An IPMA detection method and kit for African swine fever virus antibodies

Abstract

The present invention discloses a non-diagnostic African swine fever virus antibody detection method, the steps are: infecting wild boar kidney cells with a microbial deposit number of CCTCC NO: C2022258 with an African swine fever virus strain with a microbial deposit number of CCTCC NO: V202404 in a 96-well plate, fixing the infected cells in the wells of the 96-well plate, adding serum to be tested to the 96-well plate, adding HRP-labeled recombinant staphylococcal protein A to the wells, adding AEC color developing solution to the wells, washing the 96-well plate, observing the 96-well plate with an ordinary optical microscope, and judging whether the serum contains African swine fever virus antibodies based on whether reddish-brown stained cells can be observed. The present invention also discloses a virus strain and a kit for the above-mentioned detection method. The method has high sensitivity, good safety, good specificity, convenient operation, and can be promoted and applied.

Description

Technical Field

[0001] The invention belongs to the field of veterinary diagnostics and relates to an IPMA detection method and a kit for African swine fever virus antibodies. Background Art

[0002] African swine fever (ASF) has a history of 100 years since it was discovered in Kenya in 1921. According to WOAH statistics, ASF has broken out and spread in nearly 50 countries in Africa, Europe, Asia and the Caribbean, causing huge economic losses and seriously endangering the stable development of the global pig industry and related industries. In 2018, ASF was first introduced to China, and the ASFV HLJ / 18 strain was isolated for the first time. The whole genome sequence determination and evolutionary analysis proved that the ASFV HLJ / 18 strain was a genotype II virulent strain, and the mortality rate of inoculated pigs was 100%. With the prevalence of ASFV in my country, a low-virulence genotype II strain (HLJ / HRB1 / 20 strain) with no blood adsorption activity appeared in the fields in my country in 2020, a low-virulence genotype I strain (SD / DY-I / 21 strain) appeared in 2021, and a naturally recombinant strong strain of genotype I and genotype II (JS / LG / 21 strain) was reported in 2023, posing more severe challenges to my country's ASF prevention and control.

[0003] ASFV low-virulence strains have the characteristics of long incubation period, strong concealment, no obvious clinical symptoms and irregular excretion of toxins, which can easily lead to missed detection in nucleic acid detection, resulting in the spread of the disease. Therefore, WOAH recommends antibody monitoring in ASFV low-virulence epidemic areas or low-prevalence areas, aiming to detect ASFV-infected pigs at an early stage through regular antibody screening, providing important guidance for the prevention and control of ASF.

[0004] The serological detection methods recommended by WOAH and my country's current national standard for African swine fever diagnostic technology (GB / T 18648-2020) include enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA) and immunoperoxidase monolayer assay (IPMA). IFA and IPMA use live viruses as antigens to infect cells, do not require a large amount of (purified) antigens, have a more complete antigen structure, and the results of detecting antibodies are more accurate and representative. They are recommended by WOAH as the gold standard for confirming serological detection methods. Other antibody detection methods, especially ELISA methods, need to be compared with them to determine their specificity and sensitivity. In 2023, the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences established the IFA detection method for African swine fever antibodies. IFA requires expensive fluorescence microscopes, and its local application is limited. For this reason, the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences established the IPMA method.

[0005] IPMA is fast, sensitive, and specific. The results can be observed and determined under an ordinary optical microscope, which reduces the technical requirements for the operator. The experimental results can be stored for a long time and are suitable for grassroots promotion and use. IPMA requires the use of live viruses to infect susceptible cells. For the virulent African swine fever virus, there is a risk of spreading the virus, which limits the application of this method. Therefore, it is particularly necessary to screen for weak strains of African swine fever with significantly reduced virulence and stable growth in cell culture for use in IPMA antibody detection methods. Summary of the invention

[0006] The present invention uses the method of in vitro continuous passage to obtain the BK2258 cell-adapted strain ASFV HLJ18 / BK33 strain and establishes the IPMA detection method of ASFV antibodies. This method overcomes the difficulties of being limited by fluorescence microscopy and provides a gold standard for the diagnosis of ASFV antibodies and the evaluation of other antibody detection methods.

[0007] More specifically, the present invention uses the ASFV HLJ / 18 strain to continuously passage F33 generations on wild boar kidney cells (Boar kidney cells, BK2258) to obtain a cell-adapted strain, named HLJ18 / BK33 strain. The adapted strain can stably proliferate and form typical CPE on BK2258. An IPMA detection method for ASFV antibodies with high sensitivity, strong specificity, good repeatability, simple operation and lower cost has been established. It provides a gold standard that is easy to operate and feasible for other detection methods of ASFV antibodies (such as ELISA, etc.). If the kit assembled by this method is used for serological diagnosis and epidemiological investigation of ASF, it will provide a new and effective means for ASF monitoring and prevention and control in my country.

[0008] The first aspect of the present invention provides a kit, which contains African swine fever virus susceptible cells and an African swine fever virus strain or a passaged virus strain thereof with a microbial preservation number of CCTCC NO: V202404.

[0009] In some embodiments, the African swine fever virus susceptible cells are wild boar kidney cells with a microbial preservation number of CCTCC NO: C2022258.

[0010] In some embodiments, the kit further comprises any one or more combinations of the following materials:

[0011] Serum positive for African swine fever virus antibody;

[0012] African swine fever virus antibody-negative serum;

[0013] 96-well cell culture plates;

[0014] Paraformaldehyde;

[0015] Trypsin;

[0016] Fetal bovine serum;

[0017] Triton-X100 solution;

[0018] HRP-labeled recombinant Staphylococcus protein A;

[0019] PBS solution;

[0020] DMEM culture medium;

[0021] AEC color developing solution.

[0022] The second aspect of the present invention provides a method for detecting antibodies to African swine fever virus for non-diagnostic purposes, the detection method comprising the following steps:

[0023] P1: Infecting African swine fever virus susceptible cells with the African swine fever virus strain with the microbial deposit number of CCTCC NO: V202404 or its passaged virus strain to obtain infected cells;

[0024] P2: fixing the infected cells on the surface of a solid phase carrier to obtain immobilized cells;

[0025] P3: adding the sample to be tested to the immobilized cells for incubation to obtain the immobilized cells of the first incubation;

[0026] P4: adding the porcine IgG specific binding substance connected with the label to the immobilized cells of the first incubation for incubation to obtain the immobilized cells of the second incubation;

[0027] P5: Characterize the marker that is indirectly bound to the fixed cells of the second incubation, and determine whether African swine fever virus antibodies exist in the sample to be tested based on the presence or absence of the marker.

[0028] In some embodiments, in step P1, the African swine fever virus susceptible cells are wild boar kidney cells with a microbial preservation number of CCTCC NO: C2022258.

[0029] In some embodiments, in step P2, the surface of the solid support is the inner surface of a well of a microplate.

[0030] In some embodiments, in step P3, the sample to be tested is pig serum.

[0031] In some embodiments, in step P4, the labeled porcine IgG specific binder is HRP-labeled recombinant Staphylococcus protein A. In step P5, the HRP-labeled recombinant Staphylococcus protein A is characterized by AEC colorimetric solution, and whether the fixed cells are stained red-brown is determined to determine whether African swine fever virus antibodies are present in the sample to be tested.

[0032] In some embodiments, in step P1, the infection concentration of the African swine fever virus strain is 10 3.00 TCID 50 / ml to 10 4.50 TCID 50 / ml, the African swine fever virus susceptible cells are in a monolayer cell state.

[0033] In some embodiments, in step P2, the infected cells are fixed to the solid surface using a PBS solution containing 3-5 w / w% paraformaldehyde.

[0034] In some embodiments, between step P2 and step P3, the method further comprises the following steps:

[0035] P2-1: treating the immobilized cells with a membrane permeabilization solution;

[0036] P2-2: The fixed cells treated with the permeabilization solution are blocked with a blocking solution.

[0037] In some embodiments, in step P3, African swine fever virus antibody-positive serum and / or African swine fever virus antibody-negative serum are set as control samples in parallel with the sample to be tested.

[0038] In some embodiments, after step P5, double distilled water is added to terminate the reaction between the AEC color development solution and the HRP-labeled recombinant Staphylococcus protein A.

[0039] In some embodiments, in step P1, the infection is carried out using DMEM culture medium at 36-38° C. and 4-7% carbon dioxide, and the infection time is 30-40 hours.

[0040] In some embodiments, in step P2-1, the surface of the solid phase carrier is treated with a PBS solution containing 0.2-0.3 v / v% Triton-X100 and allowed to stand at room temperature for 10-20 min.

[0041] In some embodiments, in step P2-2, the surface of the solid support is treated with a PBS solution containing 8-12 v / v% FBS at 35-38° C. for 1.5-3 h.

[0042] In some embodiments, in step P3, the pig serum is diluted 20-50 times before use.

[0043] In some embodiments, in step P3, the incubation is carried out at 36-38° C. for 20-40 min.

[0044] In some embodiments, in step P4, the concentration of the HRP-labeled recombinant Staphylococcus protein A is 3-7 mg / mL.

[0045] In some embodiments, in step P4, the incubation is carried out at 36-38° C. for 40-60 min.

[0046] In some embodiments, the reaction time of the AEC color developing solution and the HRP-labeled recombinant Staphylococcus protein A is 15-25 min.

[0047] In some embodiments, before operation step P2, the surface of the solid phase carrier is washed with PBS solution.

[0048] In some embodiments, after completing step P2, the surface of the solid phase carrier is washed with a PBS solution.

[0049] In some embodiments, after completing step P2-1, the surface of the solid phase carrier is washed with a PBS solution.

[0050] In some embodiments, after completing step P2-2, the surface of the solid phase carrier is washed with a PBS solution.

[0051] In some embodiments, after completing step P3, the surface of the solid phase carrier is washed with a PBS solution.

[0052] In some embodiments, after completing step P4, the surface of the solid phase carrier is washed with a PBS solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Photographs showing ASFV HLJ18 / BK33 strain producing CPE on BK2258 cells.

[0054] Figure 2 Shown is the survival of HLJ18 / BK33 pigs after vaccination.

[0055] Figure 3 Shown is the change in body temperature of pigs after inoculation with the HLJ18 / BK33 strain.

[0056] Figure 4 The results of qPCR detection of anticoagulated blood after pigs were inoculated with HLJ18 / BK33 strain are shown.

[0057] Figure 5The results of antibody detection after pigs were inoculated with HLJ18 / BK33 strain are shown.

[0058] Figure 6 Photos showing the IPMA reaction results of ASF positive serum and negative serum under different ASFV inoculation conditions.

[0059] Figure 7 The photos show the IPMA reaction results of ASF positive serum and negative serum cultured at different times after ASFV inoculation.

[0060] Figure 8 The photos show the IPMA reaction results of the ASFV inoculation wells and ASF positive and negative sera at different dilutions.

[0061] Fig. 9 Photos of the IPMA reaction results of ASFV inoculated wells and secondary antibodies of different dilutions are shown.

[0062] Fig.10 A photograph of the IPMA reaction results showing the incubation time of the primary and secondary antibodies in the ASFV inoculation wells.

[0063] Fig.11 A photograph of the IPMA reaction results of the color development time and temperature of the ASFV inoculated well is shown.

[0064] Fig.12 A photograph showing the results of IPMA titer determination of ASFV antibody strongly positive serum.

[0065] Fig.13 Photographs of ASFV IPMA method-specific experimental results are shown. DETAILED DESCRIPTION

[0066] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0067] Materials and instruments not described in the present invention are conventional materials and instruments in the art, operation details not described in the present invention are conventional operations in the art, the software used in the present invention is operated by conventional methods with reference to the instructions of the software provider, and the kit used in the present invention is operated by conventional methods with reference to the kit instruction manual.

[0068] One of the application scenarios of African swine fever virus antibody testing for non-diagnostic purposes is to study the cross-immunity between African swine fever virus and other viruses.

[0069] Strains and cells

[0070] The naturally isolated strong strain of African swine fever (ASFV HLJ / 18 strain, abbreviated as HLJ / 18 strain) is a genotype II African swine fever virus strain, which was isolated, identified and preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The corresponding GenBank sequence number of this strain is MK333180.1, and the full name is Pig / HLJ / 2018.

[0071] Boar kidney cells (BK2258) were prepared and preserved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The preparation method of BK2258 cells is recorded in the application documents of the Chinese patent application with application number CN202211137583.3. The isolated and cultured BK2258 strain F20 cells were submitted to a patent procedure recognized depository for preservation. The depository is the China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial collection number is CCTCCNO: C2022258; the culture name is wild boar kidney cells BK2258; the Chinese classification name is: wild boar kidney cells; the English classification name is: Boar kidney cell; the preservation time is August 10, 2022; the identified survival time is August 17, 2022.

[0072] Example 1: ASFV HLJ / 18 strain passaged in BK2258 cells

[0073] ASFV HLJ / 18 strain was taken and diluted to 10 in DMEM medium (containing 3% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin). 6.0 TCID 50 / ml, inoculated with BK2258 cells grown into a well-grown monolayer, cultured in DMEM supplemented with 10 (v / v)% FBS, and placed at 37°C, 5% CO 2 The cells were cultured in an incubator and observed every day for cytopathic effect (CPE). If CPE did not occur, blind passage was performed to obtain a cell-adapted strain that could cause obvious CPE. The characteristics of CPE were cell aggregation, contraction, lysis, and finally the formation of circular plaques. The strain was further passaged to F33 and named ASFV HLJ18 / BK33. Figure 1 The following are photos of CPE of ASFV HLJ18 / BK33 strain on BK2258 cells. A is a photo of the control culture of BK2258 cells, and B is a photo of ASFV HLJ18 / BK33 strain inoculated with BK2258 cells 72 hours later. The scale bar is 100 μm. It can be seen that ASFV HLJ18 / BK33 strain can proliferate well in BK2258 cells.

[0074] The African swine fever virus HLJ18 / BK33 strain obtained by passage was submitted to a depository recognized by the patent procedure for preservation. The depository is the China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial deposit number is CCTCCNO: V202404; the culture name is African swine fever virus ASFV HLJ18 / BK33 African swine fever virusASFV HLJ18 / BK33; the Chinese classification name is: African swine fever virus; the English classification name is: African Swine Fever Virus; the preservation time is January 5, 2024; the identification survival time is January 11, 2024.

[0075] Example 2: ASFV HLJ18 / BK33 strain titer determination

[0076] The ASFV HLJ18 / BK33 strain was maintained in DMEM and serially diluted 10-fold to 10 -8 Dilution, take 10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 Five dilutions were prepared, and BK2258 cells grown in a good monolayer were inoculated in 96-well plates. Eight wells were replicated for each dilution. Uninoculated normal cells were used as controls, 100 μl / well, and the cells were placed at 37°C with 5% CO. 2 The cells were cultured in an incubator and the cytopathic effect was observed every day for 7 days. The number of cytopathic wells was recorded and the TCID was calculated according to the Reed-Muench method. 50 The results showed that the titer of ASFV HLJ18 / BK33 strain was 10 7.29 TCID 50 / ml.

[0077] Example 3: ASFV HLJ18 / BK33 strain genome full sequence determination and analysis

[0078] The genome of the cell-passaged HLJ18 / BK33 strain was segmented and spliced ​​to obtain the whole genome sequence. The SnapGene software was used to compare the whole genome sequence of the ASFV HLJ / 18 strain (GenBank: MK333180.1) and the cell-passaged HLJ18 / BK33 strain. The results showed that the whole genome length of the HLJ18 / BK33 strain was 183,487 bp, and that of the HLJ / 18 strain was 189,405 bp. Compared with the whole genome sequence of HLJ / 18, there was a large deletion at positions 181,027-187,188 in the genome of HLJ18 / BK33, specifically 11 ORFs (I8L, ASFV_G_ACD_01870, I9R, I10L, L11L, MGF_360-18R, DP71L, DP96R, ASFV_G_ACD_01940, MGF_360-19R, and AS FV_G_ACD_01960), the 5' end 9nt of I7L upstream of I8L and the 5' end 160nt of MGF_360-21R downstream of ASFV_G_ACD_01960, there are 3 truncated ORFs (ASFV_G_ACD_00120, ASFV_G_ACD_00350 and A179L) and 1 fusion expressed ORF (MGF_110-14L fused with MGF_110-11L).

[0079] Further analysis showed that compared with the whole genome of the HLJ / 18 strain, five genes of the HLJ18 / BK33 strain had nucleotide deletions, four of which had frameshift mutations, resulting in changes in multiple amino acids, early or late stop codons, and shortened or extended ORFs. Specifically, one nucleotide A was deleted at the 7830 position of ASFV_G_ACD_00120ORF, and the ORF expression was shortened by 48AAs; four nucleotide Cs were deleted at the 13,276-13,279 positions of MGF_110-14LORF, MGF_110-14L and MGF_110-11L were fused and expressed, and the expression of MGF_110-14L was extended by 36AAs; two nucleotide Gs were deleted at the 19,045-19,046 positions of ASFV_G_ACD_00350ORF, and the ORF expression was shortened by 19AAs; one nucleotide T was deleted at the 53817 position of A179LORF, and the ORF expression was shortened by 2AAs; one nucleotide A was deleted at the 24,034 position of the non-coding region. Single nucleotide mutations occurred in the four genes, all of which were missense mutations, resulting in changes in a single amino acid. Specifically, the nucleotide GA (amino acid GD) at position 47,573 of A104R ORF, the nucleotide TA (amino acid NK) at position 138,113 of D250R ORF, the nucleotide GA (amino acid TI) at position 166,237 of E199L ORF, and the nucleotide GA (amino acid DN) at position 168,093 of E120R ORF.

[0080] Example 4: Safety study of HLJ18 / BK33 strain inoculated pigs

[0081] Dilute the BK258 culture of the HLJ18 / BK33 strain to 10 6 TCID 50 / ml, 5 pigs aged 6-8 weeks (SPF pigs provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were inoculated intramuscularly, 1ml / head. After inoculation, the rectal temperature of each inoculated pig was measured daily; the clinical symptoms such as feeding, drinking, mental state, and body surface were observed; anticoagulant blood, oral swabs and anal swabs were collected every 5 days, and the ASFV virus B646L gene content was detected by qPCR using the method recommended by WOAH. The results showed that within 28 days after inoculation, all pigs survived healthily ( Figure 2 ) and normal body temperature ( Figure 3 ). The feeding, drinking, mental state, etc. were normal, and no adverse clinical symptoms were observed. The oral and anal swab qPCR tests were negative, and only one pig had a qPCR Ct value of 36.0 and 37.0 at 5 and 20 days after inoculation, respectively. The other time points of this pig and all time points of the other 4 pigs were negative ( Figure 4). This indicates that the HLJ18 / BK33 strain is completely attenuated and is very safe for inoculated pigs. The level of virus in inoculated pigs is extremely low, and there is no risk of horizontal transmission.

[0082] Example 5: Study on immunogenicity of HLJ18 / BK33 strain

[0083] Dilute the BK258 culture of the HLJ18 / BK33 strain to 10 6 TCID 50 / ml, 5 pigs aged 6-8 weeks (SPF pigs provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were injected intramuscularly, 1ml / pig. After inoculation, procoagulant blood was collected every 5 days to prepare serum, and the p22 protein blocking ELISA antibody detection method was used to determine the p22 antibody blocking rate. The detection procedure is as follows:

[0084] 1. Coating: Dilute the prokaryotic expressed African swine fever virus p22 protein antigen into 4 μg / ml antigen dilution with antigen coating solution (carbonate buffer), add to 96-well plate, 50 μl / well, and coat overnight at 4°C.

[0085] 2. Washing: Wash 5 times with 300 μl / well of PBST (phosphate buffered saline containing 0.05 v / v% Tween 20), each time for 1 minute.

[0086] 3. Blocking: Block with 5 w / v% skim milk, 200 μl / well, incubate at 37°C for 2 hours.

[0087] 4. Washing: Same as 2.

[0088] 5. Sample addition: dilute each serum with PBS at a volume ratio of 1:1, add to a 96-well plate, 50 μl / well, and incubate at 37°C for 30 minutes.

[0089] 6. Washing: Same as 2.

[0090] 7. Enzyme-labeled antibody: Use secondary antibody diluent (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., catalog number: HRP-SD-001) to dilute the enzyme-labeled antibody (mouse anti-African swine fever virus p22 protein pig IgG monoclonal antibody labeled with horseradish peroxidase) at a volume ratio of 1:2000, 50 μl / well, and incubate at 37°C for 30 minutes.

[0091] 8. Washing: Same as 2.

[0092] 9. Color development: Add 50 μl TMB substrate to each well for color development and react at 37°C in the dark for 10 minutes.

[0093] 10. Stop: Add 50 μl stop solution (2M H 2 SO 4 ).

[0094] 11. Reading: Determine OD 450nm value.

[0095] According to the following formula, the blocking rate % = [(negative control average OD 450nm - Sample OD 450nm ) / mean OD of negative control 450nm ] × 100. The judgment criteria are: a blocking rate higher than 47% is positive, and a blocking rate lower than 47% is negative.

[0096] The results showed that all pigs were negative for antibodies before and 5 days after vaccination; 10 days after vaccination, the antibodies of 2 pigs, No. 6 and No. 585, turned positive, but the blocking rate was less than 70%, which was weakly positive, and the antibodies of the other 3 pigs were negative; 25 days after vaccination, the antibody of No. 6 pig showed a downward trend, the blocking rate of No. 585 antibody reached more than 90%, which was strongly positive, the antibody of No. 492 pig had just turned positive, and the antibodies of the other 2 pigs were still negative ( Figure 5 The above results show that HLJ18 / BK33 strain can only induce some pigs to produce antibodies, and the antibody level is low, which proves that its replication level in the inoculated pigs is extremely low and the induced antibody response is weak.

[0097] Example 6. Preliminary establishment of an IPMA detection method for African swine fever virus antibodies based on wild boar kidney cells and exploration of the optimal inoculation dose of the virus

[0098] (1) Materials

[0099] The sources of materials used in the present invention are introduced as follows.

[0100] Standard positive serum of ASF: provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, among which the ASFV antibody ELISA titer is 1:12800.

[0101] ASF standard negative serum: provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, in which the ASFV antibody titer is 0.

[0102] Paraformaldehyde: purchased from Solarbio, product number P1112.

[0103] HRP-rSPA: HRP-labeled recombinant Staphylococcus aureus protein A, purchased from Thermo, catalog number 32400, wherein the solvent is sterile double distilled water, and the concentration of recombinant protein A is 5 mg / mL.

[0104] AEC color developing solution: purchased from Solarbio, product number A2010, wherein the solvent is sterile double distilled water.

[0105] (2) Virus inoculation

[0106] The ASFV HLJ18 / BK33 strain obtained in Example 1 was serially diluted 10-fold to 10 using DMEM culture medium supplemented with 3% FBS. -4 The dilution was 10 -2 , 10 -3 and 10 -4 BK2258 cells grown into a well-grown monolayer in a 96-well plate were inoculated with 100 μl / well of each dilution, and 4 wells were inoculated in parallel for each dilution.

[0107] The 96-well plate was incubated at 37°C and 5% CO 2 After culturing for 36 h under the same conditions, the culture medium was discarded, and the cells were washed three times with PBS (pH 7.4, the same below), 300 μl / well, 5 min / time, and the PBS was discarded.

[0108] (3) Cell fixation

[0109] Pre-cooled 4% (w / w) paraformaldehyde fixative (solvent: PBS) (100 μl / well) was added to each well of a 96-well plate, and the plates were fixed at room temperature for 20 min. The fixative was discarded, and the plates were washed three times with PBS, 300 μl / well, 5 min / time, and the PBS was discarded.

[0110] (4) Membrane treatment

[0111] Add 0.25% (v / v) Triton-X100 permeabilization solution (solvent: PBS) (200 μl / well) to each treated well of the 96-well plate, permeabilize for 15 min at room temperature, discard the permeabilization solution, wash three times with PBS, 300 μl / well, 5 min / time, and discard PBS.

[0112] (5) Closed

[0113] Add uninactivated 10% (v / v) FBS (solvent is PBS) (200 μl / well) and block for 2 h at 37° C. Discard the blocking solution and wash three times with PBS, 300 μl / well, 5 min / time, and discard PBS.

[0114] (6) Add primary antibody (serum)

[0115] The standard positive serum of ASF and the standard negative serum of ASF were diluted 1:100 (volume ratio) with PBS and used. For each virus dilution inoculated 4 wells, the diluted standard positive serum of ASF and the standard negative serum of ASF were added to 2 wells respectively, with an inoculation volume of 100 μl / well. The samples were incubated at 37°C for 2 h, and the samples were discarded. The samples were washed 3 times with PBS, 300 μl / well, 5 min / time, and the PBS was discarded.

[0116] (7) Add enzyme-labeled secondary antibody

[0117] HRP-rSPA was diluted with PBS at 1:10000 (volume ratio) before use, and added to each operating well of a 96-well plate at an addition volume of 100 μl / well. Incubate at 37°C for 1 h, and wash with PBS three times, 300 μl / well, 5 min / time, and discard PBS.

[0118] (8) Color rendering

[0119] Add 100 μL AEC color developing solution to each well for color development and react at room temperature in the dark for 20 min.

[0120] (9) Termination

[0121] The color developing solution was discarded and 100 μL of double distilled water was added to each well.

[0122] (10) Result determination

[0123] Specific red-brown staining can be observed in the positive serum control wells, which is characterized by bright red-brown and clustered; there is no specific red-brown staining in the negative serum control wells, which means that the detection method is established, there are no problems with the instrument and reagents, and the test results are credible. Specific red-brown staining observed under an ordinary optical microscope is determined to be positive for African swine fever virus antibodies, and no specific red-brown staining is determined to be negative for African swine fever virus antibodies.

[0124] Select one photo of various operating microscopes in the IPMA test of this example, and refer to the summary results for Figure 6 It can be seen that the ASFV positive serum reactions in the three different dilution inoculation wells all showed specific red-brown staining, while no reaction occurred in the negative serum wells. -3 The positive serum in the dilution inoculation wells was red-brown in color, bright, with clear boundaries and structures, and the number of virus plaques was moderate, which was easy to judge. Therefore, 10 was selected. -3 Dilution (equivalent to 10 4.29 TCID 50 / ml) as the optimal inoculation dose.

[0125] Example 7. Determination of the optimal inoculation time

[0126] ASFV HLJ18 / BK33 strain was 10 4.29 TCID 50 / ml titer inoculated BK2258 cells, 100 μl / well. 37°C, 5% CO 2 The cells were cultured for 24 h, 36 h, and 48 h, respectively, and other operation steps and parameters were the same as those in Example 6 to carry out IPMA test.

[0127] Select one photo of various operating microscopes in the IPMA test of this example, and refer to the summary results for Figure 7. It can be seen that for 36h post-inoculation, the red-brown staining produced by the positive serum reaction in the inoculation well is clustered, bright, with clear boundaries and structures, which is easy to judge; for 24h post-inoculation, the red-brown staining is not obvious enough, which is not conducive to observation; for 48h post-inoculation, due to virus infection, CPE is observed in the cells, and the cells aggregate, shrink, lyse, and form circular plaques, which in turn consumes serum antibodies, affecting the sensitivity of the result judgment and may miss the sample. Therefore, 36h post-inoculation incubation is selected as the optimal inoculation time.

[0128] Example 8. Determination of the optimal serum dilution

[0129] ASFV HLJ18 / BK33 strain was 10 4.29 TCID 50 / ml titer inoculated BK2258 cells, 100 μl / well. 37°C, 5% CO 2 The ASF standard positive serum and ASF standard negative serum were diluted with PBS at 1:10, 1:20, 1:40, 1:80, 1:160, and 1:320 (volume ratio), respectively, and the other operation steps and parameters were the same as those in Example 6 to perform IPMA test.

[0130] Select one photo of various operating microscopes in the IPMA test of this example, and refer to the summary results for Figure 8 . Results The positive sera at the above 6 different serum dilutions all showed specific red-brown staining with the ASFV inoculation wells, while the negative sera did not react in the ASFV inoculation wells. Analysis found that when the positive serum was diluted 1:10 and 1:20, the background in the cell well was darker, the non-specificity was stronger, and it was not easy to observe. At a dilution of 1:40, the red-brown staining was the brightest, with clear boundaries and structures, and the background was weaker; although there was no specific red-brown staining when the negative serum was diluted 1:10 and 1:20, there was some background. Therefore, a dilution of 1:40 was selected as the optimal serum dilution.

[0131] Example 9. Determination of the optimal secondary antibody dilution

[0132] ASFV HLJ18 / BK33 strain was 10 4.29 TCID 50 / ml titer inoculated BK2258 cells, 100 μl / well. 37°C, 5% CO 2 The ASF standard positive serum and ASF standard negative serum were diluted with PBS at a volume ratio of 1:40, and HRP-labeled Pierce TMRecombinant protein A (secondary antibody) was diluted with PBS at 1:5000, 1:10000, 1:20000, and 1:40000 (volume ratio), respectively. Other operating steps and parameters were the same as those in Example 6 to carry out IPMA test.

[0133] Select one photo of various operating microscopes in the IPMA test of this example, and refer to the summary results for Fig. 9 . As can be seen, the results show that with the increase of the dilution of the secondary antibody, the intensity of the specific red-brown staining of the ASF standard positive serum and the ASFV inoculation well is significantly weakened, and the red-brown staining is brightest at a dilution of 1:10000, and the background is weak, which is easy to judge; negative sera of different dilutions do not react with the ASFV inoculation well. Therefore, the dilution of 1:10000 was selected as the optimal dilution of the secondary antibody.

[0134] Example 10. Determination of the optimal primary and secondary antibody action time

[0135] ASFV HLJ18 / BK33 strain was 10 4.29 TCID 50 / ml titer inoculated BK2258 cells, 100 μl / well. 37°C, 5% CO 2 The ASF standard positive serum, ASF weak positive serum and ASF standard negative serum (primary antibody) were diluted with PBS at 1:40 (volume ratio) and HRP-labeled Pierce TM Recombinant protein A (secondary antibody) was diluted with PBS at 1:10000 (volume ratio). The primary antibody was incubated at 37°C for 30 min, 1 h and 2 h, and the secondary antibody was incubated at 37°C for 30 min, 45 min and 60 min, respectively. Other operating steps and parameters were the same as those in Example 6, and IPMA test was performed.

[0136] Select one photo of various operating microscopes in the IPMA test of this example, and refer to the summary results for Fig.10 . It can be seen that the specific red-brown staining intensity of the positive serum and the ASFV inoculation wells at the above three different action times of the primary antibody is not significantly different. The red-brown staining of the secondary antibody for 30 minutes is weak, and it is easy to miss weak positive samples. The specific red-brown staining intensity of the secondary antibody for 45 minutes and 60 minutes is not significantly different, and 45 minutes is enough to detect weak positive samples. Negative sera did not react with the ASFV inoculation wells. In order to save time and maintain better stability, the primary antibody was exposed at 37°C for 30 minutes and the secondary antibody was exposed at 37°C for 45 minutes as the optimal action time.

[0137] Example 11. Determination of the Optimal Color Development Time and Temperature

[0138] ASFV HLJ18 / BK33 strain was 10 4.29TCID 50 / ml titer inoculated BK2258 cells, 100 μl / well. 37°C, 5% CO 2 The ASF standard positive serum, ASF weak positive serum and ASF standard negative serum (primary antibody) were diluted with PBS at 1:40 (volume ratio) and HRP-labeled Pierce TM Recombinant protein A (secondary antibody) was diluted with PBS at 1:10000 (volume ratio). The primary antibody was treated at 37°C for 30 min, the secondary antibody was treated at 37°C for 45 min, and the color was developed at 37°C and room temperature for 15 min, 20 min, 25 min, and 30 min, respectively. The other operating steps and parameters were the same as those in Example 6, and the IPMA test was performed.

[0139] Select one photo of various operating microscopes in the IPMA test of this example, and refer to the summary results for Fig.11 . It can be seen that the color development effect at 37°C is not much different from that at room temperature, but the background color at 37°C is darker. After 15 minutes of color development, the red-brown staining is weak, and weak positive samples are not easy to judge. The effects of color development at 20 minutes, 25 minutes, and 30 minutes are not much different, and negative sera do not react with the ASFV inoculation holes. In order to save time, 20 minutes of room temperature color development was selected as the optimal color development time.

[0140] Example 12. ASF antibody IPMA detection procedure

[0141] Based on the results of the various test conditions in Examples 6-11 above, the IPMA operation optimization procedure is summarized and determined as follows:

[0142] (1)Planning

[0143] BK2258 cells cultured in DMEM supplemented with 10% FBS were grown to full growth in the culture flask, digested and resuspended in 0.25% (0.25 g / 100 ml) trypsin digestion solution, and then the digested cells were plated in a 96-well plate, with 100 μl inoculated in each well, and then placed in a CO 2 The cells were cultured in an incubator at 37° C. for 48 h. When the cells in the 96-well plate grew to about 90%, the 96-well plate was taken out.

[0144] (2) Virus inoculation

[0145] ASFV HLJ18 / BK33 strain was diluted with DMEM culture medium supplemented with 2% FBS at 10 4.29 TCID 50 / ml titer inoculated BK2258 cells, 100 μl / well. 37°C, 5% CO 2 Culture for 36 hours.

[0146] (3) Cell fixation

[0147] Discard the culture medium, wash with 37°C preheated PBS 3 times, 300 μl / well, 5 min / time, and discard PBS; add precooled 4% (w / w) paraformaldehyde fixative (solvent is PBS) (100 μl / well), let stand at room temperature for 20 min. Discard the fixative, wash with PBS 3 times, 300 μl / well, 5 min / time, and discard PBS.

[0148] (4) Membrane treatment

[0149] Add 0.25% (v / v) Triton-X100 permeabilization solution (solvent is PBS) (200 μl / well) to each treated well of the 96-well plate and let stand at room temperature for 15 min. Discard the permeabilization solution and wash with PBS 3 times, 300 μl / well, 5 min / time, and discard PBS.

[0150] (5) Closed

[0151] Add uninactivated 10% (v / v) FBS (solvent is PBS) (200 μl / well) and block for 2 h at 37° C. Discard the blocking solution and wash three times with PBS, 300 μl / well, 5 min / time, and discard PBS.

[0152] (6) Add primary antibody

[0153] Add the sample to be tested, dilute it with PBS at 1:40 (volume ratio), 100 μl / well, and set up a positive control (ASF standard positive serum) and a negative control (ASF standard negative serum) for incubation at 37°C for 30 minutes. Discard the sample, wash with PBS 3 times, 300 μl / well, 5 minutes / time, and discard the PBS.

[0154] (7) Add enzyme-labeled secondary antibody

[0155] HRP-labeled Pierce TM Recombinant protein A was diluted with PBS at 1:10000 (volume ratio) and the diluted HRP-labeled Pierce TM Recombinant protein A was added to each well of the 96-well plate at a volume of 100 μl / well and incubated at 37°C for 45 min. The secondary antibody was discarded and the cells were washed three times with PBS at 300 μl / well for 5 min / times, and the PBS was discarded.

[0156] (8) Color rendering

[0157] Add 100 μL AEC color development solution to each well for color development and react at room temperature in the dark for 20 min.

[0158] (9) Termination

[0159] Discard the color developing solution and add 100 μL of double distilled water to each well for long-term storage.

[0160] (10) Result determination

[0161] Specific red-brown staining can be observed in the positive serum control wells, which is characterized by bright red-brown staining in clusters; there is no specific red-brown staining in the negative serum control wells, which means that the detection method is valid, there are no problems with the instrument and reagents, and the test results are credible.

[0162] If specific red-brown staining is observed in the serum sample to be tested, it is determined to be positive for African swine fever virus antibodies; if no specific red-brown staining is observed, it is determined to be negative for African swine fever virus antibodies.

[0163] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

[0164] Example 13. Sensitivity test

[0165] 1. Antibody detection results of pigs infected with low-virulence ASFV at different times

[0166] Three pigs were inoculated with low-virulence ASFV SD / DY-I / 21 strain (GenBank sequence number: MZ945537.1) at a dose of 10 6 TCID 50 / head. Blood was collected and serum was separated on the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 13th days after inoculation with ASFV SD / DY-I / 21 strain, and IPMA detection was performed according to the operating procedure of Example 12. At the same time, a commercial ELISA detection kit was used for detection according to the process of the instruction manual. Three pigs were inoculated with ASFV SD / DY-I / 21 strain. The IPMA results showed that one pig turned positive for ASF antibodies on the 6th day after inoculation, and the other two pigs turned positive on the 7th day after inoculation; the commercial ELISA test results showed that two pigs turned positive for ASF on the 8th day after inoculation, and the other one turned positive on the 9th day after inoculation (Table 1). It can be seen that the IPMA detection method of the present invention can detect the positive antibody conversion of ASFV-infected pigs earlier than the conventional ELISA detection method, and can diagnose the disease earlier.

[0167] Table 1. Antibody ELISA test results of pigs infected with low-virulence ASFV at different times

[0168]

[0169]

[0170] Note: S / P = (sample OD 450nm Value - average OD of negative control 450nm value) / (average OD of positive control 450nm Value - average OD of negative control 450nm S / P ≥ 0.4, judged as positive; S / P < 0.3, judged as negative; 0.3 ≤ S / P < 0.4, judged as suspected. IPMA results "+" for positive, "-" for negative, and "±" for suspected.

[0171] 2. Determination of sensitivity of ASF antibody strong positive serum

[0172] ASFV HLJ18 / BK33 strain was 10 4.29 TCID 50 / ml titer inoculated BK2258 cells, 100 μl / well. 37°C, 5% CO 2 After 36 hours of culture, one serum with strong positive ASF antibody was selected and diluted with PBS at 1:40, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200 and 1:102400 (volume ratio). The other operation steps and parameters were the same as those in Example 6 to perform IPMA test. At the same time, a commercial kit was used for detection according to the instructions.

[0173] Select one photo of various operating microscopes in the IPMA test of this example, and refer to the summary results for Fig.12 The IPMA results showed that when the serum dilution was between 1:100 and 1:25600, the ASFV inoculation wells all showed specific red-brown staining, indicating a positive reaction. The commercial kit results showed that the serum dilution was positive at 1:100 to 1:6400 (Table 2). This indicates that the IPMA titer of the serum is 1:25600 and the ELISA titer is 1:6400, indicating that IPMA has a higher sensitivity.

[0174] Table 2 ELISA titer determination of ASF antibody strong positive serum

[0175]

[0176]

[0177] Note: S / P = (sample OD 450nm Value - average OD of negative control 450nm value) / (average OD of positive control 450nm Value - average OD of negative control 450nmS / P ≥ 0.4, judged as positive; S / P < 0.3, judged as negative; 0.3 ≤ S / P < 0.4, judged as suspected. IPMA result "+" is positive, "-" is negative.

[0178] Example 14. Specificity test

[0179] ASFV HLJ18 / BK33 strain was 10 4.29 TCID 50 / ml titer inoculated BK2258 cells, 100 μl / well. 37°C, 5% CO 2 Culture for 36 hours. Seven positive sera for antibodies to important porcine pathogens, including African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), foot-and-mouth disease virus type O (FMDV / O) and porcine epidemic diarrhea virus (PEDV), were selected, and SPF pig (SPF) serum was used as a negative control. According to the operating procedure of Example 12, the above eight sera were used as the primary antibodies in step (6) and IPMA detection was performed respectively.

[0180] Select one photo of various operating microscopes in the IPMA test of this example, and refer to the summary results for Fig.13 The results showed that the above six important swine pathogen antibody-positive sera did not react with the ASFV inoculation wells, indicating that the IPMA method established in the present invention has high specificity.

[0181] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A kit comprising cells susceptible to African swine fever virus and an African swine fever virus strain with a microbial collection number of CCTCC NO: V202404.

2. The kit according to claim 1, characterized in that The African swine fever virus susceptible cells are wild boar kidney cells with a microbial preservation number of CCTCC NO: C2022258.

3. The kit according to claim 1, characterized in that The kit also contains any one or more combinations of the following materials: Serum positive for African swine fever virus antibody; African swine fever virus antibody-negative serum; 96-well cell culture plates; Paraformaldehyde; Trypsin; Fetal bovine serum; Triton-X100 solution; HRP-labeled recombinant Staphylococcus protein A; PBS solution; DMEM culture medium; AEC color developing solution.

4. A method for detecting African swine fever virus antibodies for non-diagnostic purposes, the detection method comprising the following steps: P1: Infecting African swine fever virus susceptible cells with the African swine fever virus strain with a microbial deposit number of CCTCC NO: V202404 to obtain infected cells; P2: fixing the infected cells on the surface of a solid phase carrier to obtain immobilized cells; P3: adding the sample to be tested to the immobilized cells for incubation to obtain the immobilized cells of the first incubation; P4: adding the porcine IgG specific binding substance connected with the label to the immobilized cells of the first incubation for incubation to obtain the immobilized cells of the second incubation; P5: Characterize the marker that is indirectly bound to the fixed cells of the second incubation, and determine whether African swine fever virus antibodies exist in the sample to be tested based on the presence or absence of the marker.

5. The detection method according to claim 4, characterized in that: In step P1, the African swine fever virus susceptible cells are wild boar kidney cells with a microbial preservation number of CCTCC NO: C2022258.

6. The detection method according to claim 4, characterized in that: In step P2, the surface of the solid phase carrier is the inner surface of the well of the microplate.

7. The detection method according to claim 4, characterized in that: In step P3, the sample to be tested is pig serum.

8. The detection method according to claim 4, characterized in that: In step P4, the labeled pig IgG specific binder is HRP-labeled recombinant Staphylococcus protein A. In step P5, the HRP-labeled recombinant Staphylococcus protein A is characterized by AEC colorimetric solution, and whether the fixed cells are stained red-brown is determined to determine whether African swine fever virus antibodies are present in the sample to be tested.

9. The detection method according to claim 4, characterized in that: In step P1, the infection concentration of the African swine fever virus strain is 10 3.00 TCID 50 / ml to 10 4.50 TCID 50 / ml, the African swine fever virus susceptible cells are in a monolayer cell state.

10. The detection method according to claim 4, characterized in that: In step P2, a mixture containing 3-5 w / w The infected cells were fixed to the solid surface by adding 5% paraformaldehyde in PBS.

11. The detection method according to claim 4, characterized in that: Between step P2 and step P3, the method further comprises the following steps: P2-1: treating the immobilized cells with a membrane permeabilization solution; P2-2: The fixed cells treated with the permeabilization solution are blocked with a blocking solution.

12. The detection method according to claim 4, characterized in that: In step P3, African swine fever virus antibody-positive serum and / or African swine fever virus antibody-negative serum are set as control samples in parallel with the sample to be tested.

13. The detection method according to claim 8, characterized in that: After step P5, double distilled water is added to terminate the reaction between the AEC color developing solution and the HRP-labeled recombinant Staphylococcus protein A.

14. The detection method according to claim 4, characterized in that: In step P1, the infection is carried out using DMEM culture medium at 36-38° C. and 4-7% carbon dioxide for 30-40 hours.

15. The detection method according to claim 11, characterized in that: In step P2-1, 0.2-0.3 v / v The surface of the solid phase carrier is treated with PBS solution containing % Triton-X100 and allowed to stand at room temperature for 10-20 minutes.

16. The detection method according to claim 11, characterized in that: In step P2-2, the v / v The surface of the solid phase carrier is treated with % FBS in PBS at 35-38° C. for 1.5-3 h.

17. The detection method according to claim 7, characterized in that: In step P3, the pig serum is diluted 20-50 times before use.

18. The detection method according to claim 7, characterized in that: In step P3, incubate at 36-38°C for 20-40 min.

19. The detection method according to claim 8, characterized in that: In step P4, the concentration of the HRP-labeled recombinant Staphylococcus protein A is 3-7×10 -4 mg / mL.

20. The detection method according to claim 8, characterized in that: In step P4, incubate at 36-38°C for 40-60 min.

21. The detection method according to claim 8, characterized in that: The reaction time of the AEC color developing solution and the HRP-labeled recombinant Staphylococcus aureus protein A is 15-25 minutes.

22. The detection method according to claim 6, characterized in that: Before operation step P2, the surface of the solid phase carrier is washed with PBS solution.

23. The detection method according to claim 6, characterized in that: After completing step P2, the surface of the solid phase carrier is washed with a PBS solution.

24. The detection method according to claim 11, characterized in that: After completing step P2-1, the surface of the solid phase carrier is washed with a PBS solution.

25. The detection method according to claim 11, characterized in that: After completing step P2-2, the surface of the solid phase carrier is washed with a PBS solution.

26. The detection method according to claim 18, characterized in that After completing step P3, the surface of the solid phase carrier is washed with a PBS solution.

27. The detection method according to claim 20, characterized in that After completing step P4, the surface of the solid phase carrier is washed with a PBS solution.

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