solution to separate, open the stopper at the lower end of the nickel column, release the upper protective solution, then wash the column with five times the column volume of ultrapure water. After washing, equilibrate the nickel column with ten times the column volume of non-denaturing lysis buffer, open the stopper and slowly release it after a few minutes.

[0090] (2) Mix the supernatant of pet-28a-N bacterial lysate with the balanced nickel and place in a 50 ml centrifuge tube. Slowly shake on a shaker at 4°C for 120 min to allow the protein to fully bind to the nickel.

[0091] (3) Then, the protein was passed through the column to mix the bacterial lysate and BeyoGold TMThe mixture of His-tag Purification Resin (reduction-resistant chelating type) was loaded into the empty column tube of the affinity chromatography column. After standing for 5 minutes, the stopper at the lower end of the nickel column was opened, and the protein flow-through that had passed through the column was collected. The collected protein flow-through was passed through the column again. Repeated 4 times, which is conducive to the full binding of the His-tagged recombinant protein to the nickel column and reduced protein loss.

[0092] (4) Wash the column with 15 column volumes of Solution Binding Buffer (formula as shown in Table 3) to remove impurities and collect 100 ul of the washing solution.

[0093] (5) Use five times the volume of Solution Elution Buffer (the formula is shown in Table 3) to elute the target protein, adjust the flow rate to a slower rate to achieve sufficient elution of the protein, and collect each 1 ml of the eluate into different centrifuge tubes. Perform SDS-PAGE identification on the collected samples, collect the protein eluate with better purification effect, determine the protein concentration with BCA, and store it at -80°C.

[0094] Table 3 Buffer formula for soluble protein purification

[0095]

[0096] The results of SDS-PAGE identification are as follows Figure 3 As shown, after the N protein was purified, the bands were obvious and there were fewer miscellaneous bands.

[0097] 1.5 Renaturation and dialysis purification of inclusion body proteins pET-28a-IRBD and pET-32a-IIRBD

[0098] After ultrasound, a large amount of inclusion body proteins pET-28a-IRBD and pET-32a-IIRBD expressed in the precipitate were obtained. The precipitate was collected and washed with urea solutions of different concentrations (1M-8M, prepared with protein washing solution as the mother solution, and the specific components are shown in Table 4). The inclusion body proteins will be dissolved in urea solutions of different gradients, and SDS-PAGE analysis is performed. The target protein washing solution is collected by selecting a suitable urea concentration, and then several gradients of urea-containing target protein washing solutions are added to the dialysis bag for gradient dialysis (the dialysis solution is TGE solution, and the specific components are shown in Table 5. The initial dialysis concentration is 1M lower than the urea concentration of the collected target protein washing solution, and finally dialyzed in a TGE solution without urea). The specific steps are as follows:

[0099] (1) The protein solution after ultrasound was centrifuged at 8000 rpm for 20 minutes, the supernatant and precipitate were collected, and the precipitate was dissolved with 10 ml of 1 M urea solution;

[0100] (2) Centrifuge the solution at 8000 rpm for 10 minutes, collect the supernatant and precipitate, and dissolve the precipitate with 10 ml of 3 M urea;

[0101] (3) Centrifuge the solution at 8000 rpm for 10 minutes, collect the supernatant and precipitate, and dissolve the precipitate with 10 ml of 6 M urea;

[0102] (4) Centrifuge the solution at 8000 rpm for 10 minutes, collect the supernatant and precipitate, and dissolve the precipitate with 10 ml of 8 M urea;

[0103] (5) Centrifuge the solution at 8000 rpm for 10 minutes, discard the precipitate and collect the supernatant;

[0104] (6) The supernatants of the inclusion body proteins dissolved in the gradient were analyzed by SDS-PAGE, and then the expression was verified by Coomassie Brilliant Blue staining.

[0105] (7) Take out the dialysis bag soaked in 70% EtOH and sterilize it by boiling it in pure water for 10 min;

[0106] (8) If the bands are correct, use a 50 ml centrifuge tube to mix the (clearly expressed) gradient urea-dissolved inclusion body solution, add it to the dialysis bag, and seal both ends;

[0107] (9) The dialysis bag is placed in a low concentration of TGE solution containing urea for gradient dialysis, and finally dialyzed in a TGE solution without urea. The formula of the TGE solution is shown in Table 5. Generally, one gradient is performed for 12 hours.

[0108] (10) After dialysis, the cells were concentrated with sucrose and the protein concentration was determined according to the instructions of the BCA kit and stored in aliquots at -80°C.

[0109] Table 4 Protein washing solution formula

[0110] Components Usage Tris-HCl pH = 8.0 6.5g Sodium chloride 5.8g EDTA 3.7g Triton-x-100 5mL Single distilled water Make up to 1L

[0111] Table 5 TGE solution formula (pH = 7.9)

[0112] Components Usage Tris 6g Sodium chloride 2.9g EDTA 0.186g glycerin 5％ Single distilled water Make up to 1L

[0113] The results of SDS-PAGE analysis were as follows Figure 4 As shown, the inclusion bodies of the recombinant IRBD protein were dissolved at 6 and 8 M, and the inclusion bodies of the recombinant IIRBD protein were dissolved at 6 and 8 M.

[0114] 1.6 Identification of the reactogenicity of recombinant proteins

[0115] To verify whether the target protein reacts with FIPV-positive cat serum, an ELISA test was performed. The specific steps are as follows: the purified pET-28a-N, pET-28a-IRBD, and pET-32a-IIRBD proteins (10 μg / mL) were coated with carbonate buffer, and the coated target proteins were added to a 96-well plate at 100 μL / well and coated overnight at 4°C.

[0116] The next morning, discard the target protein coating solution in the 96-well plate, wash 3 times with prepared PBST, and block with 5% skim milk powder (50g / L, prepared with PBST) for 2h; shake off the 5% skim milk powder, wash 4 times with PBST, and finally pat dry to ensure that there is no PBST residue in the well; then dilute the FIPV-positive cat serum with PBS at 1:500, 100μL of the diluted sample per well, and the control group is FIPV-negative cat serum, incubated in a 37°C incubator for 1h; shake off the diluted serum sample, wash 5 times with PBST, shake on a micro-oscillator for 5min, and finally pat dry to ensure that there is no PBST residue in the well; then dilute the rabbit anti-cat IgG-HRP at 1:2000 (the diluent is PBST), add 100μL to each well, and incubate in a 37°C incubator for 1h; shake off the liquid in the well, wash 5 times with PBST, and finally pat dry to ensure that there is no PBST residue in the well; at room temperature, add 100μL to each well TMB colorimetric solution was added and reacted for 10 min in the dark. Finally, 50 μL of stop solution was added to each well to stop the reaction. The absorbance of each well sample at OD450 nm was detected using an ELISA reader.

[0117] The results are as follows Figure 5 As shown, the reactivity of the purified N, IRBD, and IIRBD recombinant proteins was analyzed by ELISA, which proved that they had good reactivity and could be used as antigens in the kit.

[0118] Example 2 Establishment and optimization of indirect ELISA antibody detection method

[0119] 2.1 Establishment of indirect ELISA antibody detection method

[0120] (1) Dilute the purified recombinant protein in ELISA 1x coating buffer to a final concentration of 5 μg / mL. Add 100 μL per well to a 96-well ELISA plate in triplicate and coat overnight at 4°C.

[0121] (2) Discard the coating solution, wash three times with PBST (PBS containing 0.05% Tween 20), and pat dry on absorbent paper.

[0122] (3) Block with PBST containing 5% skimmed milk powder, 100 μL / well, at 37°C for 2 h.

[0123] (4) Discard the blocking solution, wash four times with PBST, pat dry, and dilute the feline FCOV positive and negative sera at a ratio of 1:500 with PBST. Add the diluted sera to the ELISA plate at 100 μL / well and incubate at 37°C for 2 h.

[0124] (5) Discard cat serum, wash with PBST five times, pat dry, add 1:2000 diluted HRP-labeled rabbit anti-cat IgG enzyme-labeled antibody, 100 μL / well, and incubate at 37°C for 1 h.

[0125] (6) Discard the enzyme-labeled antibody, wash with PBST five times, pat dry, add TMB colorimetric solution, 100 μL / well, incubate at 37°C in the dark for a period of time, and then add 50 μL ELISA stop solution to each well for reaction.

[0126] (7) Read the OD450 nm value on an ELISA reader and record the result.

[0127] (8) Result determination: The antigen coating concentration with the largest ratio (P / N) of positive serum to negative serum and the lowest negative value is the optimal antigen coating concentration.

[0128] 2.2 Optimization of antigen coating concentration

[0129] The purified recombinant protein was diluted in ELISA 1x coating solution to a final concentration of 1, 2, 3, 4, 5 ug / mL. The detection was performed according to the basic operation procedure of indirect ELISA described in 2.1. OD450 nm was read. By comparing the P / N value, the antigen coating concentration with the largest P / N ratio between positive serum and negative serum and the lowest negative value was selected.

[0130] The results are as follows Figure 6 As shown, in view of the detection of the optimal antigen coating concentration of each protein and in order to ensure uniform conditions and convenience of operation during detection, 3ug / mL was selected as the coating concentration of the three proteins.

[0131] 2.3 Optimization of antigen coating conditions.

[0132] Four coating conditions were selected: overnight coating at 4°C, coating at 37°C for 2h, coating at 37°C for 1h, and coating at 37°C for 0.5h, and the detection was performed according to the basic operating procedures of indirect ELISA.

[0133] The results are as follows Figure 7 As shown, in view of the detection of the best antigen coating conditions for each protein and in order to ensure uniform conditions and convenience of operation during detection, 37°C for 2h was selected as the coating condition for the three proteins.

[0134] 2.4 Screening of blocking solution

[0135] 2% skim milk, 5% skim milk, 2% BSA and 5% BSA were selected as ELISA blocking solutions, and the detection was carried out according to the basic operating procedures of indirect ELISA.

[0136] The results are as follows Figure 8 As shown, in view of the screening results of the best blocking solution for each protein, and in order to ensure uniform conditions and convenience of operation during detection, 2% skim milk was selected as the blocking solution for the three proteins.

[0137] 2.5 Optimization of closure conditions

[0138] The blocking time was set at 37°C for 0.5 h, 37°C for 1 h, 37°C for 2 h and 37°C for 3 h. Four different blocking time conditions were used for detection according to the basic operating procedures of indirect ELISA.

[0139] The results are as follows Fig. 9 As shown, in view of the detection of the optimal blocking conditions for each protein and in order to ensure uniform conditions and convenience of operation during detection, 37°C for 3h was selected as the blocking condition for the three proteins.

[0140] 2.6 Optimization of serum dilution

[0141] Cat positive serum and cat negative serum were diluted with PBS solution at dilution multiples of 1:250, 1:500, 1:1000 and 1:2000, and tested according to the basic operating procedures of indirect ELISA.

[0142] The results are as follows Fig.10 As shown, in view of the detection of the optimal serum dilution of each protein and in order to ensure uniform conditions and convenience of operation during detection, 1:1000 was selected as the serum dilution of the three proteins.

[0143] 2.7 Optimization of serum incubation conditions

[0144] The incubation time of cat positive and negative serum was set to 30 min, 1 h, 2 h and 4 h, four time conditions, and the detection was carried out according to the basic operation process of indirect ELISA.

[0145] The results are as follows Fig.11 As shown, in view of the detection of the optimal serum incubation conditions for each protein and in order to ensure uniform conditions and convenience of operation during detection, 37°C for 1 h was selected as the serum incubation condition for the three proteins.

[0146] 2.8 Optimization of enzyme-labeled antibody dilution

[0147] Rabbit anti-cat IgG enzyme-labeled antibody was diluted with PBST solution at four different dilutions of 2000 times, 4000 times, 6000 times and 8000 times, and the detection was carried out according to the basic operation process of indirect ELISA.

[0148] The results are as Fig.12 shown. Considering the detection of the optimal dilution of the enzyme-labeled antibody for each protein, and to ensure the uniformity of conditions and the convenience of operation during detection, a 4000-fold dilution was selected as the dilution factor of the enzyme-labeled antibody for the three proteins.

[0149] 2.9 Optimization of the incubation conditions of the enzyme-labeled antibody

[0150] The incubation time of the rabbit anti-cat IgG enzyme-labeled antibody was set at 15 min, 30 min, 1 h, and 1.5 h, four time conditions, and the detection was carried out according to the basic operation procedure of indirect ELISA.

[0151] The results are as Fig.13 shown. Considering the detection of the optimal incubation conditions of the enzyme-labeled antibody for each protein, and to ensure the uniformity of conditions and the convenience of operation during detection, 1 h at 37 °C was selected as the incubation conditions of the enzyme-labeled antibody for the three proteins.

[0152] 2.10 Optimization of the color development time

[0153] The action time of the TMB color development solution was set at 2 min, 4 min, 6 min, and 8 min, and the detection was carried out according to the basic operation procedure of indirect ELISA.

[0154] The results are as Fig.14 shown. Considering the detection of the optimal color development time for each protein, and to ensure the uniformity of conditions and the convenience of operation during detection, 4 min was selected as the action time of the TMB color development solution for the three proteins.

[0155] 2.11 Determination of the critical value

[0156] Using the above-established and optimized ELISA method, 24 cat negative sera stored in this laboratory were detected, the OD450 nm was measured, the mean value (X) and the standard deviation (SD) were calculated. According to the statistical principle, the value of X + 3SD was used as the critical value (cutoff value). When the OD450 nm of the sample ≥ X + 3SD, it was determined as positive; when OD450 nm < X + 3SD, it was determined as negative.

[0157] The results are as Fig.15 shown. The critical value of the recombinant N protein was 0.264, that is, when the OD450 nm of the tested serum ≥ 0.264, it was positive; the critical value of the recombinant IRBD protein was 0.254, that is, when the OD450 nm of the tested serum ≥ 0.254, it was positive; the critical value of the recombinant IIRBD protein was 0.222, that is, when the OD450 nm of the tested serum ≥ 0.222, it was positive.

[0158] Example 3 Performance evaluation of the indirect ELISA antibody detection method

[0159] 3.1 Specificity experiments

[0160] The positive sera of feline calicivirus, feline herpesvirus and feline panleukopenia virus stored in our laboratory were selected, and the positive sera of FCOV were used as positive control to analyze the specificity of the established ELISA method. Fig.16 As shown, the established ELISA method has no specific reaction to these three common feline viral infectious diseases in clinical practice, and only shows specificity to FCOV positive serum.

[0161] 3.2 Repeatability Experiment

[0162] Four confirmed FCOV positive and negative cat sera were randomly selected, and the same batch of ELISA plates were used for intra-batch repeatability experiments. Six replicate wells were set up for each sample, and the OD450 nm was measured. The coefficient of variation of the same sample was calculated to test the intra-batch repeatability of the method. Four confirmed FCOV positive and negative cat sera were randomly selected, and different batches of ELISA plates were used for inter-batch repeatability experiments. Six replicate wells were set up for each sample, and the OD450 nm was measured. The coefficient of variation of the same sample was calculated to test the inter-batch repeatability of the method.

[0163] The coefficient of variation is obtained by calculating the mean and standard deviation of the test values ​​of the same serum using the formula CV (coefficient of variation) = (standard deviation SD / mean X) × 100%.

[0164] The results are as follows Fig.17 and Fig.18 As shown, the intra-batch coefficient of variation of the N recombinant protein ELISA coated plate was 3.436%-7.319%, and the inter-batch coefficient of variation was 3.220%-8.264%, both of which were less than 10%; the intra-batch coefficient of variation of the IRBD recombinant protein ELISA coated plate was 4.986%-8.413%, and the inter-batch coefficient of variation was 5.260%-8.501%, both of which were less than 10%; the intra-batch coefficient of variation of the IIRBD recombinant protein ELISA coated plate was 1.802%-6.121%, and the inter-batch coefficient of variation was 3.346%-8.948%, both of which were less than 10%, indicating that the ELISA method has good repeatability.

[0165] 3.3 Sensitivity experiment

[0166] The FCOV positive serum was diluted in multiples (1000, 4000, 16,000, 32,000, 64,000, 128,000, 256,000, and 512,000 times with PBS) and tested using the above optimized conditions. Cat negative serum was used as the corresponding negative control, and the critical value was used as the standard to analyze the sensitivity of the method.

[0167] The results are as follows Fig.19 As shown, when the dilution of positive serum for N, IRBD and IIRBD proteins was 1:64,000, 1:64,000 and 1:32,000, respectively, the detection result values ​​were still greater than the critical value, and P / N ≥ 2.

[0168] Example 4 Clinical application of indirect ELISA antibody detection method based on N, IRBD, and IIRBD proteins

[0169] The N, IRBD and IIRBD proteins purified in this study were used as coating antigens, and the established indirect ELISA method was used to detect antibodies in 123 cat sera collected in Guangzhou. The detection of each antibody was statistically analyzed, serotype analysis was performed, and the correlation between antibody levels and the occurrence of FCOV symptoms was observed.

[0170] Statistics on the positive detection rate of 4.1N, IRBD and IIRBD protein antibodies

[0171] The results are as follows Fig. 20 As shown, 83 samples among the 123 samples were detected to be positive for N antibody, and the positive rate of FCOV-N antibody was 67.5%; 84 samples were positive for IRBD antibody, and the positive rate of FCOV-IRBD antibody was 68.3%; there were 3 samples that were positive for IIRBD antibody, and the positive rate of FCOV-IIRBD antibody was 2.4%.

[0172] 4.2 Statistical analysis of combined antibody detection

[0173] The results of the three antibodies (N, IRBD and IIRBD) of a single individual were statistically analyzed. Fig.21 The results showed that among the 123 samples, 33 samples did not detect any of the N, IRBD, and IIRBD antibodies, indicating that 26.83% of the samples were negative for FCOV antibodies; 90 samples detected one or more of the N, IRBD, and IIRBD antibodies, indicating that 73.17% of the samples were positive for FCOV antibodies. Among these positive samples, type 1 infection accounted for 91.11%, type 2 infection accounted for 1.11%, and the mixed infection rate accounted for 2.22%.

[0174] 4.3 Correlation between antibodies and the occurrence of FCOV symptoms

[0175] According to the presence or absence of FCOV symptoms, 105 clinically healthy cats and 18 cats clinically diagnosed with FIP were divided into the following groups. The antibody test results were statistically analyzed. Fig. 22As shown in the figure, the detection rate of N antibody in cats without FCOV symptoms was 61.9%, the detection rate of IRBD antibody was 62.86%, and the detection rate of IIRBD antibody was 2.86%. The positive rate of N antibody and IRBD antibody in cats clinically diagnosed with FIP was 100%.

[0176] 4.4 Correlation between antibody levels and the occurrence of FCOV symptoms

[0177] According to the OD450 nm of the sample detection, the antibody level was divided into high level (OD450 nm>0.8), medium level (0.8>OD450 nm>0.5), low level (0.5>OD450 nm>critical value) and negative (OD450nm<critical value), and the antibody level detection results were statistically analyzed.

[0178] The results show Fig.23 As shown, the antibody levels of 65 cats with detectable N antibodies among 105 clinically healthy cats were statistically analyzed, of which 50.77% detected high levels of N antibodies, 16.92% detected medium levels of N antibodies, and 32.31% detected low levels of N antibodies. The antibody levels of 66 cats with detectable IRBD antibodies among 105 clinically healthy cats were statistically analyzed, of which 36.36% detected high levels of IRBD antibodies, 27.27% detected medium levels of IRBD antibodies, and 36.36% detected low levels of IRBD antibodies. The antibody levels of 3 cats with detectable IIRBD antibodies among 105 clinically healthy cats were statistically analyzed, and only low levels of IIRBD antibodies were detected in all of them.

[0179] All 18 cats clinically diagnosed with FIP had high levels of N antibodies. In addition, 61.11% of cats had high levels of IRBD antibodies, 16.67% had medium levels of IRBD antibodies, and 22.22% had low levels of IRBD antibodies. None of the 18 FIP cats had IIRBD antibodies.

[0180] Judging from the test results, the method established in this study has good sensitivity, specificity and repeatability, and has achieved good results in preliminary applications, demonstrating that the kit can detect cats infected with FCOV in large quantities and has a good effect in identifying serotypes.

[0181] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antigen for detecting type I and type II feline coronavirus antibodies, characterized in that The antigen is one or more groups of purified recombinant type I N protein, type I IRBD protein and type II IIRBD protein of feline coronavirus; the amino acid sequences of the recombinant type I N protein, type I IRBD protein and type II IIRBD protein are shown in SEQ ID NO.1-3.

2. The nucleotide sequences encoding the recombinant type I N protein, type I IRBD protein and type II IIRBD protein according to claim 1 are shown in SEQ ID NOs. 4-6.

3. An indirect ELISA test kit for detecting type I and type II feline coronavirus antibodies, characterized in that, The ELISA plate of the kit is coated with the antigen according to claim 1.

4. The kit according to claim 3, characterized in that: The kit also includes a positive control sample, a negative control sample, a sample diluent, a washing solution, an HRG-labeled rabbit anti-cat IgG antibody, a color developing solution and a stop solution.

5. The kit according to claim 4, characterized in that: The positive control sample is FCOV positive cat serum; the negative control sample is FCOV negative cat serum; the sample diluent is PBS; the washing solution is PBST containing 0.05% Tween-20; the color developing solution is TMB solution; and the stop solution is 2M sulfuric acid solution.

6. An indirect ELISA method for detecting type I and type II feline coronavirus antibodies, characterized in that: The following steps are involved: S1. Incubation of serum to be tested: dilute the sample serum and add it to the ELISA plate coated with recombinant N protein, IRBD protein (type I) and IIRBD protein (type II). Incubate at 37°C for 1 hour, then wash 5 times with PBST and pat dry. S2, enzyme-labeled antibody incubation: add 4000-fold diluted HRP-labeled rabbit anti-cat IgG antibody to the ELISA plate obtained in step S1, incubate at 37° C. for 1 h, discard the solution, wash with PBST 5 times and pat dry; S3, color development: add TMB single-component color development solution to each well and color development for 4 minutes in the dark; S4. Stop: Add stop solution to each well to stop the reaction and measure the OD450 nm value of the sample to be tested.

7. The method according to claim 6, characterized in that: The method also includes determining whether the sample to be tested contains feline coronavirus antibodies.

8. The method according to claim 7, characterized in that: The standard for determining whether the sample to be tested contains feline coronavirus antibodies is: the critical value of the recombinant N protein is 0.264, that is, the OD450 nm of the serum to be tested ≥0.264 was positive; recombinant IRBD protein The critical value of white is 0.254, that is, the OD450 nm of the tested serum is ≥ 0.254 The critical value for positive recombinant IIRBD protein is 0.222, that is, the tested serum OD450 nm ≥ 0.222 is positive.

9. Use of the antigen described in claim 1 or the test kit described in claim 3 in detecting Type I and Type II feline coronavirus antibodies.

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