Competitive chemiluminescence detection kit for detecting African swine fever virus p30 antibodies and its application

Abstract

The present invention discloses a competitive chemiluminescence detection kit for detecting African swine fever virus (ASFV) p30 antibodies and its application. The present invention utilizes the principle that LgBiT and HiBiT complement each other to form a luciferase-catalyzed substrate furimazine to produce chemiluminescence. After ASFVp30 monoclonal antibody 16-5E7E8 is fused with HiBiT for expression, ASFVp30 antibodies in serum are detected using competitive chemiluminescence. The kit of the present invention is not only highly sensitive and specific for detecting ASFVp30 antibodies, but also simple to operate and has a short reaction time. It only takes 20 minutes to quickly and effectively detect whether the serum to be tested contains ASFVp30 antibodies. It has a good application prospect in ASFV antibody detection and provides technical means for the comprehensive prevention and treatment of ASF.

Description

Technical Field

[0001] The present invention relates to a kit for detecting antibodies to African swine fever virus and its application, and in particular to a competitive chemiluminescence detection kit for detecting antibodies to African swine fever virus and its application. The present invention belongs to the technical field of virus detection. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious, and lethal animal disease caused by the African swine fever virus (ASFV). The mortality rate of pigs infected with the hyperacute and acute forms of ASFV is nearly 100%. The World Organization for Animal Health (OIE) lists it as a notifiable animal disease, and my country classifies it as a Category I animal disease. ASFV is a large, linear, double-stranded DNA arbovirus, the only member of the Assurviridae family. Its genome ranges from 170 to 194 kbp in length and encodes 68 structural proteins and over 100 nonstructural proteins. p30 is one of the membrane proteins produced early during ASFV infection of host animals. Encoded by the CP204L gene, it is located in the viral envelope membrane and is highly conserved, making it one of the most immunogenic proteins. Expression of p30 can be observed approximately 2 to 4 hours after infection and persists until the end of the viral life cycle. p30 is a key structural protein of ASFV and can induce high levels of antibodies in animals. Therefore, p30 protein can be used for early detection of ASFV and has been widely used in ASFV serological detection.

[0003] Shimomura et al. first isolated OLuc from the deep-sea shrimp Oplophorus gracilirostris in 1978. Subsequent studies demonstrated that OLuc is structurally stable, exhibits high activity and quantum yield, and is ATP-independent in its catalytic production of blue light from coelenterazine. OLuc is composed of two heterodimeric subunits, 35 kDa and 19 kDa. cDNA cloning revealed that bioluminescent activity is exclusively associated with the smaller subunit (Oluc-19). However, Oluc-19 is unstable and exhibits low expression levels in the absence of its 35 kDa partner. In 2012, Hall et al. subjected OLuc to three rounds of mutagenesis to generate NanoLuc (NLuc). NLuc catalyzes the chemiluminescence produced by furimazine, which is 150 times more intense than both FLuc and RLuc. It also possesses advantages such as high stability, a small molecular weight (19.1 kDa), and a long luminescence duration. In 2016, Dixon et al. cut and mutated NLuc and screened out LgBiT (17.6 kDa) and HiBiT (11 aa) with high affinity, with an affinity constant Kd of 0.7 nM.

[0004] Based on this, the present invention connects the gene sequence of ASFV p30 monoclonal antibody 16-5E7E8 with HiBiT through a linker and expresses it in CHO. After purification, it is used as a competitive antibody for detecting ASFV p30 antibodies in serum. Summary of the Invention

[0005] The purpose of the present invention is to provide a competitive chemiluminescence detection kit for rapid detection of ASFV p30 antibodies and its application. The kit has good sensitivity, high specificity, and short time required. The entire experiment only takes 20 minutes.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] The present invention provides a competitive chemiluminescent detection kit for detecting African swine fever virus p30 antibodies. The kit comprises a chemiluminescent plate coated with an African swine fever virus p30 recombinant protein, a fusion protein 16-5E7E8-HiBiT, an LgBiT protein, and a furimazine compound. The fusion protein 16-5E7E8-HiBiT is obtained by sequentially connecting the light chain, linker, HiBiT, P2A, and heavy chain of the anti-African swine fever virus p30 protein monoclonal antibody 16-5E7E8. The amino acid sequence of the fusion protein 16-5E7E8-HiBiT is shown in SEQ ID NO.3.

[0008] Among them, preferably, the African swine fever virus p30 recombinant protein is obtained by inserting the nucleotide sequence of the CP204L gene encoding the African swine fever virus p30 protein into a prokaryotic expression vector, and then expressing and purifying it through a prokaryotic expression system.

[0009] Among them, preferably, the chemiluminescent plate coated with African swine fever virus p30 recombinant protein is prepared by the following method: the purified p30 recombinant protein is diluted to 0.125 μg / mL with pH 9.6 carbonate buffer, and added to a white detachable polystyrene 96-well plate at 100 μL / well, and kept at 4°C overnight; after drying, 200 μL / well of blocking solution is added, and the blocking solution is 0.01 mol / L phosphate buffer containing 1% w / w BSA, 2% w / w sucrose, 0.05% v / v Tween-20, 0.1% w / w proclin 300, and 6% v / v horse serum, and the plate is blocked at 37°C for 1.5h.

[0010] Preferably, the LgBiT is obtained by sequentially connecting the kozak sequence, signal peptide, LgBiT, linker, and 6×His, and its amino acid sequence is shown in SEQ ID NO.4.

[0011] Preferably, the furimazine compound is dissolved in a mixed solution of DMSO:H2O with a volume ratio of 4:1 and then stored at -20°C with a storage concentration of 10 mM.

[0012] Among them, preferably, the kit also includes serum diluent, PBST washing solution, ASFV standard positive serum and ASFV standard negative serum.

[0013] Among them, preferably, the serum diluent is a phosphate buffer containing 1% w / w casein, 0.05% v / v Tween-20, 0.1% v / v proclin 300, and 2% w / w sucrose with a concentration of 0.01 mol / L; the PBST washing solution is a phosphate buffer containing 0.05% v / v Tween-20, pH 7.2-7.4, and a concentration of 0.01 mol / L.

[0014] Furthermore, the present invention also proposes the use of the competitive chemiluminescence detection kit in the preparation of a reagent for detecting African swine fever virus p30 antibodies.

[0015] Furthermore, the present invention also proposes a method for detecting African swine fever virus p30 antibodies using the competitive chemiluminescence detection kit, wherein the method does not include disease diagnosis and treatment methods, and the method comprises the following steps:

[0016] (1) Room temperature equilibration: Take out the competitive chemiluminescence detection kit from 4°C and return it to room temperature for later use;

[0017] (2) Serum dilution: In a U-shaped dilution plate, dilute the serum to be tested and the positive and negative control sera at a 1:5 ratio with serum diluent. Set up 3 positive control sera and 3 negative control sera on each plate; shake to mix;

[0018] (3) Dilution of fusion protein 16-5E7E8-HiBiT: dilute the fusion protein 16-5E7E8-HiBiT to 0.125 μg / mL using serum diluent;

[0019] (4) Sample addition: Transfer the serum in the dilution plate in step (2) to the chemiluminescent plate coated with African swine fever virus p30 recombinant protein in order, 50 μL / well, and then add 50 μL of the diluted fusion protein 16-5E7E8-HiBiT in turn, shake and mix, and react at room temperature for 10 minutes;

[0020] (5) Washing: Discard the reaction solution, add 300 μL of PBST to each well, wash 5 times, and pat dry for the last time;

[0021] (6) Chemiluminescence reaction: LgBiT protein was diluted to 2 μg / mL with PBST, 25 μL / well, and then 25 μL of 1:250 diluted furimazine compound was added in sequence, shaken to mix, and reacted at room temperature for 10 min;

[0022] (7) Detection: After the reaction, the chemiluminescence (CLIA) value was detected using a chemiluminescence detector;

[0023] (8) Calculation of blocking rate (PI): PI = (1-test sample CLIA value / negative control average CLIA value) × 100%

[0024] (9) The conditions for the test to be established: the chemiluminescence value of the standard negative serum should be ≥15,000,000, and the blocking rate (PI) of the standard positive serum should be ≥95%;

[0025] (10) Judgment criteria: When testing pig serum samples, if PI ≥ 30%, it is judged as ASFV p30 antibody positive; if PI < 30%, it is judged as ASFV p30 antibody negative.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention sequences mouse monoclonal antibody cells screened for p30 antibody detection, links the antibody sequence to HiBiT, and expresses it on CHO cells. This avoids the batch-to-batch variability and animal welfare issues associated with traditional mouse monoclonal antibody expression via intraperitoneal immunization of mice. Furthermore, HiBiT is only 11 aa in size, and linking it to the end of the monoclonal antibody light chain via a linker does not affect the antibody structure or cause steric hindrance to antibody recognition.

[0028] 2. Traditional chemical conjugation-labeled antibodies are disordered, have uncertain coupling positions, and have differences in coupling efficiency between different batches. However, the present invention connects HiBiT to the end of the monoclonal antibody light chain through a linker, with a deterministic direction and position. Each monoclonal antibody light chain end carries HiBiT.

[0029] 3. The present invention is used to detect ASFV p30 antibodies with not only high sensitivity and good specificity, but also simple operation and short reaction time. It only takes 20 minutes to quickly and effectively detect whether the serum to be tested contains ASFV p30 antibodies. It has a good application prospect in ASFV antibody detection and provides a technical means for the comprehensive prevention and control of ASF. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 For the expression and purification of p30 recombinant protein;

[0031] Wherein, M: marker, 1: bacterial lysate before induction, 2: bacterial lysate after induction, 3: supernatant after bacterial ultrasonic lysis, 4: precipitate after bacterial ultrasonic lysis, 5: purified p30 recombinant protein;

[0032] Figure 2 For the expression and purification of 16-5E7E8-HiBiT;

[0033] Wherein, M: marker, 1: expression of 16-5E7E8-HiBiT in CHO cell supernatant, 2: flow-through after 16-5E7E8-HiBiT loading, 3: PBS washing, 4: purified 16-5E7E8-HiBiT;

[0034] Figure 3 For the expression and purification of LgBiT;

[0035] Wherein, M: marker, 1: supernatant of CHO cells not transfected with LgBiT plasmid, 2: expression of LgBiT in CHO cell supernatant, 3: purified LgBiT;

[0036] Figure 4 To determine the optimal reaction time between 16-5E7E8-HiBiT and serum;

[0037] Figure 5 To determine the cut-off value, diagnostic sensitivity and diagnostic specificity of the method of the present invention;

[0038] Among them, A: ROC curve, each point on the graph represents Dsn and Dsp under a specific threshold; B: background interaction point graph, 0 is a negative serum sample, and 1 is a positive serum sample

[0039] Figure 6 Comparison of analytical sensitivity between the method of the present invention (MAb-HiBiT) and commercial kits;

[0040] Among them, A: two methods were used to detect the gradient dilution of ASFV standard positive serum; B: two methods were used to detect 2 gradient dilutions of ASFV positive serum. DETAILED DESCRIPTION

[0041] The following embodiments and examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the present invention. Where specific conditions are not specified, conventional conditions or those recommended by the manufacturer shall be followed.

[0042] Example 1 Construction, expression and purification of ASFV p30 recombinant plasmid

[0043] The CP204L gene sequence encoding the p30 protein of ASFV SY18 strain (GenBank: MH766894.1) was optimized and synthesized according to the codon preference of Escherichia coli, and inserted into the pET-28a vector through EcoR I and SalI to construct the recombinant plasmid pET-28a-p30. Then, the constructed pET-28a-p30 recombinant plasmid was transformed into the competent cell BL21 (DE3) by heat shock method, plated, and single colonies were selected for shaking. When the bacterial solution concentration OD 600 When the pH reached 0.6-0.8, IPTG was added for induction and cultured at 16°C with shaking for 20 hours. The bacterial solution was then centrifuged, and the collected cells were ultrasonicated and centrifuged. The supernatant and precipitate of the bacterial lysate were collected to verify the expression pattern.

[0044] The p30 inclusion bodies were resuspended in IB solubilization buffer (20 mM Tris, 500 mM NaCl, 8 M urea, pH 8.0) and dissolved overnight at 4°C. The supernatant was centrifuged and applied to a Ni-NTA column for purification. Impurities were eluted and the p30 recombinant protein was collected. The purified p30 recombinant protein was placed in a dialysis bag and subjected to gradient renaturation in renaturation buffer 1 (4 M urea, 200 mM imidazole, 20 mM Tris, 250 mM NaCl buffer, 0.1% Triton-100, pH 8.0), renaturation buffer 2 (2 M urea, 100 mM imidazole, 20 mM Tris, 250 mM NaCl, 0.1% Triton-100, pH 8.0), and renaturation buffer 3 (20 mM Tris, 250 mM NaCl, 0.1% Triton-100, 10% glycerol, 0.4 mM arginine, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, pH 8.0).

[0045] The results are as follows Figure 1 As shown, the protein was expressed in the form of inclusion bodies with a molecular weight of approximately 30 kDa (p30 approximately 24 kDa, carrier protein approximately 6 kDa), which was consistent with the expected size. After purification, a p30 recombinant protein with high purity was obtained.

[0046] Example 2 Preparation of monoclonal antibodies against ASFV p30

[0047] The renatured p30 recombinant protein (prepared in Example 1) was emulsified with an equal volume of Freund's complete adjuvant and intramuscularly immunized 8-week-old Balb / c mice. 21 days after the first immunization, Freund's incomplete adjuvant was emulsified with an equal volume of p30 recombinant protein and then immunized three times, once every two weeks, with an immunization dose of 20 μg / head. 7 days after the last immunization, 20 μg of p30 recombinant protein was injected intraperitoneally. 3 days later, the spleen cells of the mice were taken and fused with SP2 / 0 cells, and the positive hybridomas were subcloned three times by limiting dilution to obtain 20 monoclonal antibodies. The epitopes recognized by the proteins were identified, and 16-5E7E8 was finally selected for subsequent experiments. The epitope recognized by monoclonal antibody 16-5E7E8 is "APDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKL".

[0048] Example 3 Construction, expression and purification of 16-5E7E8-HiBiT and LgBiT expression plasmids

[0049] 1. Construction of 16-5E7E8-HiBiT and LgBiT

[0050] RNA from hybridoma cell 16-5E7E8 was extracted using an RNA-easy kit and reverse-transcribed into cDNA. The VH and VL cDNAs were amplified using rapid amplification of cDNA ends (RACE) and ligated into a cloning vector for sequencing. The Kozak sequence, the light chain of 16-5E7E8, the linker (GGSSGG), HiBiT, P2A, and the heavy chain of 16-5E7E8 were sequentially ligated and synthesized. BamHI and XhoI restriction sites were added to the 5' and 3' ends of the sequence, respectively. The resulting sequence, designated 16-5E7E8-HiBiT, has a nucleotide sequence shown in SEQ ID NO. 1. This cDNA was then inserted into the pcDNA3.1(+) vector via BamHI and XhoI to construct the recombinant plasmid 16-5E7E8-HiBiT-pcDNA3.1.

[0051] The kozak sequence, signal peptide (MRLSVCLLLLTLALCCYRANA), LgBiT, linker (GGSSGG), and 6×His were sequentially ligated and synthesized. BamHI and XhoI restriction sites were added to the 5' and 3' ends of the sequence, respectively. The resulting sequence, designated LgBiT, has a nucleotide sequence shown in SEQ ID NO. 2. The recombinant plasmid, LgBiT-pcDNA3.1, was then inserted into the pcDNA3.1(+) vector via BamHI and XhoI restriction enzymes.

[0052] 2. Expression and purification of 16-5E7E8-HiBiT and LgBiT

[0053] The recombinant plasmids 16-5E7E8-HiBiT-pcDNA3.1 and LgBiT-pcDNA3.1 were transfected into CHO cells (7×10 6 ~1.0×10 7 Dilute to a final density of 6 × 10 viable cells / mL. 6 18-22 hours after transfection, add ExpiFectamine TM CHO Enhancer and ExpiCHO TM The supplementary materials were added and the culture flask was returned to the 37°C incubator containing 8% CO2 for shaking culture. The cell supernatant was collected 10 days after transfection.

[0054] 16-5E7E8-HiBiT was purified by protein G affinity chromatography, and LgBiT was purified by His affinity chromatography. Figure 2 、 Figure 3 Results indicate that highly pure 16-5E7E8-HiBiT (50 kDa heavy chain and 25 kDa light chain) and LgBiT proteins (19 kDa) were obtained, with sizes consistent with expectations. The amino acid sequences of 16-5E7E8-HiBiT and LgBiT are shown in SEQ ID NOs. 3 and 4, respectively. The concentrations of 16-5E7E8-HiBiT and LgBiT were 1 mg / mL and 200 μg / mL, respectively, as measured by Bradford ELISA.

[0055] Example 4 Establishment of a competitive chemiluminescence assay for detecting AFSV p30 antibodies

[0056] 1. Optimization of optimal p30 coating concentration, optimal 16-5E7E8-HiBiT detection concentration, and serum dilution

[0057] The checkerboard titration method was used to screen the optimal coating concentration of p30 recombinant protein, the optimal detection concentration of 16-5E7E8-HiBiT, and the serum dilution. Briefly, the purified p30 recombinant protein was diluted to 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, and 0.03125 μg / mL in carbonate buffer (CBS, pH 9.6), 100 μL / well, and incubated at 4°C overnight. After drying, 200 μL / well of blocking solution (containing 1% w / w BSA, 2% w / w sucrose, 0.1% w / w proclin 300, 0.05% v / v Tween-20, and 6% v / v horse serum in 0.01 mol / L phosphate buffer) was added and blocked at 37°C for 1.5 h. Then, 50 μL of serum diluent (containing 1% w / w casein, 0.05% v / v Tween-20, 0.1% v / v proclin 300, and 6% v / v horse serum) was added. AFSV standard positive serum and standard negative serum were diluted at a volume ratio of 1:5 to 1:40 (300, 2% w / w sucrose in 0.01 mol / L phosphate buffer), and 16-5E7E8-HiBiT at concentrations of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and 0.06125 μg / mL were added with 50 μL of serum diluent at the same time, and the reaction was carried out at 37°C for 30 min. After washing five times with PBST, 25 μL of LgBiT diluted to 2 μg / mL with PBST (0.01 mol / L phosphate buffer containing 0.05% v / v Tween-20, pH 7.2-7.4) and 25 μL of furimazine compound diluted at a volume ratio of 1:250 (furimazine was dissolved in a mixed solution of DMSO:H2O volume ratio = 4:1 and stored at -20°C with a storage concentration of 10 mM. It was diluted 1:250 with PBST before use) were added, reacted at room temperature for 10 min, and the chemiluminescence value was detected.

[0058] Taking into account N / P and economic factors, the optimal coating concentration of AFSV p30 was 0.125 μg / mL, the optimal detection concentration of 16-5E7E8-HiBiT enzyme-labeled antibody was 0.125 μg / mL, and the optimal serum dilution was 1:5.

[0059] 2. Determination of the optimal reaction time between 16-5E7E8-HiBiT and serum

[0060] After determining the optimal reaction conditions, we optimized the reaction time between 16-5E7E8-HiBiT and serum. The following steps were performed: chemiluminescent plates were coated with 0.125 μg / mL p30 protein (100 μL / well) at 4°C overnight. After drying, the plates were added with 200 μL / well blocking buffer and blocked at 37°C for 1.5 hours. Then, 50 μL of a 1:5 diluted standard positive serum and standard negative serum were added, along with 50 μL of 0.125 μg / mL 16-5E7E8-HiBiT. The plates were incubated at 37°C for 10, 20, 30, 40, 50, and 60 minutes. After washing five times with PBST, 25 μL of 2 μg / mL LgBiT and 25 μL of a 1:250 diluted furimazine compound were added. The plates were incubated at room temperature for 10 minutes, and chemiluminescence was detected.

[0061] The results are as follows Figure 4 As shown, after a 10-minute competitive reaction between the standard positive serum and 16-5E7E8-HiBiT, the chemiluminescence value stabilizes. However, after 10 to 60 minutes of competitive reaction between the standard negative serum and 16-5E7E8-HiBiT, the chemiluminescence value continues to rise. Therefore, the N / P ratio increases with increasing reaction time. To demonstrate the rapid detection capability and sample accuracy of the present invention, a 10-minute competitive reaction was selected for subsequent testing. Therefore, the present invention can complete detection in a total reaction time of only 20 minutes.

[0062] Example 5 Assembly of a competitive chemiluminescent detection kit for detecting AFSV p30 antibodies

[0063] The kit comprises:

[0064] 1. Chemiluminescent plate coated with African swine fever virus p30 recombinant protein;

[0065] 2. Fusion protein 16-5E7E8-HiBiT, the amino acid sequence of which is shown in SEQ ID NO.3;

[0066] 3. LgBiT protein, the amino acid sequence of which is shown in SEQ ID NO. 4;

[0067] 4. Furimazine compound: Dissolve furimazine in a 4:1 DMSO:H2O mixture and store at -20°C (storage concentration 10 mM).

[0068] 5. Serum diluent: 0.01 mol / L phosphate buffer containing 1% w / w casein, 0.05% v / v Tween-20, 0.1% v / v proclin 300, and 2% w / w sucrose, pH 7.2-7.4;

[0069] 6. PBST washing solution: 0.01 mol / L phosphate buffer containing 0.05% v / v Tween-20, pH 7.2-7.4;

[0070] 7. ASFV standard positive serum and ASFV standard negative serum.

[0071] Example 6 Determination of Cut-off Value, Diagnostic Sensitivity and Diagnostic Specificity

[0072] The kit assembled in Example 5 was used to detect 42 ASFV-positive sera and 112 ASFV-negative sera with clear background according to the method established in Example 4. The specific steps are as follows:

[0073] 1. Room temperature balance: Take out the test kit from 4℃ and return it to room temperature for later use.

[0074] 2. Serum Dilution: In a U-shaped dilution plate, dilute the test serum and the positive and negative control sera 1:5 in serum diluent (0.01 mol / L phosphate buffer containing 1% w / w casein, 0.05% v / v Tween-20, 0.1% v / v proclin 300, and 2% w / w sucrose). Set up three positive control sera and three negative control sera per plate. Vortex to mix thoroughly.

[0075] 3. Dilution of fusion protein 16-5E7E8-HiBiT: Dilute 16-5E7E8-HiBiT to 0.125 μg / mL using serum diluent.

[0076] 4. Sample addition: Transfer the serum from the dilution plate in step 2 to the chemiluminescent plate coated with p30 antigen in sequence, 50 μL / well, and then add 50 μL of diluted 16-5E7E8-HiBiT in sequence, shake and mix, and react at room temperature for 10 minutes.

[0077] 5. Wash the plate: discard the reaction solution, add 300 μL of PBST (0.01 mol / L phosphate buffer containing 0.05% v / v Tween-20, pH 7.2-7.4) to each well, wash 5 times, and pat dry for the last time.

[0078] 6. Chemiluminescent reaction: Dilute LgBiT to 2 μg / mL with PBST, add 25 μL / well, then add 25 μL of 1:250 diluted furimazine compound, shake to mix, and react at room temperature for 10 minutes.

[0079] 7. Detection: After the reaction, use a chemiluminescence detector to detect the chemiluminescence (CLIA) value.

[0080] 8. The blocking rate (PI) was used as the evaluation standard for the test results, that is, PI = (1-test sample CLIA / standard negative average CLIA value) × 100%.

[0081] 9. Conditions for the test to be established:

[0082] (1) The chemiluminescence value of the standard negative serum should be ≥15,000,000

[0083] (2) The blocking rate (PI) of the standard positive serum is ≥95%.

[0084] The test results were analyzed by ROC curve analysis using MedCalc software to determine the cut-off value, diagnostic sensitivity, and diagnostic specificity of the method. Figure 5 As shown in the figure, when the cut-off value is 30%, the diagnostic sensitivity is 100% and the diagnostic specificity is 100%.

[0085] Example 7 Comparison of analytical sensitivity between competitive chemiluminescence detection kit and commercial kit

[0086] After three ASFV-positive sera were serially diluted (1:4 to 1:2048), they were detected using the competitive chemiluminescence detection kit assembled in Example 5 (the detection steps were the same as in Example 6) and a commercial kit.

[0087] The results are as follows Figure 6 As shown in A, the highest dilution of the commercial kit for detecting ASFV standard positive serum is 1:512, while the highest dilution of the method of the present invention for detecting ASFV standard positive serum is 1:1024. In order to further determine the analytical sensitivity of the method, two serially diluted ASFV positive sera (P3354 and P3355) were tested using the above two kits. The results are shown in Figure 6 As shown in Figure B, the analytical sensitivity of the kit of the present invention is 1 to 2 dilutions higher than that of the commercial kit, and the time used is much shorter than that of the commercial kit (20 min vs 100 min).

[0088] In summary, the competitive chemiluminescence detection kit established by the present invention can be used for the detection of ASFV p30 antibodies, and its detection sensitivity is higher than that of commercial kits.

Claims

1. A competitive chemiluminescence detection kit for detecting African swine fever virus p30 antibodies, characterized in that: The kit comprises a chemiluminescent plate coated with African swine fever virus p30 recombinant protein, a fusion protein 16-5E7E8-HiBiT, an LgBiT protein, and a furimazine compound, wherein the fusion protein 16-5E7E8-HiBiT is obtained by sequentially connecting the light chain, linker, HiBiT, P2A, and heavy chain of the anti-African swine fever virus p30 protein monoclonal antibody 16-5E7E8, and the amino acid sequence of the fusion protein 16-5E7E8-HiBiT is shown in SEQ ID NO.

3.

2. The competitive chemiluminescence detection kit according to claim 1, wherein The African swine fever virus p30 recombinant protein is a protein encoding the African swine fever virus p30 protein. CP204L The nucleotide sequence of the gene is inserted into a prokaryotic expression vector, and then expressed and purified through a prokaryotic expression system.

3. The competitive chemiluminescence detection kit according to claim 1, wherein The chemiluminescent plate coated with the African swine fever virus p30 recombinant protein is prepared by the following method: the purified p30 recombinant protein is diluted to 0.125 μg / mL with a pH 9.6 carbonate buffer, and 100 μL / well is added to a white detachable polystyrene 96-well plate and incubated at 4°C overnight; after drying, 200 μL / well of blocking solution is added, and the blocking solution is a 0.01 mol / L phosphate buffer containing 1% w / w BSA, 2% w / w sucrose, 0.05% v / v Tween-20, 0.1% w / w proclin 300, and 6% v / v horse serum, and the plate is blocked at 37°C for 1.5 h.

4. The competitive chemiluminescence detection kit according to claim 1, wherein The LgBiT protein is obtained by sequentially connecting the kozak sequence, signal peptide, LgBiT, linker, and 6×His, and its amino acid sequence is shown in SEQ ID NO.

4.

5. The competitive chemiluminescence detection kit according to claim 1, wherein The furimazine compound is dissolved in a mixed solution of DMSO:H2O with a volume ratio of 4:1 and then stored at -20°C with a storage concentration of 10 mM.

6. The competitive chemiluminescence detection kit according to claim 1, wherein The kit also includes serum diluent, PBST washing solution, ASFV standard positive serum and ASFV standard negative serum.

7. The competitive chemiluminescence detection kit according to claim 6, wherein The serum diluent is a 0.01 mol / L phosphate buffer containing 1% w / w casein, 0.05% v / v Tween-20, 0.1% v / v proclin 300, and 2% w / w sucrose; the PBST washing solution is a phosphate buffer containing 0.05% v / v Tween-20, pH 7.2-7.4, and a concentration of 0.01 mol / L.

8. Use of the competitive chemiluminescence detection kit according to any one of claims 1 to 7 in the preparation of a reagent for detecting African swine fever virus p30 antibodies.

9. A method for detecting African swine fever virus p30 antibodies using the competitive chemiluminescence detection kit according to any one of claims 1 to 7, wherein the method does not include a method for diagnosing and treating the disease, and the method comprises the following steps: (1) Room temperature equilibration: Take out the competitive chemiluminescence detection kit according to any one of claims 1 to 7 from 4°C and return it to room temperature for later use; (2) Serum dilution: In a U-shaped dilution plate, dilute the serum to be tested and the positive and negative control sera at a ratio of 1:5 with serum diluent. Set up 3 positive control sera and 3 negative control sera on each plate; shake to mix; (3) Dilution of fusion protein 16-5E7E8-HiBiT: Dilute the fusion protein 16-5E7E8-HiBiT to 0.125 μg / mL using serum diluent; (4) Sample addition: Transfer the serum from the dilution plate in step (2) to the chemiluminescent plate coated with African swine fever virus p30 recombinant protein in order, 50 μL / well, and then add 50 μL of the diluted fusion protein 16-5E7E8-HiBiT in turn, shake and mix, and react at room temperature for 10 min; (5) Washing: Discard the reaction solution, add 300 μL of PBST to each well, wash 5 times, and pat dry for the last time; (6) Chemiluminescence reaction: Dilute LgBiT protein to 2 μg / mL with PBST, 25 μL / well, then add 25 μL of 1:250 diluted furimazine compound, shake and mix, and react at room temperature for 10 min; (7) Detection: After the reaction, the chemiluminescence value is detected using a chemiluminescence detector; (8) Calculation of blocking rate: blocking rate = (1-test sample chemiluminescence value / negative control average chemiluminescence value) × 100% (9) The conditions for the test to be established: the chemiluminescence value of the standard negative serum should be ≥15,000,000, and the blocking rate of the standard positive serum should be ≥95%; (10) Judgment criteria: When testing pig serum samples, if the blocking rate is ≥30%, it is judged as ASFV p30 antibody positive; if the blocking rate is <30%, it is judged as ASFV p30 antibody negative.

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