Kit for quantitatively detecting African swine fever virus based on chemiluminescence method and application thereof

Abstract

The invention discloses a kit for quantitatively detecting African swine fever virus based on a chemiluminescence method and application of the kit. The invention belongs to the technical field of virus detection. The kit comprises a chemiluminiscence plate, a fusion protein 16-5E7E8-HiBiT, an LgBiT and a furimazine compound, wherein the chemiluminiscence plate is coated with a monoclonal antibody 2-1B3G3 for resisting the African swine fever virus p30 protein, and the fusion protein 16-5E7E8-HiBiT, the LgBiT and the furimazine compound. Wherein the fusion protein 16-5E7E8-HiBiT is obtained by sequentially connecting a light chain of an anti-African swine fever virus p30 protein monoclonal antibody 16-5E7E8, a linker, HiBiT, P2A and a 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 as shown in SEQ ID NO. 4. The kit disclosed by the invention has high sensitivity and a wide linear range, the detection limit of the p30 protein is 15pg / mL, and the detection limit of the ASFV is 12.2 HAD50. In addition, the kit can quantitatively detect the content of ASFVp30 in tissues, blood and nasal swabs of ASFV infected pigs, and has a good application prospect 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 method 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 African swine fever virus (ASFV). The mortality rate of pigs infected with hyperacute and acute ASFV is nearly 100%, which seriously affects the development of the pig industry and the international trade of pig products. ASFV is a large linear double-stranded DNA arbovirus with a genome length of 170-190kb, encoding more than 150 proteins, which play an important role in virus assembly, virus replication, virus-host interaction and immune escape. Studies have found that the ASFVp30 structural protein encoded by the CP204L gene is located in the inner membrane of the viral envelope and can be secreted and expressed in large quantities in the initial stage of infection, usually produced 2-4h after infection, and continuously expressed throughout the infection period. p30 is an important structural protein of ASFV, which can induce animals to produce high levels of antibodies and has been widely used in ASFV serological detection. In addition, p30 can induce neutralizing antibodies in animals and plays a key role in virus endocytosis, making it an important structural protein of ASFV. These characteristics make p30 an ideal antigen for serological diagnosis and immunological detection.

[0003] Ancient people discovered luminous animals such as mollusks, purple jellyfish, and fireflies very early, and described them in depth. In 1885, DuBois first discovered the reaction between luciferase and luciferin. It was not until the late 1940s that Green and McElroy extracted and purified luciferase FLuc from fireflies. FLuc has a molecular weight of 61kDa and uses ATP to catalyze the substrate luciferin (D-luciferin). After that, scientists conducted in-depth research on the luminescence principle of firefly luciferase FLuc and applied it to the biomedical field. In the 1970s, MJCormier purified Renilla luciferase (RLuc) from Renilla serrata. RLuc has a molecular weight of 36kDa and catalyzes coelenterazine to produce chemiluminescence. In the past 30 years, scientists have isolated luciferase GLuc (20kDa) from the marine copepod Gaussiaprinceps and OLuc (19kDa) from the deep-sea shrimp Oplophorus gracilirostris. The catalytic substrates of GLuc and OLuc are both coelenterazine. OLuc is composed of two heterodimeric subunits of 35kDa and 19kDa, of which the 19kDa subunit (OLuc-19) plays a catalytic role, but its expression level is low and unstable. Therefore, Hall et al. conducted three rounds of mutagenesis on OLuc to produce NanoLuc (NLuc). The chemiluminescence intensity of furimazine produced by NLuc catalyzed by NLuc is 150 times that of FLuc and RLuc, and it has the advantages of high stability, small molecular weight (19.1KDa) and long luminescence time. It has been widely used in protein-protein and protein-ligand interactions, gene regulation and cell signaling pathways, protein stability monitoring, bioluminescence energy resonance 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 with 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 object 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 discloses a kit for quantitatively detecting African swine fever virus based on chemiluminescence method, comprising 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 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.

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

[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: dilute the purified anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 to 2 μg / mL with PBS, add 100 μL / well to a white detachable polystyrene 96-well plate, and coat overnight at 4°C; after drying, add 200 μL / well blocking solution, block at 37°C for 1.5 hours, and then pat dry; after drying at room temperature for 2 hours, vacuum package with an aluminum foil bag, and store at 4°C.

[0012] Preferably, the blocking solution is a phosphate buffer with a pH of 7.2 to 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] 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 followed:

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

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

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

[0018] 4) Washing: discard the reaction solution, add 300 μL PBST to each well, wash 5 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, 100 μl / well, react at 37°C for 30 min;

[0020] 6) Washing: discard the reaction solution, add 300 μL PBST to each well, wash 5 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, 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, a linear regression equation for detecting high and low concentrations of p30 was established;

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

[0025] 11) The conditions for the test to be valid: The chemiluminescence value of the 200ng / mL p30 standard should be ≥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, with a storage concentration of 10 mM, and is diluted with PBST to a concentration of 40 μM 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 that can be used for p30 detection, and expresses the antibody sequence on CHO after connecting it with HiBiT, thereby avoiding the batch-to-batch differences and animal welfare issues of traditional mouse monoclonal antibodies expressed through intraperitoneal immunization of mice; in addition, HiBiT is only 11aa in size, and connecting it to the end of the monoclonal antibody light chain through a linker will not affect the structure of the antibody, nor will it cause steric hindrance to the antibody's recognition.

[0030] 2. Traditional chemical coupling labeling of antibodies is disordered, has uncertainty in coupling position, and has differences in coupling efficiency between different batches. However, the present invention connects HiBiT to the end of the light chain of the monoclonal antibody through a linker, and the direction and position are deterministic. The light chain end of each monoclonal antibody carries HiBiT.

[0031] 3. The linear range of the detection of prokaryotic expression of p30 antigen using the kit of the present invention 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 is 12.2HAD 50 , indicating that the kit has high sensitivity and a wide linear range. In addition, the kit can quantitatively detect the ASFVp30 content in tissues, blood and nasal swabs of ASFV-infected pigs. Therefore, the kit of the present invention has a good application prospect in ASFV pathogen detection and provides a technical means for the comprehensive prevention and treatment 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 SDS-PAGE analysis of the purified monoclonal antibody 2-1B3G3;

[0035] Among them, 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] Among them, M: marker, 1: CHO cell supernatant without LgBiT plasmid transfection, 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 concentration 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] Fig. 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 (2μg / mL~7.6pg / mL); B: When the p30 concentration is 0.9765ng / mL~125ng / 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 0.488ng / mL;

[0047] Fig.10 The linear range and detection limit of ASFV detection by chemiluminescence method based on HiBiT;

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

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

[0050] Fig.12 Determination of ASFVp30 content in tissue, blood, nasal, pharyngeal and anal swab samples of 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 only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified, the reaction is carried out according to conventional conditions or conditions recommended by the manufacturer.

[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 optimized and synthesized according to codon preference, and connected to the pET-28a vector to construct a recombinant plasmid, and then the recombinant plasmid (pET-28a-p30) was transferred into Escherichia coli BL21 (DE3). When the OD value was 0.8, a final concentration of 1mM IPTG was added for induction (37°C, 8h). The cells were harvested after centrifugation at 5000g for 5 minutes and purified using Ni affinity chromatography. Then, the purified p30 protein was renatured by gradually reducing the concentrations of urea and imidazole, and finally dissolved in tris buffer (20mM tris, 250mM 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), the size was as expected, and high-purity p30 recombinant protein was obtained, which can be used to immunize Balb / c mice to prepare monoclonal antibodies.

[0057] 2. Preparation of monoclonal antibodies

[0058] The p30 recombinant protein expressed in prokaryotes was renatured and emulsified with an equal volume of Freund's complete adjuvant and then 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 2 weeks, with an immunization dose of 20μg / mouse. 7 days after the last immunization, 20μg p30 recombinant protein was injected intraperitoneally. 3 days later, the spleen cells of the mice were taken and fused with SP2 / 0 cells respectively, and the positive hybridomas were subcloned three times by limiting dilution method to obtain 20 monoclonal antibodies, and the epitopes recognized by the proteins were identified. Finally, monoclonal antibodies 2-1B3G3 and 16-5E7E8 that recognize 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 injection of hybridoma cells, 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 a high-purity monoclonal antibody (50 kDa heavy chain and 25 kDa light chain) is obtained, which meets the requirements of subsequent experiments. The concentration of monoclonal antibody 2-1B3G3 is 5.5 mg / mL using Bradford assay.

[0061] RNA of hybridoma cell 2-1B3G3 was extracted using RNA-easy kit and reverse transcribed into cDNA, and then the heavy chain (VH) and light chain VL (VH) of the antibody were amplified using rapid amplification of cDNA ends (RACE) and connected to the 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 of hybridoma cell 16-5E7E8 was extracted using RNA-easy kit and reverse transcribed into cDNA. Then, VH and VL of the antibody were amplified using cDNA end rapid cloning technology and connected to a cloning vector for sequencing. The kozak sequence, light chain of 16-5E7E8, linker (GGSSGG), HiBiT, P2A and heavy chain sequence of 16-5E7E8 were connected and synthesized in sequence, and BamHI and Xho I restriction sites were added to the 5' end and 3' end of the sequence, respectively. The obtained sequence was recorded as 16-5E7E8-HiBiT, and its nucleotide sequence is shown in SEQ ID NO.3. The above sequence 16-5E7E8-HiBiT was inserted into the pcDNA3.1(+) vector through BamHI and Xho I to construct the recombinant plasmid 16-5E7E8-HiBiT-pcDNA3.1.

[0065] After the kozak sequence, signal peptide (MRLSVCLLLLTLALCCYRANA), LgBiT, linker (GGSSGG), and 6×His were sequentially synthesized in series, BamHI and XhoI restriction sites were added to the 5' and 3' ends of the sequence, respectively, and the resulting sequence was recorded as LgBiT, and its nucleotide sequence is shown in SEQ ID NO. 5. The above sequence LgBiT was inserted into the pcDNA3.1(+) vector through 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 As shown, the results show that 16-5E7E8-HiBiT (50 kDa heavy chain and 25 kDa light chain) and LgBiT proteins (19 KDa) with high purity are obtained, and the size is consistent with the expected size. The amino acid sequences of 16-5E7E8-HiBiT and LgBiT proteins are shown in SEQ ID NO. 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.

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

[0070] The monoclonal antibody 2-1B3G3 was used as the coating antibody and 16-5E7E8-HiBiT was used as the detection antibody to detect ASFV p30 using the double antibody sandwich method. The principle is that 16-5E7E8-HiBiT and LgBiT can combine to form NanoLuc enzyme, catalyze the luminescent substrate furimazine, and generate luminescent signals. In order to improve the detection sensitivity of p30, the concentration of LgBiT, the dissolution method of the luminescent substrate furimazine, the concentration and diluent used, the coating antibody concentration, and the detection antibody concentration were optimized.

[0071] 1. Dissolution of Furimazine Compounds and Optimization of Their Usage Concentration

[0072] The purified monoclonal antibody 2-1B3G3 was diluted to 2 μg / mL with PBS, added to a white detachable polystyrene 96-well plate (100 μL / well), and incubated at 4°C overnight; after drying, 200 μL / well 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; then serum diluent (containing 1% w / w casein, 0.05% v / v Tween-20, 0.1% v / v proclin 300, 6% v / v horse serum, 0.05% v / v Tween-20, 0.1% v / v proclin 300) was 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, react at 37°C for 30 min; after washing 5 times with PBST (0.01 mol / L phosphate buffer containing 0.05% v / v Tween-20, pH 7.2-7.4), add serum diluent to dilute to a concentration of 1 μg / mL 16-5E7E8-HiBiT, react at 37°C for 30 min; after washing 5 times with PBST, add LgBiT protein (2μg / mL) diluted 1:100 with PBST, 25μL / 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 10mM). When used, dilute it with PBST at 1:15.625, 1:31.25, 1:62.5, 1:125, 1:250, 1:500, 25μL / 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] The purified monoclonal antibody 2-1B3G3 was diluted to 2 μg / mL with PBS, coated on the chemiluminescent plate (100 μL / well), and incubated at 4°C overnight; after drying, 200 μL / well 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 5 times with PBST, 16-5E7E8-HiBiT diluted with serum diluent at a concentration of 1 μg / mL was added, and reacted at 37°C for 30 min; then different concentrations of LgBiT protein diluted with PBST were added at 25 μL / well; and furimazine compound diluted with PBST at a concentration of 40 μM 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 2μg / mL.

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

[0078] After determining 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, the 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 solution 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 in serum diluent was added, and the cells were reacted at 37°C for 30 min; the cells were washed with PBST for 5 times, 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 in serum diluent was added, and the cells were reacted at 37°C for 30 min. After washing with PBST for 5 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 respectively, reacted at room temperature for 10 min, 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 above 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 2μg / mL monoclonal antibody 2-1B3G3, 100μL / well, overnight at 4℃; after drying, 200μL / well blocking solution was added, and the plate was blocked at 37℃ for 1.5h; then 100ng / mL p30 antigen was added, and the plate was reacted at 37℃ for 30min; after washing 5 times with PBST, 0.5μg / mL 16-5E7E8-HiBiT was added, 100μl / well, and the plate was reacted at 37℃ for 30min; after washing 5 times with PBST, 50μL / well PBS, PBST, CBS, TBST, Tris-HCl (pH 8.0), Tris-HCl+1mM EDTA (pH 8.0), and Tris-Hcl+5mM EDTA (pH 8.0) LgBiT (final concentration of 1 μg / mL) and furimazine compound (final concentration of 20 μM) diluted with seven buffers were then placed at room temperature for 5 min, 10 min, 20 min, 30 min, and 60 min, and then read to screen out the best buffer for diluting LgBiT and furimazine and determine the optimal reaction time.

[0082] The results are as follows Figure 7 As shown in the figure, all seven buffers can be used to dilute LgBiT and furimazine. Among them, the chemiluminescence value of LgBiT and furimazine diluted with TBST is the highest, but its error is also the largest. In view of the convenience of use, this study selected PBST for subsequent detection. After adding LgBiT and furimazine, the chemiluminescence value first increased (from 5min to 10min) and then decreased (after 10min), so the optimal reaction time was selected as 10min.

[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: the purified monoclonal antibody 2-1B3G3 was diluted to 2 μg / mL with PBS, 100 μL / well was coated on the chemiluminescent plate, and it was kept at 4°C overnight; after drying, 200 μL / well 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 it was blocked at 37°C for 1.5h; after drying at room temperature for 2h, it was vacuum-packed with an aluminum foil bag and stored at 4°C.

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

[0086] The coated chemiluminescent plate was taken out and returned to room temperature. The p30 antigen was diluted 2-fold with serum diluent (2μg / mL~0.238pg / mL), 100μL / well, added to the chemiluminescent plate, and reacted at 37°C for 30 minutes; after washing 5 times with PBST, 0.5μg / mL 16-5E7E8-HiBiT diluted with serum diluent was added, and reacted at 37°C for 30 minutes; after washing 5 times with PBST, LgBiT diluted with PBST at a concentration of 2μg / mL and furimazine compound diluted with PBST at a concentration of 40μM were added, 25μL / well each, and 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 the chemical method of HRP catalyzing luminol. Specific steps: restore the coated chemiluminescent plate to room temperature, dilute the p30 antigen 2-fold (2μg / mL~7.6pg / mL), 100μL / well, add to the chemiluminescent plate, react at 37℃ for 30min; after washing 5 times with PBST, add 0.5μg / mL 16-5E7E8-HRP, 100μl / well, react at 37℃ for 30min; after washing 5 times with PBST, add 100μL of 1:1 mixed chemiluminescent A solution and B solution, and detect the chemiluminescent value with a chemiluminescent detector after 5min.

[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 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 concentration 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( Figure 8 C). The linear regression equation for detecting p30 protein based on HRP-catalyzed luminol chemiluminescence 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 ( Fig. 9 ), and its linear range is much smaller than that of HiBiT chemiluminescence method, which further indicates that the chemiluminescence method based on HiBiT has high sensitivity and 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 with 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 Fig.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)( Fig.10 A) To detect low HAD 50 The results are more accurate and the detection of 48.8~6250HAD is established 50 The standard curve is Y = 1210*X + 45508 (R 2 =0.9992), the detection limit is 12.2HAD50( Fig.10 B).

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

[0093] Example 5 Determination of ASFVp30 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, and blood and heart, liver, spleen, lung, kidney, submandibular lymph nodes, mesenteric lymph nodes, and hilar lymph nodes were collected. Then, the blood was centrifuged in a biosafety level 3 laboratory to separate the serum; the heart, liver, spleen, lung, kidney, submandibular lymph nodes, mesenteric lymph nodes, and hilar lymph nodes (about 1 g) were homogenized and placed at 4°C overnight before centrifugation to obtain the supernatant. Finally, the nasal, pharyngeal, and anal swabs, serum, and tissue supernatant were placed in a 65°C water bath for inactivation (30 min).

[0096] The serum and tissue sample supernatants were diluted with serum diluent at a volume ratio of 1:5, and the nasal, pharyngeal, and anal swab samples were diluted at a volume ratio of 1:2 and added to the U-shaped plate; at the same time, the p30 protein was diluted 4 times with serum diluent in the U-shaped plate (200ng / mL~48.8pg / ml) as a standard. Then the liquid in the U-shaped plate was moved to the chemiluminescent plate coated with monoclonal antibody 2-1B3G3 and reacted at 37℃ for 30min. After washing 5 times with PBST, the fusion protein 16-5E7E8-HiBiT at a concentration of 0.5μg / mL diluted with serum diluent was added, 100μL / well, and incubated at 37℃ for 30min. After washing 5 times with PBST, LgBiT at a concentration of 2μg / mL diluted with PBST and furimazine compound at a concentration of 40μM diluted with PBST were added, 25μL / well each, reacted at room temperature for 10min, and the chemiluminescence value was measured. The p30 protein standard was used to establish a linear regression equation for detecting high and low concentrations of p30, which was used to calculate the ASFVp30 content in diluted tissue samples, blood, and swabs. Finally, the ASFVp30 content in the sample was obtained by multiplying it by the dilution factor.

[0097] The results are as follows Fig.12 As shown in the figure, 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 ( Fig.12 ASFVp30 can be detected in the serum of ASFV-infected pigs, but not in the negative serum ( Fig.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 the furimazine compound in a mixed solution of DMSO:H2O with a volume ratio of 4:1 and store at -20°C (storage concentration is 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 proclin300, 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] Method for using the kit for quantitative detection of African swine fever virus based on chemiluminescence:

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

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

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

[0112] 4) Washing: discard the reaction solution, add 300 μL PBST to each well, wash 5 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, 100 μl / well, react at 37°C for 30 min;

[0114] 6) Washing: discard the reaction solution, add 300 μL PBST to each well, wash 5 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, 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, a linear regression equation for detecting high and low concentrations of p30 was established;

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

[0119] 11) The conditions for the test to be valid: The chemiluminescence value of the 200ng / mL p30 standard should be ≥100,000,000.

Claims

1. A kit for quantitatively detecting African swine fever virus (ASFV) based on chemiluminescence, characterized in that: The kit comprises 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 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, characterized in that The kit also includes serum diluent, PBST washing solution and p30 protein standard.

3. The kit according to claim 2, characterized in that The serum diluent is a phosphate buffer containing 1% w / w casein, 0.05% v / v Tween-20, 0.1% v / v proclin 300, 2% w / w sucrose, and 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 and purifying.

4. The kit according to claim 1, characterized in that 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, characterized in that The steps for preparing the chemiluminescent plate coated with the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 are as follows: diluting the purified anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3 to 2 μg / mL with PBS, adding 100 μL / well to a white detachable polystyrene 96-well plate, and coating 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 h, the samples were vacuum-packed in aluminum foil bags and stored at 4 °C.

6. The kit according to claim 5, characterized in that 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, characterized in that 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.

6.

8. The kit according to any one of claims 1 to 7, characterized in that When the kit is used for quantitative detection of African swine fever virus, the following steps are followed: 1) Room temperature balance: The test kit taken out from 4°C needs to be restored to room temperature for use; 2) Sample dilution: In a U-shaped dilution plate, dilute the sample to be tested with serum diluent, and dilute the p30 protein standard in a 4-fold gradient, with 2 wells for each dilution; 3) Sample addition: transfer the samples in the dilution plate of step 2) and the serially diluted standards in order to the chemiluminescent plate coated with the anti-African swine fever virus p30 protein monoclonal antibody 2-1B3G3, 100 μL / well, and react at 37°C for 30 min; 4) Washing: discard the reaction solution, add 300 μL PBST to each well, wash 5 times, and pat dry for the last time; 5) Detection antibody: dilute the fusion protein 16-5E7E8-HiBiT to 0.5 μg / mL with serum diluent, 100 μl / well, react at 37°C for 30 min; 6) Washing: discard the reaction solution, add 300 μL PBST to each well, wash 5 times, and pat dry for the last time; 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, react at room temperature for 10 min; 8) Detection: After 10 minutes, the chemiluminescence (CLIA) value was detected using a chemiluminescence detector; 9) Establishing a regression equation: Using p30 protein standards, a linear regression equation for detecting high and low concentrations of p30 was established; 10) ASFVp30 content in samples: The ASFVp30 content in diluted tissue samples, blood and swabs was calculated using a linear regression equation, and then multiplied by the dilution factor to obtain the ASFVp30 content in the sample; 11) The conditions for the test to be valid: The chemiluminescence value of the 200ng / mL p30 standard should be ≥100,000,000.

9. The kit according to claim 8, characterized in that 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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