A kit for quantitative detection of African swine fever virus based on chemiluminescence and its application

Abstract

The present invention discloses a kit for quantitatively detecting African swine fever virus based on chemiluminescence and its application. The present invention belongs to the technical field of virus detection. The kit includes a chemiluminescent plate coated with anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3, fusion protein 16-5E7E8-HiBiT, LgBiT and furimazine compound; wherein the fusion protein 16-5E7E8-HiBiT is obtained by sequentially connecting the light chain, linker, HiBiT, P2A and heavy chain of 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.4. The kit of the present invention has high sensitivity and a wide linear range, with a detection limit of 15 pg / mL for p30 protein and a detection limit of 12.2 HAD for ASFV. 50 In addition, the kit can quantitatively detect the ASFVp30 content in the tissues, blood, and nasal swabs of ASFV-infected pigs, and has good application prospects in ASFV pathogen detection.

Description

Technical Field

[0001] The present invention relates to a kit for quantitatively detecting African swine fever virus and its application, and in particular to a kit for quantitatively detecting African swine fever virus based on chemiluminescence 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). Hyperacute and acute forms of ASFV infection in pigs have a mortality rate approaching 100%, severely impacting the pig industry and the international trade of pig products. ASFV is a large, linear, double-stranded DNA arbovirus with a genome length of 170 to 190 kb, encoding over 150 proteins that play important roles in viral assembly, replication, virus-host interactions, and immune evasion. Studies have shown that the ASFV p30 structural protein, encoded by the CP204L gene, is located in the inner membrane of the viral envelope and is secreted in high quantities during the initial infection phase, typically 2 to 4 hours after infection, and remains expressed throughout the infection. p30 is a key structural protein of ASFV that can induce high levels of antibodies in animals and has been widely used in ASFV serological testing. In addition, p30 can induce the production of neutralizing antibodies in animals and plays a key role in viral endocytosis. It is an important structural protein of ASFV. These characteristics make p30 an ideal antigen for serological diagnosis and immunological detection.

[0003] Ancient peoples discovered and described bioluminescent animals such as mollusks, purple jellyfish, and fireflies long ago. In 1885, DuBois first discovered the reaction between luciferase and luciferin. It wasn't until the late 1940s that Green and McElroy extracted and purified the luciferase FLuc from fireflies. FLuc has a molecular weight of 61 kDa and uses ATP to catalyze the production of its substrate, luciferin (D-luciferin). Since then, scientists have conducted in-depth research on the luminescence mechanism of firefly luciferase FLuc and applied it to biomedical research. In the 1970s, MJ Cormier purified Renilla luciferase (RLuc) from sea bream. RLuc has a molecular weight of 36 kDa and catalyzes the production of coelenterazine to produce chemiluminescence. Over the past 30 years, scientists have isolated luciferases GLuc (20 kDa) from the marine copepod Gaussia princeps and OLuc (19 kDa) from the deep-sea shrimp Oplophorus gracilirostris. Both GLuc and OLuc catalyze coelenterazine as their substrates. OLuc is composed of two heterodimeric subunits, 35 kDa and 19 kDa. The 19 kDa subunit (OLuc-19) plays a catalytic role, but its expression level is low and unstable. Therefore, Hall et al. subjected OLuc to three rounds of mutagenesis to generate NanoLuc (NLuc). The chemiluminescence produced by NLuc upon furimazine is 150 times more intense than that of FLuc and RLuc. NLuc also possesses advantages such as high stability, a small molecular weight (19.1 kDa), and a long luminescence duration. It has been widely used to investigate protein-protein and protein-ligand interactions, investigate gene regulation and cell signaling pathways, monitor protein stability, and develop bioluminescence resonance energy transfer sensors and in vivo bioluminescence imaging. In 2016, Dixon cut and mutated NLuc and screened out 11S (LgBiT: 17.6 kDa) and 86 (HiBiT: 11 aa) with high affinity (Kd = 0.7 nM).

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

[0005] The purpose of the present invention is to provide a kit for quantitatively detecting African swine fever virus based on chemiluminescence and its application, wherein the kit has high sensitivity and a wide linear range.

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

[0007] The present invention provides a kit for quantitatively detecting African swine fever virus based on a chemiluminescence method, comprising a chemiluminescent plate coated with an anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3, a fusion protein 16-5E7E8-HiBiT, LgBiT, 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.4.

[0008] Preferably, the kit further comprises serum diluent, PBST washing solution and p30 protein standard.

[0009] 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, with a concentration of 0.01 mol / L; the p30 protein standard is obtained by transferring the p30 gene sequence of the African swine fever virus SY18 strain into a prokaryotic expression system, expressing and purifying it.

[0010] Among them, preferably, the heavy chain amino acid sequence of the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2.

[0011] Among them, preferably, the preparation steps of the chemiluminescent plate coated with the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 are: diluting the purified anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 to 2 μg / mL with PBS, adding it to a white detachable polystyrene 96-well plate at 100 μL / well, and coating it at 4°C overnight; after drying, adding 200 μL / well blocking solution, blocking at 37°C for 1.5 hours, and then patting dry; after drying at room temperature for 2 hours, vacuum-sealing it with an aluminum foil bag, and storing it at 4°C.

[0012] Among them, preferably, the blocking solution is a phosphate buffer solution with a pH of 7.2-7.4 and a concentration of 0.01 mol / L, containing 1% w / w BSA, 2% w / w sucrose, 0.1% v / v proclin 300, 6% v / v horse serum and 0.05% v / v Tween-20.

[0013] Among them, preferably, the LgBiT is obtained by sequentially connecting the kozak sequence, signal peptide (MRLSVCLLLLTLALCCYRANA), LgBiT, linker (GGSSGG), and 6×His, and its amino acid sequence is shown in SEQ ID NO.6.

[0014] Wherein, preferably, when the kit is used for quantitative detection of African swine fever virus, the following steps are performed:

[0015] 1) Room temperature equilibration: The test kit taken out from 4°C needs to be restored to room temperature before use;

[0016] 2) Sample dilution: In a U-shaped dilution plate, dilute the sample to be tested with serum diluent and perform a 4-fold serial dilution of the p30 protein standard, with 2 wells for each dilution;

[0017] 3) Sample addition: Transfer the samples and serially diluted standards from the dilution plate in step 2) to a chemiluminescent plate coated with the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 in order, 100 μL / well, and react at 37°C for 30 min;

[0018] 4) Washing: Discard the reaction solution, add 300 μL of PBST to each well, wash five times, and pat dry for the last time;

[0019] 5) Detection Antibody: Dilute the fusion protein 16-5E7E8-HiBiT to 0.5 μg / mL with serum diluent, add 100 μl / well, and react at 37°C for 30 min.

[0020] 6) Washing: Discard the reaction solution, add 300 μL of PBST to each well, wash five times, and pat dry for the last time;

[0021] 7) Chemiluminescent reaction: Dilute LgBiT to 2 μg / mL with PBST, add 25 μL / well, then add 40 μM furimazine compound, 25 μL / well, shake and mix, and react at room temperature for 10 min;

[0022] 8) Detection: After 10 minutes, the chemiluminescence (CLIA) value was detected using a chemiluminescence detector;

[0023] 9) Establishing a regression equation: Using p30 protein standards, establish a linear regression equation for detecting high and low concentrations of p30;

[0024] 10) ASFVp30 content in samples: Calculate the ASFVp30 content in diluted tissue samples, blood, and swabs using a linear regression equation, and then multiply by the dilution factor to obtain the ASFVp30 content in the sample;

[0025] 11) The test is valid if the chemiluminescence value of the 200 ng / mL p30 standard is ≥ 100,000,000.

[0026] Among them, preferably, the gradient concentration of the p30 protein standard is 200 ng / mL, 50 ng / mL, 12.5 ng / mL, 3.125 ng / mL, 781 pg / mL, 195 pg / mL, 48.8 pg / mL and 0 pg / mL; the furimazine compound is dissolved in a mixed solution of DMSO and H2O in a volume ratio of 4:1 and stored at -20°C. The storage concentration is 10 mM and the concentration is diluted to 40 μM with PBST when used.

[0027] Furthermore, the present invention also proposes the use of the kit in the preparation of reagents for detecting African swine fever virus.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention sequences the mouse monoclonal antibody cells screened for p30 detection and expresses the antibody sequence on a CHO cell after linking it to HiBiT. 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.

[0030] 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.

[0031] 3. The linear range of the detection kit of the present invention for prokaryotic expression of p30 antigen is 30pg / mL to 250ng / mL, and the detection limit is 15pg / mL; the linear range of the detection of ASFV is 48.8 to 25000HAD 50 , the detection limit was 12.2HAD 50 , demonstrating that the kit has high sensitivity and a wide linear range. Furthermore, the kit can quantitatively detect ASFV p30 levels in tissues, blood, and nasal swabs of ASFV-infected pigs. Therefore, the kit of the present invention has promising application prospects in ASFV pathogen detection and provides a technical means for the comprehensive prevention and control of ASF. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1For the expression and purification of p30 recombinant protein;

[0033] 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;

[0034] Figure 2 This is the SDS-PAGE analysis of the purified monoclonal antibody 2-1B3G3;

[0035] Wherein, M: marker, 1: 2-1B3G3 ascites, 2: flow-through after 2-1B3G3 ascites loading, 3: purified 2-1B3G3;

[0036] Figure 3 For the expression and purification of 16-5E7E8-HiBiT;

[0037] 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;

[0038] Figure 4 For the expression and purification of LgBiT;

[0039] Wherein, M: marker, 1: supernatant of CHO cells not transfected with LgBiT plasmid, 2: expression of LgBiT in CHO cell supernatant, 3: purified LgBiT;

[0040] Figure 5 To optimize the dilution and concentration of Furimazine compound;

[0041] Figure 6 For the optimization of LgBiT concentration;

[0042] Figure 7 Screening of the most suitable buffer for LgBiT and furimazine compounds and optimization of their reaction time;

[0043] Figure 8 The linear range and detection limit of the chemiluminescence method for detecting p30 protein based on HiBiT;

[0044] Among them, A: detection of 2-fold gradient dilution of p30 protein (2μg / mL~0.238pg / mL); B: when detecting high concentration of p30, the linear regression equation is Y=712.7*X+1385877(3.9ng / mL-250ng / mL), R 2=0.9984; C: When detecting low concentrations of p30, the linear regression equation is Y=971.1*X+53831(30pg / mL-7.8ng / mL), R 2 =0.9999, detection limit is 15pg / mL;

[0045] Figure 9 The linear range and detection limit of p30 protein were determined by chemiluminescence assay based on HRP-catalyzed luminol.

[0046] Among them, A: Detection of 2-fold gradient dilution of p30 protein (2 μg / mL to 7.6 pg / mL); B: When the p30 concentration is 0.9765 ng / mL to 125 ng / ml, the measured chemiluminescence intensity is linearly related to the p30 concentration, and the linear regression equation is Y = 92891*X + 394945 (R 2 =0.9898), the detection limit was 0.488 ng / mL;

[0047] Figure 10 The linear range and detection limit of the chemiluminescence method for ASFV detection based on HiBiT were established;

[0048] Wherein, A: Linear regression equation is Y=1239*X-8983(48.8-25000HAD 50 ), R 2 =0.999; B: When detecting low concentrations of ASFV (48.8~6250HAD 50 ), the linear regression equation is Y=1210*X+45508(R 2 =0.9992), the detection limit was 12.2HAD 50 ;

[0049] Figure 11 HAD for ASFV 50 Linear regression analysis with p30;

[0050] Figure 12 Determination of ASFV p30 content in tissue, blood, nasal, pharyngeal, and anal swab samples from ASFV-infected pigs;

[0051] Among them, A: tissue sample; B: blood sample; C: nasal, pharyngeal and anal swab samples. DETAILED DESCRIPTION

[0052] 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.

[0053] Example 1 Preparation and purification of monoclonal antibodies against ASFVp30

[0054] 1. Expression and purification of ASFVp30 protein

[0055] The p30 gene sequence of ASFV SY18 strain (GenBank: MH766894.1) was synthesized based on codon preference optimization and ligated into the pET-28a vector to construct a recombinant plasmid. The recombinant plasmid (pET-28a-p30) was then transformed into Escherichia coli BL21(DE3) and induced (at an OD value of 0.8) with the addition of 1 mM IPTG (37°C, 8 h). The cells were harvested after centrifugation at 5000 g for 5 minutes and purified using Ni affinity chromatography. The purified p30 protein was then renatured by gradually decreasing the concentrations of urea and imidazole and finally dissolved in tris buffer (20 mM tris, 250 mM NaCl, 10% glycerol).

[0056] The expression and purification results of p30 recombinant protein are shown in Figure 1 As shown, the results showed that the p30 recombinant protein was successfully expressed in Escherichia coli BL21 (DE3) with the expected size, and high-purity p30 recombinant protein was obtained, which could be used to immunize Balb / c mice to prepare monoclonal antibodies.

[0057] 2. Preparation of monoclonal antibodies

[0058] Prokaryotically expressed p30 recombinant protein was renatured and emulsified with an equal volume of Freund's complete adjuvant and then immunized intramuscularly into 8-week-old Balb / c mice. Twenty-one days after the first immunization, mice were immunized three more times with an equal volume of p30 recombinant protein emulsified with Freund's incomplete adjuvant, every two weeks at a dose of 20 μg per mouse. Seven days after the final immunization, 20 μg of p30 recombinant protein was injected intraperitoneally. Three days later, splenocytes from the mice were fused with SP2 / 0 cells. Positive hybridomas were subcloned three times by limiting dilution, yielding 20 monoclonal antibodies. The epitopes recognized by the proteins were then identified, and the monoclonal antibodies 2-1B3G3 and 16-5E7E8, which recognized different epitopes, were selected for subsequent experiments.

[0059] 3. Preparation and purification of ascites

[0060] 0.3-0.5 mL of ascites adjuvant was injected into the peritoneal cavity of 12-week-old female BALB / c mice. 14 days later, hybridoma cells 2-1B3G3 (approximately 1.0×10 6 ) were injected into the mouse peritoneal cavity. 9-10 days after the hybridoma cell injection, the mouse ascites was collected and purified by protein G affinity chromatography. The purification results of 2-1B3G3 are shown in Figure 2As shown, the results show that the monoclonal antibody with high purity (50kDa heavy chain and 25kDa light chain) was obtained, which meets the requirements of subsequent experiments. The concentration of monoclonal antibody 2-1B3G3 was 5.5mg / mL using Bradford assay.

[0061] RNA from hybridoma cell line 2-1B3G3 was extracted using an RNA-easy kit and reverse-transcribed into cDNA. The heavy chain (VH) and light chain (VL) of the antibody were amplified using rapid amplification of cDNA ends (RACE) and ligated into a cloning vector for sequencing. The amino acid sequence of the heavy chain of monoclonal antibody 2-1B3G3 is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain is shown in SEQ ID NO. 2.

[0062] Example 216-5E7E8-Construction, Expression and Purification of HiBiT and LgBiT Expression Plasmids

[0063] 1. Construction of 16-5E7E8-HiBiT and LgBiT

[0064] RNA from hybridoma cell 16-5E7E8 was extracted using an RNA-easy kit and reverse-transcribed into cDNA. The VH and VL fragments of the antibody were amplified using the rapid cDNA end cloning technique 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 sequence 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 was designated 16-5E7E8-HiBiT, and its nucleotide sequence is shown in SEQ ID NO. 3. The 16-5E7E8-HiBiT sequence was inserted into the pcDNA3.1(+) vector via BamHI and XhoI to construct the recombinant plasmid 16-5E7E8-HiBiT-pcDNA3.1.

[0065] The kozak sequence, signal peptide (MRLSVCLLLLTLALCCYRANA), LgBiT, linker (GGSSGG), and 6×His were sequentially synthesized in series, and BamHI and XhoI restriction sites were added to the 5' and 3' ends of the sequence, respectively. The resulting sequence was designated LgBiT, and its nucleotide sequence is shown in SEQ ID NO. 5. The LgBiT sequence was inserted into the pcDNA3.1(+) vector via BamHI and XhoI to construct the recombinant plasmid LgBiT-pcDNA3.1.

[0066] 2. Expression and purification of 16-5E7E8-HiBiT and LgBiT

[0067] The recombinant plasmids 16-5E7E8-HiBiT-pcDNA3.1 and LgBiT-pcDNA3.1 were transfected into Chinese hamster ovary cell lines (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.

[0068] 16-5E7E8-HiBiT was purified by protein G affinity chromatography, and LgBiT was purified by His affinity chromatography. Figure 3 、 Figure 4 The results showed that highly pure 16-5E7E8-HiBiT (50 kDa heavy chain and 25 kDa light chain) and LgBiT proteins (19 kDa) were obtained, and their sizes were consistent with expectations. The amino acid sequences of 16-5E7E8-HiBiT and LgBiT proteins are shown in SEQ ID NOs. 4 and 6, respectively. The concentrations of 16-5E7E8-HiBiT and LgBiT were 1 mg / mL and 200 μg / mL, respectively, as measured by Bradford elution.

[0069] Example 3 Establishment of a double antibody sandwich chemiluminescence method for detecting p30 antigen

[0070] A double-antibody sandwich assay was used to detect ASFV p30, using monoclonal antibody 2-1B3G3 as the coating antibody and 16-5E7E8-HiBiT as the detection antibody. The principle is that 16-5E7E8-HiBiT and LgBiT bind to form the NanoLuc enzyme, which catalyzes the luminescent substrate furimazine to produce a luminescent signal. To improve p30 detection sensitivity, the concentration of LgBiT, the dissolution method and dilution of the luminescent substrate furimazine, the coating antibody concentration, and the detection antibody concentration were optimized.

[0071] 1. Dissolution of Furimazine Compound and Optimization of Its Usage Concentration

[0072] The purified monoclonal antibody 2-1B3G3 was diluted to 2 μg / mL with PBS and added to a white detachable polystyrene 96-well plate (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% v / v proclin 300, 6% v / v horse serum, 0.05% v / v Tween-20 in 0.01 mol / L phosphate buffer, pH 7.2-7.4) was added and blocked at 37°C for 1.5 h. Serum diluent (containing 1% w / w casein, 0.05% v / v Tween-20, 0.1% v / v proclin 300) was then added. 300, 2% w / w sucrose in 0.01 mol / L phosphate buffer) diluted to a concentration of 10 ng / mL p30 antigen, 100 μL / well, at 37°C for 30 min; after washing five times with PBST (0.01 mol / L phosphate buffer containing 0.05% v / v Tween-20, pH 7.2-7.4), serum diluent was added to dilute the concentration to 1 μg / mL 16-5E7E8-HiBiT, react at 37°C for 30 min; after washing five times with PBST, add LgBiT protein (2 μg / mL) diluted 1:100 with PBST at 25 μL / well; dissolve the furimazine compound in a mixed solution of DMSO:H2O with a volume ratio of 4:1 or 1:1 and store at -20°C (storage concentration is 10 mM). When used, dilute it with PBST at 1:15.625, 1:31.25, 1:62.5, 1:125, 1:250, and 1:500 at 25 μL / well; react at room temperature for 10 min, and measure the chemiluminescence value.

[0073] The results are as follows Figure 5 As shown, the optimal dissolution condition of furimazine compound is DMSO:H2O=4:1, and the optimal dilution is 1:250, that is, the concentration is 40 μM.

[0074] 2. Optimization of LgBiT concentration

[0075] Purified monoclonal antibody 2-1B3G3 was diluted to 2 μg / mL with PBS and coated on a chemiluminescent plate (100 μL / well) at 4°C overnight. After drying, 200 μL / well of blocking solution was added and the plate was blocked at 37°C for 1.5 h. Then, p30 antigen (500 ng / mL, 125 ng / mL, 31.25 ng / mL, 7.8125 ng / mL) diluted in serum diluent was added at 100 μL / well and reacted at 37°C for 30 min. After washing five times with PBST, 16-5E7E8-HiBiT was added at a concentration of 1 μg / mL diluted in serum diluent and reacted at 37°C for 30 min. Then, different concentrations of LgBiT protein diluted with PBST were added at 25 μL / well. Finally, 40 μM furimazine compound diluted with PBST was added at 25 μL / well. The plate was reacted at room temperature for 10 min, and the chemiluminescence value was measured.

[0076] The results are as follows Figure 6 As shown, considering the signal-to-noise ratio and economic factors, the optimal dilution of LgBiT is 1:100, that is, the concentration is 2 μg / mL.

[0077] 3. Determination of the coating concentration of monoclonal antibody 2-1B3G3 and the detection concentration of 16-5E7E8-HiBiT

[0078] Based on the determination of the optimal concentration of LgBiT and the optimal dissolving solution and concentration of furimazine, the optimal coating concentration of monoclonal antibody 2-1B3G3 and the optimal detection concentration of 16-5E7E8-HiBiT were screened by the checkerboard titration method. Briefly, purified monoclonal antibody 2-1B3G3 was diluted with PBS to 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, 100 μL / well, and incubated at 4°C overnight. After drying, 200 μL / well blocking buffer was added and the cells were blocked at 37°C for 1.5 h. Then, p30 antigen (937.5 ng / mL, 187.5 ng / mL, 37.5 ng / mL, 7.5 ng / mL, 1.5 ng / mL, 300 pg / mL, and 60 pg / mL) diluted serially in serum diluent was added and reacted at 37°C for 30 min. The cells were washed five times with PBST, and enzyme-labeled monoclonal antibody 16-5E7E8-HiBiT (2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL) diluted serially in serum diluent was added and reacted at 37°C for 30 min. After washing with PBST five times, LgBiT diluted with PBST at a concentration of 2 μg / mL and furimazine compound diluted with PBST at a concentration of 40 μM were added, 25 μL / well each, reacted at room temperature for 10 minutes, and the chemiluminescence value was detected.

[0079] The results showed that, considering the detection linear range and economic factors, the optimal coating concentration of monoclonal antibody 2-1B3G3 was 2 μg / mL, and the optimal detection concentration of 16-5E7E8-HiBiT enzyme-labeled antibody was 0.5 μg / mL.

[0080] 4. Determination of the optimal buffer and reaction time for diluting LgBiT and furimazine

[0081] After determining the optimal reaction conditions, the optimal buffer for diluting LgBiT and furimazine was screened from seven buffers, and the optimal reaction time was determined. Briefly, the chemiluminescent plate was coated with 2 μg / mL monoclonal antibody 2-1B3G3, 100 μL / well, at 4°C overnight; after drying, 200 μL / well blocking solution was added, and the plate was blocked at 37°C for 1.5 hours; then 100 ng / mL p30 antigen was added and the plate was reacted at 37°C for 30 minutes; after washing 5 times with PBST, 0.5 μg / mL 16-5E7E8-HiBiT was added, 100 μl / well, and the plate was reacted at 37°C for 30 minutes; after washing 5 times with PBST, 50 μL / well PBS, PBST, CBS, TBST, Tris-HCl (pH 8.0), Tris-HCl + 1 mM EDTA (pH 8.0), and Tris-HCl + 5 mM EDTA (pH 8.0) LgBiT (final concentration of 1 μg / mL) and furimazine (final concentration of 20 μM) were diluted in seven buffers and then incubated at room temperature for 5, 10, 20, 30, and 60 minutes before reading to identify the optimal buffer for diluting LgBiT and furimazine and determine the optimal reaction time.

[0082] The results are as follows Figure 7 As shown, all seven buffers can be used to dilute LgBiT and furimazine. TBST dilution of LgBiT and furimazine yielded the highest chemiluminescence values, but also the greatest error. For ease of use, PBST was selected for subsequent detection in this study. After adding LgBiT and furimazine, chemiluminescence values ​​initially increased (from 5 to 10 minutes) and then decreased (after 10 minutes), thus selecting a 10-minute reaction time as the optimal reaction time.

[0083] Example 4 Determination of linear range and detection limit

[0084] After determining the optimal coating concentration of the monoclonal antibody, a batch of chemiluminescent plates were coated according to the method of Example 3 for subsequent detection. Specific steps: Purified monoclonal antibody 2-1B3G3 was diluted to 2 μg / mL with PBS, and 100 μL / well of the chemiluminescent plate was coated at 4°C overnight; after drying, 200 μL / well of blocking solution (1×PBST containing 1% w / w BSA, 2% w / w sucrose, 0.1% v / v proclin 300, and 6% v / v horse serum) was added and blocked at 37°C for 1.5 hours; after drying at room temperature for 2 hours, the plates were vacuum-sealed in aluminum foil bags and stored at 4°C.

[0085] 1. Linear range and detection limit of p30 protein

[0086] The coated chemiluminescent plate was removed and returned to room temperature. The p30 antigen was diluted 2-fold with serum diluent (2μg / mL to 0.238pg / mL), 100μL / well, added to the chemiluminescent plate, and reacted at 37°C for 30 minutes; after washing five times with PBST, 0.5μg / mL 16-5E7E8-HiBiT diluted with serum diluent was added, and the plate was reacted at 37°C for 30 minutes; after washing five times with PBST, 2μg / mL LgBiT diluted with PBST and 40μM furimazine compound diluted with PBST were added, 25μL / well each, and the plate was reacted at room temperature for 10 minutes. The chemiluminescence value was detected to determine the linear range and detection limit of this method for detecting p30 protein.

[0087] In addition, the linear range and detection limit of p30 protein were compared with those of the HRP-catalyzed luminol method. Specifically, the coated chemiluminescent plate was returned to room temperature. A two-fold serial dilution of p30 antigen (2 μg / mL to 7.6 pg / mL) was added to the plate at 100 μL / well and incubated at 37°C for 30 minutes. After washing five times with PBST, 100 μL / well of 0.5 μg / mL 16-5E7E8-HRP was added and incubated at 37°C for 30 minutes. After washing five times with PBST, 100 μL of a 1:1 mixture of chemiluminescent solution A and solution B was added. After 5 minutes, the chemiluminescence value was measured using a chemiluminescence detector.

[0088] The linear range of the chemiluminescence method for detecting p30 protein based on HiBiT is 30pg / mL to 250ng / mL ( Figure 8 A), indicating that this method has a wide linear range for detecting p30 antigen, high sensitivity, and is not interfered with by other stray light. In order to ensure the accuracy of the test results, when detecting high concentrations of p30, the linear regression equation is Y = 712.7*X + 1385877 (3.9ng / mL-250ng / mL), R 2 =0.9984( Figure 8B); When detecting low concentrations of p30, the linear regression equation is Y = 971.1*X + 53831 (30pg / mL - 7.8ng / mL), R 2 =0.9999, the detection limit is 15pg / mL( Figure 8 C). The linear regression equation for the detection of p30 protein by chemiluminescence method based on HRP catalyzed luminol is Y=92891*X+394945(R 2 =0.9898), the linear range of detection was 0.9765ng / mL~125ng / mL, and the detection limit was 0.488ng / mL ( Figure 9 ), and its linear range was much smaller than that of the HiBiT chemiluminescence method, which further demonstrated that the chemiluminescence method based on HiBiT had high sensitivity and a wide linear range.

[0089] 2. Linear range and detection limit for ASFV detection

[0090] In order to verify that the method of the present invention can be used for the detection of ASFV, we inactivated 10 5.4 HAD 50 ASFV (65°C, 30 min) was diluted 2-fold from 1:10 to 1:163840, and then detected using a coated chemiluminescent plate, 100 μL / well, and reacted at 37°C for 30 min; after washing 5 times with PBST, 0.5 μg / mL16-5E7E8-HiBiT was added, 100 μl / well, and reacted at 37°C for 30 min; after washing 5 times with PBST, LgBiT diluted with PBST at a concentration of 2 μg / mL and furimazine compound diluted with PBST at a concentration of 40 μM were added, 25 μL / well each, and reacted at room temperature for 10 min. The chemiluminescence value was detected to determine the linear range and detection limit of ASFV virus detection.

[0091] The results are as follows Figure 10 As shown, when ASFV is 48.8~25000HAD 50 The measured chemiluminescence intensity was linearly related to ASFV, and the linear regression equation was Y=1239*X-8983(R 2 =0.999)( Figure 10 A) To detect low HAD 50 The results are more accurate, and the detection of 48.8~6250 HAD is established 50 The standard curve of Y=1210*X+45508(R 2 =0.9992), the detection limit was 12.2HAD50( Figure 10 B).

[0092] In addition, we will HAD ASFV 50A linear regression analysis was performed with p30, and the linear regression equation was Y = 1.277*X-83.38 (R 2 =0.9989)( Figure 11 ).

[0093] Example 5 Determination of ASFV p30 content in tissue, blood, nasal, pharyngeal and anal swab samples of ASFV-infected pigs

[0094] In order to verify the feasibility of the method of the present invention in clinical practice, we quantitatively detected the ASFVp30 content in tissue, blood, and nasal, pharyngeal, and anal swab samples of ASFV-infected pigs.

[0095] First, pigs were infected with ASFV in a biosafety level 3 animal laboratory. Seven days after infection, nasal, pharyngeal, and anal swabs were collected and placed in 1 mL of PBS. Blood, as well as samples from the heart, liver, spleen, lungs, kidneys, submandibular lymph nodes, mesenteric lymph nodes, and portal lymph nodes were collected. The blood was then centrifuged in a biosafety level 3 laboratory to separate the serum. Approximately 1 g of tissue was homogenized from the heart, liver, spleen, lungs, kidneys, submandibular lymph nodes, mesenteric lymph nodes, and portal lymph nodes, placed at 4°C overnight, and centrifuged to obtain the supernatant. Finally, the nasal, pharyngeal, and anal swabs, serum, and tissue supernatant were inactivated in a 65°C water bath for 30 minutes.

[0096] Serum and tissue sample supernatants were diluted 1:5 by volume with serum diluent, while nasal, pharyngeal, and anal swab samples were diluted 1:2 by volume and added to a U-shaped plate. Meanwhile, p30 protein was serially diluted 4-fold in serum diluent (200 ng / mL to 48.8 pg / mL) as a standard in the U-shaped plate. The solution in the U-shaped plate was then transferred to a chemiluminescent plate coated with monoclonal antibody 2-1B3G3 and incubated at 37°C for 30 minutes. After washing five times with PBST, 100 μL / well of the fusion protein 16-5E7E8-HiBiT, diluted in serum diluent to a concentration of 0.5 μg / mL, was added and incubated at 37°C for 30 minutes. After washing five times with PBST, 25 μL / well of LgBiT, diluted in PBST to a concentration of 2 μg / mL, and 40 μM furimazine, diluted in PBST, were added. The plates were incubated at room temperature for 10 minutes, and chemiluminescence was measured. A linear regression equation for detecting high and low concentrations of p30 was established using p30 protein standards to calculate the ASFVp30 content in diluted tissue samples, blood, and swabs. Finally, the ASFVp30 content in the sample was obtained by multiplying the equation by the dilution factor.

[0097] The results are as follows Figure 12 As shown in the results, among the tissue samples, the ASFVp30 content was highest in the lung, spleen, and liver, followed by the kidney and submandibular lymph nodes, and the lowest in the mesenteric lymph nodes, portal lymph nodes, and heart ( Figure 12 ASFVp30 can be detected in the serum of ASFV-infected pigs, but not in the negative serum ( Figure 12 B) In the testing of nasal, pharyngeal, and anal swabs, the detection rate of nasal swabs was 80% (8 / 10), the detection rate of pharyngeal swabs was 40% (4 / 10), and the detection rate of anal swabs was 20% (2 / 10), indicating that the detection rate of nasal swabs was the highest.

[0098] In summary, this method can be used for the quantitative detection of ASFV p30 in tissues, blood, and nasal swabs of ASFV-infected pigs.

[0099] Example 6 Assembly and Use of a Kit for Quantitative Detection of African Swine Fever Virus Based on Chemiluminescence

[0100] The kit comprises:

[0101] 1. A chemiluminescent plate coated with the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3, wherein the heavy chain amino acid sequence of the monoclonal antibody 2-1B3G3 is shown in SEQ ID NO. 1, and the light chain amino acid sequence is shown in SEQ ID NO. 2;

[0102] 2. Fusion protein 16-5E7E8-HiBiT, the amino acid sequence of which is shown in SEQ ID NO.4;

[0103] 3. LgBiT protein, the amino acid sequence of which is shown in SEQ ID NO.6;

[0104] 4. Furimazine compound: Dissolve furimazine in a 4:1 DMSO:H2O mixture and store at -20°C (storage concentration 10 mM).

[0105] 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;

[0106] 6. PBST washing solution: 0.01 mol / L phosphate buffer containing 0.05% v / v Tween-20, pH 7.2-7.4;

[0107] 7. p30 protein standard.

[0108] Instructions for use of the kit for quantitative detection of African swine fever virus based on chemiluminescence:

[0109] 1) Room temperature equilibration: The test kit taken out from 4°C needs to be restored to room temperature before use;

[0110] 2) Sample dilution: In a U-shaped dilution plate, dilute the sample to be tested with serum diluent and perform a 4-fold serial dilution of the p30 protein standard, with 2 wells for each dilution;

[0111] 3) Sample addition: Transfer the samples and serially diluted standards from the dilution plate in step 2) to a chemiluminescent plate coated with the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 in order, 100 μL / well, and react at 37°C for 30 min;

[0112] 4) Washing: Discard the reaction solution, add 300 μL of PBST to each well, wash five times, and pat dry for the last time;

[0113] 5) Detection Antibody: Dilute the fusion protein 16-5E7E8-HiBiT to 0.5 μg / mL with serum diluent, add 100 μl / well, and react at 37°C for 30 min.

[0114] 6) Washing: Discard the reaction solution, add 300 μL of PBST to each well, wash five times, and pat dry for the last time;

[0115] 7) Chemiluminescent reaction: Dilute LgBiT to 2 μg / mL with PBST, add 25 μL / well, then add 40 μM furimazine compound, 25 μL / well, shake and mix, and react at room temperature for 10 min;

[0116] 8) Detection: After 10 minutes, the chemiluminescence (CLIA) value was detected using a chemiluminescence detector;

[0117] 9) Establishing a regression equation: Using p30 protein standards, establish a linear regression equation for detecting high and low concentrations of p30;

[0118] 10) ASFVp30 content in samples: Calculate the ASFVp30 content in diluted tissue samples, blood, and swabs using a linear regression equation, and then multiply by the dilution factor to obtain the ASFVp30 content in the sample;

[0119] 11) The test is valid if the chemiluminescence value of the 200 ng / mL p30 standard is ≥ 100,000,000.

Claims

1. A kit for quantitative detection of African swine fever virus based on chemiluminescence, characterized in that: The kit includes a chemiluminescent plate coated with the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3, 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 of the anti-African swine fever virus p30 protein monoclonal antibody 16-5E7E8 and the heavy chain of 16-5E7E8, and the amino acid sequence of the fusion protein 16-5E7E8-HiBiT is shown in SEQ ID NO.

4.

2. The kit according to claim 1, wherein The kit also includes serum diluent, PBST washing solution and p30 protein standard.

3. The kit according to claim 2, wherein 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; the p30 protein standard is obtained by transferring the p30 gene sequence of the African swine fever virus SY18 strain into a prokaryotic expression system, expressing it, and purifying it.

4. The kit according to claim 1, wherein The heavy chain amino acid sequence of the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.

2.

5. The kit according to claim 1, wherein The preparation steps of the chemiluminescent plate coated with the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 are as follows: the purified anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 is diluted to 2 μg / mL with PBS, added to a white detachable polystyrene 96-well plate at 100 μL / well, and coated overnight at 4°C; after drying, 200 μL / well blocking solution is added, blocking at 37°C for 1.5 hours, and then patting dry; after drying at room temperature for 2 hours, vacuum-sealing with an aluminum foil bag, and storing at 4°C.

6. The kit according to claim 5, wherein The blocking solution is a phosphate buffer solution with a pH of 7.2-7.4 and a concentration of 0.01 mol / L, containing 1% w / w BSA, 2% w / w sucrose, 0.1% v / v proclin 300, 6% v / v horse serum, and 0.05% v / v Tween-20.

7. The 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.

6.

8. The kit according to any one of claims 1 to 7, wherein When the kit is used for quantitative detection of African swine fever virus, the following steps are followed: 1) Room temperature equilibration: The test kit taken out from 4°C needs to be restored to room temperature before use; 2) Sample dilution: In a U-shaped dilution plate, dilute the sample to be tested with serum diluent and perform a 4-fold serial dilution of the p30 protein standard, with 2 wells for each dilution. 3) Sample addition: Transfer the samples and serially diluted standards from the dilution plate in step 2) to a chemiluminescent plate coated with the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 in sequence, 100 μL / well, and react at 37°C for 30 min. 4) Washing: Discard the reaction solution, add 300 μL of PBST to each well, wash five times, and pat dry for the final wash. 5) Detection Antibody: Dilute the fusion protein 16-5E7E8-HiBiT to 0.5 μg / mL with serum diluent, add 100 μl / well, and incubate at 37°C for 30 min. 6) Washing: Discard the reaction solution, add 300 μL of PBST to each well, wash five times, and pat dry for the final wash. 7) Chemiluminescent reaction: Dilute LgBiT protein to 2 μg / mL with PBST, add 25 μL / well, then add 40 μM furimazine compound, 25 μL / well, shake well, and react at room temperature for 10 min. 8) Detection: After 10 minutes, use a chemiluminescence detector to detect the chemiluminescence value; 9) Establish a regression equation: Use p30 protein standards to establish a linear regression equation for detecting high and low concentrations of p30; 10) ASFV p30 content in samples: Calculate the ASFV p30 content in diluted tissue samples, blood, and swabs using a linear regression equation. Multiply by the dilution factor to obtain the ASFV p30 content in the sample. 11) The chemiluminescence value of the 200 ng / mL p30 standard should be ≥100,000,000.

9. The kit according to claim 8, wherein The gradient concentration of the p30 protein standard is 200 ng / mL, 50 ng / mL, 12.5 ng / mL, 3.125 ng / mL, 781 pg / mL, 195 pg / mL, 48.8 pg / mL and 0 pg / mL; the furimazine compound is dissolved in a mixed solution of DMSO and H2O in a volume ratio of 4:1 and stored at -20°C. The storage concentration is 10 mM and the concentration is diluted to 40 μM with PBST when used.

10. Use of the kit according to any one of claims 1 to 9 in the preparation of a reagent for detecting African swine fever virus.

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