Near-infrared fluorescence immunochromatography test strip for detecting African swine fever virus and application of near-infrared fluorescence immunochromatography test strip

Abstract

The invention provides a near-infrared fluorescence immunochromatography test strip for detecting African swine fever virus and application of the near-infrared fluorescence immunochromatography test strip, and belongs to the technical field of biological detection. The test strip comprises a sample pad, a combination pad, an antibody coated NC membrane and a water absorption pad, the combination pad is coated with a fluorescent conjugate, and the fluorescent conjugate is obtained by coupling an African swine fever virus p30 protein monoclonal antibody 027B and a near-infrared fluorescent dye through biotin and streptavidin; the antibody-coated NC membrane is provided with a detection line and a quality control line, and an African swine fever virus p30 protein monoclonal antibody 2-12B is fixed on the detection line; the monoclonal antibody 2-12B is secreted by the hybridoma cell strain 2-12B, and the preservation number is CCTCC (China Center For Type Culture Collection) NO: C2022116; the monoclonal antibody 027B is purchased from Wuhan original valley biotechnology limited liability company, and the product number is 027B. The test strip disclosed by the invention can realize on-site, rapid, high-sensitivity and high-specificity ASFV detection.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and specifically relates to a near-infrared fluorescent immunochromatographic test strip for detecting African swine fever virus and its application. Background Technology

[0002] African swine fever (ASF) is an acute, febrile, and contagious animal disease caused by the African swine fever virus (ASFV). It has extremely high infection and mortality rates in both domestic and wild pigs and is listed as a reportable animal disease by the World Organisation for Animal Health (WOAH), posing a devastating threat to the global pig industry. Due to its diverse transmission routes, long incubation period, and the lack of an effective commercial vaccine, establishing rapid, accurate, and sensitive early diagnostic methods is crucial for the prevention, control, eradication, and control of the epidemic.

[0003] Currently, ASFV detection methods mainly include virus isolation and identification, nucleic acid detection technologies such as quantitative polymerase chain reaction (qPCR), and immunological detection technologies such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA). Virus isolation is too time-consuming and not suitable for large-scale rapid screening. Although the qPCR method has high sensitivity and specificity, it is complex to operate, relies on expensive equipment and professional personnel, and is difficult to promote at the grassroots level and in the field.

[0004] In immunological detection methods, the ASFV p30 protein (encoded by the viral CP204L gene) is an important early-expressed structural protein with excellent immunogenicity and conservation. It is highly expressed in the cytoplasm of infected monocytes and macrophages early in viral infection, making it an ideal target for developing ASFV detection reagents.

[0005] Traditional colloidal gold immunochromatographic assay strips (GICA), while simple to operate and inexpensive, have relatively low sensitivity and the results are subjective and dependent on visual interpretation. To improve detection sensitivity, fluorescence immunochromatographic assays (FICA) were developed. By using fluorescent labels instead of colloidal gold and employing a dedicated reader, FICA can significantly improve detection sensitivity and enable quantitative analysis. However, when dealing with early-stage African swine fever virus infection samples with low viral loads, conventional FICA may still have insufficient detection limits. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a near-infrared fluorescence immunochromatographic test strip for detecting African swine fever virus, which can achieve on-site, rapid, highly sensitive and highly specific detection of ASFV.

[0007] Another object of the present invention is to provide a method for preparing the test strip and a method for using it.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A near-infrared fluorescent immunochromatographic test strip for detecting African swine fever virus includes a sample pad, a conjugate pad, an antibody-coated NC membrane, and an absorbent pad sequentially arranged on a support pad. The conjugate pad is coated with a fluorescent conjugate obtained by conjugating an African swine fever virus p30 protein monoclonal antibody 027B with a near-infrared fluorescent dye using biotin and streptavidin. The antibody-coated NC membrane has a detection line and a control line. The detection line is coated with African swine fever virus p30 protein monoclonal antibody 2-12B. The monoclonal antibody 2-12B is secreted by the hybridoma cell line 2-12B, with accession number CCTCC NO: C2022116. The monoclonal antibody 027B was purchased from Wuhan Yuangu Biotechnology Co., Ltd., product number 027B.

[0009] In this invention, the fluorescent conjugate is prepared by the following method: the African swine fever virus p30 protein monoclonal antibody 027B is labeled with biotin, streptavidin is labeled with DyLight 800 dye, and the biotin-labeled monoclonal antibody 027B is conjugated with the DyLight 800 dye-labeled streptavidin to obtain the fluorescent conjugate.

[0010] In this invention, the molar ratio of biotin-labeled monoclonal antibody 027B to streptavidin labeled with DyLight 800 dye is 1 to 4:1, preferably 4:1.

[0011] In this invention, the quality control line is coated with rabbit anti-streptavidin polyclonal antibody.

[0012] In this invention, a nitrocellulose membrane is coated with a fluorescent conjugate at a concentration of 0.05-0.2 mg / mL to obtain a conjugation pad; a detection line is obtained by spraying a p30 protein monoclonal antibody 2-12B at a concentration of 0.8-2 mg / mL onto the nitrocellulose membrane; and a control line is obtained by spraying a rabbit anti-streptavidin polyclonal antibody at a concentration of 0.02-0.04 mg / mL.

[0013] In this invention, a nitrocellulose membrane is treated with a pretreatment blocking solution to obtain a sample pad. The pretreatment blocking solution is an aqueous solution containing 0.1-0.2 g / L boric acid, 45-55 g / L trehalose, 1.8-2.2 g / L polyethylene glycol, 2.3-2.8 g / L bovine serum albumin and 4-6 g / L Tween 20.

[0014] This invention also provides a method for detecting African swine fever virus using the test strip for non-diagnostic purposes, characterized by comprising the following steps: (1) Add the sample to be tested to the lysis buffer. After lysis, take a sample and drop it into the sample pad of the test strip for chromatography. (2) The fluorescence signal intensity of the C line (C) and the fluorescence signal intensity of the T line (T) were detected using a dry fluorescence immunoassay analyzer at a wavelength of 800 nm after chromatography. (3) Result determination: If the ratio of the T / C value of the test sample to the negative control T / C value is greater than or equal to 2, then the test sample contains African swine fever virus; if only the C line of the test sample shows an obvious fluorescence signal peak and the T line does not show a fluorescence signal peak, then the test sample does not contain African swine fever virus; if only the C line of the test sample shows an obvious fluorescence signal peak and the ratio of the T / C value of the test sample to the negative control T / C value is less than 2, then the test sample does not contain African swine fever virus.

[0015] In this invention, the lysis buffer is a phosphate buffer containing 2.3-2.8 g / L Tween 20.

[0016] In this invention, the negative control is African swine fever virus-negative pig serum.

[0017] The detection principle of this invention's test strip is as follows: The lysed sample is dropped onto the sample pad. Under capillary action, the sample migrates forward to the conjugate pad, dissolving the fluorescent conjugate coated thereon. If the sample contains the p30 protein of African swine fever virus (ASFV), the p30 protein binds to the ASFV p30 protein monoclonal antibody 027B on the fluorescent conjugate, forming a "p30-fluorescent conjugate" complex. Under capillary action, the "p30-fluorescent conjugate" complex and free fluorescent conjugate migrate forward together. When it reaches the T-line, the "p30-fluorescent conjugate" complex is captured by the ASFV p30 protein monoclonal antibody 2-12B coated on the T-line, forming a sandwich immune complex of "mAb 2-12B-p30-fluorescent conjugate," resulting in enrichment of the fluorescent signal in the T-line region. Unbound or excess free fluorescent conjugates continue to migrate forward. When they reach the C-line, the streptavidin on them is captured by the rabbit anti-streptavidin polyclonal antibody immobilized at the C-line, resulting in an enrichment of the fluorescent signal in the C-line region. The reacted test strip is then placed in a dry fluorescence immunoassay analyzer for detection. By reading the fluorescence signal intensities of the C-line and T-line and calculating the ratio, qualitative or quantitative analysis of the p30 antigen in the sample can be performed.

[0018] Compared with existing technologies, this invention has the following significant advantages: This invention employs a dual signal amplification strategy, introduces a biotin-streptavidin system to construct a fluorescent conjugate, and uses monoclonal antibody 027B as the conjugate antibody and 2-12B as the T-line fixation antibody, achieving highly sensitive detection of African swine fever virus. The limit of detection for this invention's test strip is 0.01 ng / mL. Furthermore, this invention's test strip exhibits high specificity and accurate detection results. The near-infrared band (700-900 nm) is the "optical window" of biological tissues, where the absorption and autofluorescence of endogenous substances such as hemoglobin and bilirubin are very weak. Therefore, this invention's test strip can effectively resist interference from complex sample matrices (such as hemolyzed samples and jaundice samples), ensuring the stability and reliability of the results. The detection process of this invention's test strip is rapid and simple: the entire detection process requires no complex sample pretreatment, and the process from sample addition to obtaining results is usually completed within 5 minutes. It is simple to operate and suitable for large-scale rapid screening in field settings, grassroots veterinary stations, and farms. Attached Figure Description

[0019] Figure 1Electrophoresis images of the pET-28a-p30-his recombinant plasmid, where Figure A is the electrophoresis image of the amplified product of the p30 protein encoding gene (CP204L gene); Figure B is the electrophoresis image of the double enzyme digestion product of the recombinant plasmid pMD19-T-p30-his; and Figure C is the electrophoresis image of the double enzyme digestion product of the recombinant plasmid pET-28a-p30-his. In the figures, M1, M2, and M3 represent DNA markers; 1 represents the amplified product of the p30 protein encoding gene (CP204L gene); 2 represents the double enzyme digestion product of the recombinant plasmid pMD19-T-p30-his; 3 represents the double enzyme digestion product of the pET-28a vector; and 4 represents the double enzyme digestion product of the recombinant plasmid pET-28a-p30-his.

[0020] Figure 2 The results of the p30 recombinant protein solubility analysis are shown, where: lane 1 is the uninduced control bacteria; lane 2 is the whole bacterial culture of the induced control bacteria; lane 3 is the supernatant of the lysate of the induced control bacteria; lane 4 is the precipitate of the lysate of the induced control bacteria; lane 5 is the protein marker; lane 6 is the uninduced BL21-pET-28a-p30; lane 7 is the whole bacterial culture of the induced BL21-pET-28a-p30; lane 8 is the supernatant of the lysate of the induced BL21-pET-28a-p30; and lane 9 is the precipitate of the lysate of the induced BL21-pET-28a-p30.

[0021] Figure 3 SDS-PAGE electrophoresis image of purified p30 recombinant protein, where: lane 1 is protein marker; lane 2 is precipitate of lysate of recombinant bacteria BL21-pET-28a-p30 after induction; lane 3 is the pass-through liquid of the sample; lanes 4-6 are purified p30 recombinant protein.

[0022] Figure 4 Western blotting electrophoresis images of each monoclonal antibody, where: the primary antibody sources in Figures A, B and C are culture media 1-6A, 2-12B, and SP2 / 0 cell culture, respectively; lane M is the protein marker; lane 1 is the p30 recombinant protein; and lane 2 is the negative control, which is the supernatant of the control bacterial lysate.

[0023] Figure 5 The construction results of the pcDNA3.1-p30-flag recombinant plasmid are shown in Figure A, which is an electrophoresis diagram of the target gene amplification, and Figure B is an electrophoresis diagram of the recombinant plasmid double enzyme digestion. Lane M1 is the DL2000 Marker; lane 1 is the target gene amplification product p30-flag; lane M2 is the DL5000 Marker; and lane 2 is the double enzyme digestion product of the pcDNA3.1-p30-flag recombinant vector.

[0024] Figure 6 The indirect immunofluorescence identification results of p30 monoclonal antibody, from left to right, are blank cell group, empty vector group, and experimental group, respectively.

[0025] Figure 7 A schematic diagram of the test strip structure of this invention.

[0026] The preservation information for hybridoma cell line 2-12B is as follows: Classified and named African swine fever virus P30 monoclonal antibody hybridoma cell line MAb P30 2-12B, the preservation date is April 27, 2022, the full name of the depositary is China Center for Type Culture Collection (CCTCC), the address of the depositary is Wuhan University, and the preservation number is CCTCC NO: C2022116. In the name MAb P30 2-12B, "P30" refers to the African swine fever virus p30 protein. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0028] Example 1: Preparation and identification of monoclonal antibodies (1-6A, 2-12B) against African swine fever virus p30 protein 1. Preparation of p30 recombinant protein 1.1 Gene Origin and Amplification Based on the CP204L gene sequence of ASFV Wuhan 2019-2 strain (GenBank: MN393477.1) published by NCBI, primers P1 and P2, with BamHI and HindIII restriction sites at both ends respectively, were designed and synthesized. The sequence of primer P1 is as follows: 5'-CGGGATCCCGatggattttattttaaat-3'; the sequence of primer P2 is as follows: 5'-CCCAAGCTTGGGTTAgtgatggtgatggtgatgttttttttttaaaagtttaataaccatg-3'.

[0029] Using the synthesized CP204L gene (GenBank: MN393477.1) as a template, and primers P1 and P2, the CP204L gene fragment was amplified by PCR and then electrophoresis was performed. The results are shown below. Figure 1 In step A, a 585bp target fragment appeared in the amplification product, which is as expected. The target fragment was recovered according to the gel extraction kit instructions.

[0030] 1.2 Construction of expression vector The gel-recovered product of the CP204L gene fragment was mixed with the pMD19-T vector, and T4 DNA ligase and its buffer were added. The mixture was incubated at 16°C for 30 min to complete the TA cloning ligation reaction, yielding the pMD19-T-p30-his plasmid. The pMD19-T-p30-his plasmid was transformed into Trans5α competent cells using standard procedures. The cells were plated on ampicillin-resistant LB medium and incubated at 37°C for 14 hours. Single colonies were picked for PCR verification. Positive colonies were used to extract the pMD19-T-p30-his plasmid using an Omega Plasmid Mini Kit I kit. The pMD19-T-p30-his plasmid and pET-28a vector were double-digested with BamHI and HindIII, respectively. The target fragment and digested vector were recovered by gel electrophoresis. The results are shown below. Figure 1 In the double digestion product of recombinant plasmid pMD19-T-p30-his, a 585bp target fragment appeared in the swimming lane. The target fragment and the digested vector were recovered from the gel and ligated using T4 ligase at 16℃ for 12 hours. The CP204L gene was then inserted into pET-28a to obtain the recombinant plasmid pET-28a-p30-his. The recombinant plasmid pET-28a-p30-his was then transformed into Trans5α competent cells, plated on kanamycin-resistant medium, and cultured for 14 hours. Single colonies were picked for PCR verification, and plasmids were extracted from positive bacterial cultures using a kit. Double digestion with BamHI and HindIII, PCR, and sequencing confirmed that the target sequence was 100% correct and that there were no mutations in the reading frame. The double digestion verification results are shown in [link to double digestion verification]. Figure 1 In the double digestion product lane of the recombinant plasmid pET-28a-p30-his, a 585bp target fragment and a 5300bp linear vector were observed, indicating that the pET-28a-p30-his recombinant plasmid was successfully constructed and transformed.

[0031] 1.3 Induced expression and purification of p30 recombinant protein The recombinant plasmid pET-28a-p30-his was transformed into BL21(DE3) competent cells to obtain the recombinant bacterium BL21-pET-28a-p30. The target protein was expressed by IPTG induction, and the bacterial cells were collected. The cells were resuspended in PBS buffer and washed three times. Lysis-Equilibration-Wash Buffer (an aqueous solution containing 50 mM NaH2PO4 and 300 mM NaCl, pH 8.0) was added, followed by the addition of lysozyme to a final concentration of 1 mg / mL. The mixture was stirred on ice for 30 min and then sonicated. The lysis was performed by centrifugation at 10,000 r / min for 30 min at 4 °C, separating the supernatant and precipitate. Inclusion bodies were resuspended in Lysis-Equilibration-Wash Buffer. In addition, the empty pET-28a vector was transformed into BL21(DE3) competent cells to obtain control bacteria. The control bacteria were induced using the same method, and the whole bacterial cultures, supernatant of the lysate after sonication, and precipitates of both the test and control bacteria were identified by SDS-PAGE electrophoresis. Results are shown below. Figure 2 No target protein was observed in the supernatant of lane 8, while in lane 9, the induced recombinant bacterium BL21-pET-28a-p30 successfully expressed the 33 kDa target protein, indicating that the target protein mainly existed in the precipitate as inclusion bodies. Additionally, the control bacterium did not express the target protein. The induced recombinant bacterium BL21-pET-28a-p30 was lysed and centrifuged. The precipitate was dissolved in Denaturing Solubilization Buffer (lysis-equilibration-wash buffer with 8 M urea added to a final concentration, pH 8.0), filtered through a 0.45 μm filter membrane, and the filtrate was used as the sample for protein purification using a Ni-TED gravity column (purchased from MACHEY-NAGEL). The purified p30 recombinant protein was verified by SDS-PAGE electrophoresis, showing good purification results (see [link to sample]). Figure 3 The purified p30 recombinant protein was renatured by dialyzing on ice using gradient urea buffers (lysis-equilibration-wash buffer). The dialyzing was performed with a buffer containing 6M urea for 2 hours, a buffer containing 4M urea for 2 hours, a buffer containing 2M urea overnight, and a buffer without urea overnight. The renatured p30 recombinant protein concentration was determined to be 0.5 mg / mL using the BCA method and aliquoted at -80°C for storage.

[0032] 2. Preparation of p30 recombinant protein mouse hyperimmune serum Four 6-week-old female SPF BALB / c mice (purchased from the Comparative Medicine Center of Yangzhou University) were used for primary immunization. The p30 recombinant protein antigen and Freund's complete adjuvant were emulsified in equal volumes and injected at multiple sites. The p30 recombinant protein antigen and Freund's incomplete adjuvant were then emulsified in equal volumes and injected at multiple sites. Two immunizations were performed after these two treatments. The antigen dose for each immunization was 100 μg per mouse, with an interval of 14 days between immunizations.

[0033] Seven days after the third immunization, blood was collected from the eyeballs. The p30 recombinant protein was coated onto an ELISA plate. An indirect ELISA reaction was performed using serially diluted mouse immune serum as the primary antibody and HRP-labeled goat anti-mouse IgG (purchased from Wuhan Sanying Biotechnology Co., Ltd., SA00001-1) as the secondary antibody. The antibody titer was determined by reading the OD450 nm value. The highest antibody titer was 1:102400; this serum was the p30 recombinant protein mouse hyperimmune serum. Mice with the highest antibody titer were selected and given a booster immunization via intraperitoneal injection of antigen (100 μg / mouse).

[0034] 3. Preparation and identification of p30 recombinant protein monoclonal antibodies 3.1 Culture medium preparation The culture medium should be prepared in advance according to the following method: 1640 complete medium: Add 10% FBS (fetal bovine serum, purchased from Gibco, USA) to RPMI 1640 liquid medium (purchased from Gibco, USA) to a final concentration (volume percentage).

[0035] HAT / HT Complete Medium: Dissolve the corresponding HAT / HT powder in 10 mL of 1640 medium (dosage according to the instructions) to obtain HAT stock solution. Add the above HAT stock solution to 500 mL of 1640 complete medium to obtain HAT / HT complete medium.

[0036] 2×RPMI 1640 Complete Medium: Take 0.4g of 1640 powder (RPMI 1640 powder medium, purchased from Gibco, USA) and add 100mL of ddH2O to obtain a medium with twice the concentration. Filter with a 0.22µm filter, aliquot and store. When using, add FBS to a final concentration (volume percentage) of 20% to obtain 2×RPMI 1640 complete medium.

[0037] 2×HT complete medium: Take 4 mL of HAT / HT complete medium and add it to 100 mL of 2×RPMI 1640 complete medium, then mix well.

[0038] 1.2% agar: Add 100mL of ddH2O to 1.2g of agar powder and autoclave.

[0039] 3.2 Hybridoma cell preparation and screening 3.2.1 Preparation of feeder cells (peritoneal macrophages): One healthy BALB / c mouse was euthanized by cervical dislocation and soaked in 75% alcohol for 10 min. 5 mL of HAT / HT complete culture medium was drawn into the peritoneum through a syringe and injected into the peritoneal cavity. The culture medium was then aspirated back into the peritoneal cavity and placed into a centrifuge tube. This process was repeated three times. The cell suspensions obtained from the three trials were mixed and 100 μL / well was added to a 96-well plate.

[0040] 3.2.2 Preparation of spleen cells and SP2 / 0 cells: Spleen cells: Take the mice that have been boosted with intraperitoneal injection of antigen (100 μg / mouse) as described in Title 2 of this embodiment (final immunized mice), aseptically remove the spleen, place it on a cell sieve, add 1640 complete culture medium to wash and grind until completely ground, centrifuge at 2000 r / min for 5 min, take the precipitate and resuspend it in fresh 1640 complete culture medium, repeat the above centrifugation, precipitate collection, and resuspending operation 2 times (3 times in total), finally, add 30 ml of 1640 complete culture medium to the cell precipitate to obtain spleen cell suspension for later use.

[0041] SP2 / 0 cells: Take SP2 / 0 cells from 4 flasks of T75 cells in the logarithmic growth phase, discard the culture medium, add an appropriate amount of 1640 complete culture medium to wash once, blow off the cells with 1640 complete culture medium to form a single-cell suspension, combine the suspensions from 4 flasks into the same centrifuge tube for later use.

[0042] 3.2.3 Cell fusion: Following standard methods, spleen cells and SP2 / 0 cells were fused at a ratio of 1:10. The fused cells were placed in HAT / HT complete medium and seeded at 100 μL / well into 96-well plates containing feeder cells. The cells were cultured at 37°C and 5% CO2 for 10 days.

[0043] 3.2.4 Screening for positive hybridoma cells: Initial screening was performed on day 10 post-fusion: 96-well ELISA plates were coated with 0.5 μg / mL p30 recombinant protein and 0.5 μg / mL control bacterial lysate (prepared in title 1.3). High-immune mouse serum containing p30 recombinant protein (prepared in title 2) was used as the sample. Hybridoma cell supernatant was used as the primary antibody, and HRP-labeled goat anti-mouse IgG (purchased from Wuhan Sanying Biotechnology Co., Ltd., SA00001-1) was used as the secondary antibody. Positive hybridoma cells were screened using an indirect ELISA method. Wells containing positive cells that reacted with p30 recombinant protein but not with the control bacterial lysate supernatant were selected for further culture expansion.

[0044] Semi-solid culture screening: Hybridoma cells from expanded culture were screened as follows: 1.2% agar at 55℃ and 2×HT complete medium preheated at 37℃ were mixed at a volume ratio of 1:1. 2 mL of the resulting medium was added to each well of a 6-well plate and allowed to solidify at 4℃ for 6 hours to obtain the bottom agar. The 1.2% agar was diluted to a mass percentage concentration of 0.3% with ddH2O at 37℃, and then mixed with 2×HT complete medium at a volume ratio of 1:1 to obtain HT complete medium containing 0.15% agar. This medium was then used to prepare 1×10⁻⁶ hybridoma cells. 5 Three-fold dilution of hybridoma cells per mL yielded 3.3 × 10⁻⁶ cells. 4 cells / mL, 1.1×10 4 cells / mL, 3.7×10 3 Cell suspensions of three different concentrations were prepared. At 37°C, 40 μL of each of the four concentrations was aspirated and added to 4 mL of HT complete medium containing 0.15% agar. The mixture was then added to three wells of a 6-well agar plate preheated to 37°C. The plates were incubated at room temperature for 15 min to form a gel, and then cultured at 37°C in a CO2 incubator for 3 days. 1 mL of 2×HT complete medium was added to each well to keep the upper gel layer moist. Seven days later, cell clusters were located and labeled under a microscope, and then aspirated into 96-well plates using a pipette tip. Each cell cluster was placed in a separate well, and culture was started. When the wells were filled to 50% capacity, the cells were tested. The positive wells were then cultured to obtain eight monoclonal hybridoma cell lines. Two of the positive hybridoma cell lines had high titers and were named African swine fever virus P30 monoclonal antibody hybridoma cell line MAb P30 2-12B (abbreviated as hybridoma cell line 2-12B) and African swine fever virus P30 monoclonal antibody hybridoma cell line MAb P30 1-6A (abbreviated as hybridoma cell line 1-6A), respectively. The secreted monoclonal antibodies were also named p30 protein monoclonal antibody 2-12B and p30 protein monoclonal antibody 1-6A, respectively.

[0045] Preparation of ascites monoclonal antibodies (antibodies 1-6A and 2-12B): 8-week-old BALB / c mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant, followed by an injection of 1 × 10⁻⁶ oz. 1 week later. 6 Hybridoma cells were collected, and ascites fluid was collected after 10 days. The intermediate aqueous phase was collected by centrifugation at 2000 rpm as the ascites fluid monoclonal antibody. The p30 recombinant protein was coated onto an ELISA plate. An indirect ELISA reaction was performed using serially diluted ascites fluid monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse IgG (purchased from Wuhan Sanying Biotechnology Co., Ltd., SA00001-1) as the secondary antibody. OD values ​​were read. 450nm Values ​​were calculated to determine potency. Results: Ascites potency ≥ 1:51200, aliquoted and stored at -80℃.

[0046] 3.3 Monoclonal antibody specificity identification 3.3.1 Western Blot Identification: Using p30 recombinant protein and the supernatant of lysate from control bacteria (prepared in Title 1.3) as coating antigens, and the culture medium of two positive hybridoma cell lines (1-6A and 2-12B) as primary antibodies, SP2 / 0 cell culture as a blank control, and HRP-labeled goat anti-mouse IgG (purchased from Wuhan Sanying Biotechnology Co., Ltd., SA00001-1) as a secondary antibody, Western blotting was performed to examine the reactivity of monoclonal antibodies secreted by hybridoma cells 1-6A and 2-12B with p30 recombinant protein. Figure 4 It is evident that the monoclonal antibodies secreted by the two positive hybridoma cells could recognize the 33kDa target band (recombinant p30 protein) and did not react with the control bacterial protein introduced with the pET-28a empty vector. Furthermore, SP2 / 0 cells did not react with the recombinant p30 protein, indicating that the monoclonal antibodies secreted by the hybridoma cells could react with ASFV p30.

[0047] 3.3.2 Indirect Immunofluorescence (IFA) Identification Primers tagged with a flag were designed. The upstream primer contained a Hind III restriction site, with the sequence: 5'-CCCAAGCTTGGGATGGATTTTATTTTAAAT-3'; the downstream primer contained a BamHI restriction site, with the sequence: 5'-CGGGATCCCGTTACTTATCGTCGTCATCCTTGTAATCTTTTTTTTTTAAAAGTTTAATAACCATG-3'. Using the synthesized CP204L gene as a template, PCR amplification was performed using the flag-tagged primers, yielding a 631 bp amplification product p30-flag. (See below...) Figure 5 In step A, using the pcDNA3.1 vector and following the same experimental procedure as in Example 1.2, the p30-flag was inserted between the BamHI and HindIII restriction sites of the pcDNA3.1 vector to obtain the pcDNA3.1-p30-flag recombinant vector. Electrophoresis after double enzyme digestion confirmed the results as expected; see [see details]. Figure 5The correctly sequenced plasmid pcDNA3.1-p30-flag was transfected into 293T cells and cultured at 37℃ in a 5% CO2 cell culture incubator for 48 h before IFA (in vitro anabolism). The following groups were set up: ① Experimental group (pcDNA3.1-p30-flag group): 293T cells transfected with the pcDNA3.1-p30-flag recombinant plasmid; ② Empty vector group (pcDNA3.1 empty vector group): 293T cells transfected with the pcDNA3.1 empty vector; ③ Blank cell group (293T blank cell group): 293T cells not transfected with any plasmid. Each group had three experiments, using ascites fluid from hybridoma cells 1-6A and 2-12B diluted 1:1000 with PBST as primary antibodies, and a flag-tagged antibody (abcam, ab18230) as the primary antibody. After adding the primary antibody, the cells were incubated for 2 hours and washed three times. For each experiment, FITC-labeled goat anti-mouse IgG (purchased from Abcam, ab6785) was added as the secondary antibody and incubated for 1 hour. After washing with PBST, slides were mounted with anti-fluorescence quenching mounting medium and observed under a fluorescence microscope. Specific green fluorescence was observed only in the experimental groups; no fluorescence was observed in the empty vector group and the blank cell group. Results are shown in the table below. Figure 6 The above experiments demonstrate that both p30 protein monoclonal antibodies 1-6A (abbreviated as 1-6A) and 2-12B (abbreviated as 2-12B) can specifically bind to the recombinant p30 protein.

[0048] 3.4 Monoclonal antibody 1-6A and 2-12B site competition assay Indirect ELISA superposition assay: 0.125 μg / L purified p30 recombinant protein was used to coat the microplate at 100 μL / well.

[0049] Group 1: Add 100µL OD450 to each well first. nm The monoclonal antibody 1-6A with a value of 1.25 was reacted at 37°C for 1 hour, then the solution was discarded and washed three times with PBST. HRP-labeled goat anti-mouse IgG (purchased from Wuhan Sanying Biotechnology Co., Ltd., SA00001-1) was then added, reacted at 37°C for 1 hour, then the solution was discarded and washed three times with PBST. 50 µL of TMB substrate solution was added, and the mixture was incubated at 37°C in the dark for 15 minutes. The reaction was terminated by adding stop solution (2M H2SO4 aqueous solution), and the OD value was read at 450 nm using a microplate reader.

[0050] Group 2: Add 100 μL OD450 to each well first. nmThe monoclonal antibody 2-12B with a value of 1.25 was reacted at 37°C for 1 hour, then the solution was discarded and washed three times with PBST. HRP-labeled goat anti-mouse IgG (purchased from Wuhan Sanying Biotechnology Co., Ltd., SA00001-1) was then added, reacted at 37°C for 1 hour, then the solution was discarded and washed three times with PBST. 50 µL of TMB substrate solution was added, and the mixture was incubated at 37°C in the dark for 15 minutes. The reaction was terminated by adding stop solution (2M H2SO4 aqueous solution), and the OD value was read at 450 nm using a microplate reader.

[0051] Group 3: Add 100 μL OD450 to each well first. nm Monoclonal antibody 1-6A with an OD450 nm value of 1.25 was reacted at 37°C for 1 hour, then discarded and washed three times with PBST. 100 µl of monoclonal antibody 2-12B with an OD450 nm value of 1.25 was added, reacted at 37°C for 1 hour, then discarded and washed three times with PBST. HRP-labeled goat anti-mouse IgG (purchased from Wuhan Sanying Biotechnology Co., Ltd., SA00001-1) was then added, reacted at 37°C for 1 hour, then discarded and washed three times with PBST. 50 μL of TMB substrate solution was added, and the mixture was incubated at 37°C in the dark for 15 minutes. The reaction was terminated by adding stop solution (2 M H2SO4 aqueous solution), and the OD value was read at 450 nm using a microplate reader.

[0052] Results (Table 1): The OD450nm value of the third group was not significantly higher than that of the first and second groups, indicating that monoclonal antibodies 1-6A and 2-12B bind to the p30 recombinant protein at the same site.

[0053] Table 1 Results of indirect ELISA superposition assays for monoclonal antibodies 1-6A and 2-12B

[0054] Example 2: Preparation of fluorescent conjugates Fluorescent conjugates were prepared using the following method: (1) Preparation of biotin-labeled monoclonal antibodies (Biotin-mAb) The following method was used to biotinylate p30 protein monoclonal antibodies 1-6A and 2-12B (ascites antibody), African swine fever p30 protein monoclonal antibodies 027B (purchased from Wuhan Yuangu Biotechnology Co., Ltd., product number 027B) and 027A (purchased from Wuhan Yuangu Biotechnology Co., Ltd., product number 027A), respectively: 0.224 mg of EZ-Link NHS-PEG12-biotin (purchased from Thermo Fisher Scientific, product number 21313) was dissolved in 22.4 μL of anhydrous DMF to prepare a 10 mg / mL biotin stock solution. The monoclonal antibodies were diluted to 2 mg / mL with PBS buffer at pH 8.5. EZ-Link NHS-PEG12-Biotin was slowly added to the antibody solution at a molar ratio of 1:10.1, i.e., 8.9 μL of a 10 mg / mL biotin stock solution was added to every 1 mL of antibody. The reaction was carried out at room temperature for 30 min. After the reaction, unreacted biotin was removed by ultrafiltration or gel size exclusion chromatography to obtain biotin-labeled monoclonal antibodies. Biotin-labeled monoclonal antibodies 1-6A are designated Biotin-mAb-1-6A, 2-12B as Biotin-mAb-2-12B, 027B as Biotin-mAb-027B, and 027A as Biotin-mAb-027A.

[0055] Preparation of streptavidin-dye conjugate (SA-DyLight 800) Take a 70 mg / mL streptavidin solution and dilute it to 2 mg / mL with labeling buffer (pH 8.5, 0.05 M sodium borate buffer). Equilibrate DyLight 800 dye (Thermo Fisher Scientific, product number 46421) at room temperature for 30 min, then dissolve it in anhydrous DMF to prepare a 25 mg / mL DyLight stock solution. Slowly add 63 μL of the DyLight 800 stock solution to 1 mL of a 2 mg / mL streptavidin solution, pipette to mix, and incubate at room temperature in the dark for 1 h, gently mixing every 20 min. After the reaction is complete, remove the free dye by dialysis or ultrafiltration to obtain DyLight 800-labeled streptavidin, denoted as SA-DyLight 800.

[0056] Formation of fluorescent conjugates Mix 102 μL of biotin-labeled monoclonal antibody at a concentration of 1.89 mg / mL and 10 μL of SA-DyLight 800 at a concentration of 2.12 mg / mL (the molar ratio of Biotin-mAb to SA-DyLight 800 is 4:1), and incubate with low-speed shaking at 200 rpm for 30 min to allow for complete binding and the formation of a stable fluorescent conjugate. The fluorescent conjugates corresponding to Biotin-mAb-1-6A are designated Biotin-mAb-1-6A-SA-DyLight 800, Biotin-mAb-2-12B-SA-DyLight 800, Biotin-mAb-027B-SA-DyLight 800, and Biotin-mAb-027A-SA-DyLight 800. Each conjugate is diluted to 0.1 mg / mL with PBS buffer (pH 7.4) containing 1% (w / v) BSA (bovine serum albumin), 5% (w / v) sucrose, and 0.1% (v / v) Tween 20.

[0057] Example 3: Assembly of near-infrared fluorescence immunochromatographic test strips Sample pads: Place a 60mm (width) × 300mm (length) nitrocellulose membrane (purchased from Millipore, GFCP203000) in a dish, pour in 50mL of pretreatment blocking solution, soak for 30min, remove, dry in a 37℃ oven for 12h, then vacuum dry for 24h, and cut into 17mm × 300mm sample pads using a CNC strip cutter. The pretreatment blocking solution is an aqueous solution containing 0.1237g / L boric acid, 50g / L trehalose, 2g / L polyethylene glycol 20000 (PEG20000), 2.5g / L bovine serum albumin (BSA), and 5g / L Tween 20, with a pH of 8.0, and ultrapure water as the solvent.

[0058] Conjugation pad: A nitrocellulose membrane (purchased from Millipore, product code GFCP203000) was cut to a length of 7.5 mm (width) × 300 mm. Using a gold spraying apparatus (Biodot XYZ3060), with the coating solution volume set to 4.4 μL / cm, 0.1 mg / mL of the fluorescent conjugate Biotin-mAb-027B-SA-DyLight800 (prepared in Example 2) was uniformly coated onto the nitrocellulose membrane along its length. The membrane was then vacuum dried at 37 °C for 2 h to obtain the conjugation pad for later use.

[0059] Antibody-coated NC membrane: A 20mm (width) × 300mm (length) nitrocellulose membrane was precisely streaked using a streak scribing machine. The detection line (T line) was streaked using 1 mg / mL p30 protein monoclonal antibody 2-12B as the streaking solution. The control line (C line) was streaked approximately 5mm from the T line using 0.03 mg / mL rabbit anti-streptavidin polyclonal antibody solution (purchased from Beijing Bosi Technology Co., Ltd., product number B1285). After streaking, the membrane was dried at 37℃ for 24 hours, and then aged in a 60℃ oven for 6 days to obtain the antibody-coated NC membrane.

[0060] Assembly ( Figure 7 First, the antibody-coated NC membrane is pasted onto the middle of the PVC support pad, with the C line at the top. The antibody-coated NC membrane is 15mm away from the upper edge of the PVC support pad. The absorbent paper is pasted onto the PVC support pad on the side near the C line and overlaps with the antibody-coated NC membrane by 2mm. The conjugate pad is pasted onto the PVC support pad on the side near the T line and overlaps with the antibody-coated NC membrane by 1mm. The sample pad is pasted onto the PVC support pad on the other side of the conjugate pad and overlaps with the conjugate pad by 1.5mm. Then, the strips are cut into 3.4mm wide test strips using a strip cutter. The upper and lower caps are installed, and the strips are assembled using a shell presser to obtain a near-infrared fluorescent immunochromatographic test strip for detecting African swine fever virus, which is referred to as the test strip of this invention.

[0061] Example 4: Method of using and result determination of the test strip of the present invention 1. Method of using the test strip of this invention Test samples: Take whole blood or serum from pigs as test samples; or prepare a 100g / L homogenate of suspected infected tissue (such as spleen or lymph nodes) with sterile PBS solution and use the homogenate as the test sample; or directly use oral and nasal fluid as the test sample.

[0062] Negative control: Pig serum that tested negative for African swine fever antibodies using the Jinno African Swine Fever Virus ELISA Antibody Detection Kit (purchased from Beijing Jinno Biotech Co., Ltd.).

[0063] Sample loading: Add Tween 20 to a final concentration of 2.5 g / L in 10 mM phosphate buffer (pH 7.4) as lysis buffer. Add 10 μL of the test sample to 190 μL of lysis buffer, lyse for 10 min, and then drop 70 μL into the sample pad of the test strip of this invention. Simultaneously, process the negative control using the same method and load the sample accordingly.

[0064] Chromatography and reaction: Start the timer and let the chromatography stand at room temperature (15-30℃) for 5 min.

[0065] 2. Result Determination Insert the reacted test strip into a BF2100 dry fluorescence immunoassay analyzer (purchased from Baifu Biotechnology (Taizhou) Co., Ltd., Jiangsu Medical Device Registration Certificate No. 20242222390), with a detection wavelength of 800 nm. The instrument will automatically detect the fluorescence signal intensity (i.e., fluorescence value RFU) of the C-line and T-line, and calculate the T / C value, where T refers to the fluorescence signal intensity of the T-line and C refers to the fluorescence signal intensity of the C-line.

[0066] Positive result: A positive result is defined as a T / C ratio of the test sample to the negative control T / C ratio greater than or equal to 2, indicating that the test sample contains African swine fever virus. The fluorescence value of the T line in a positive sample is positively correlated with the concentration of p30 antigen in the sample.

[0067] Negative result: If the test sample shows a clear fluorescence signal peak only in the C line and no fluorescence signal peak in the T line, then the test sample is a negative sample, that is, it does not contain African swine fever virus; if the test sample shows a clear fluorescence signal peak only in the C line and the ratio of the T / C value of the test sample to the T / C value of the negative control is less than 2, it is judged as negative, that is, it does not contain African swine fever virus.

[0068] Invalid result: If no fluorescence signal peak appears on line C, it indicates that the test strip is invalid or the operation is improper, and the test needs to be repeated.

[0069] Example 5: Paired Screening of Monoclonal Antibodies in Pathogen Detection Using Near-Infrared Fluorescence Immunochromatographic Test Strips Two monoclonal antibodies against the African swine fever virus p30 protein, 1-6A and 2-12B (targeting the same epitope), and two commercially available monoclonal antibodies against African swine fever p30 targeting different epitopes (027B and 027A, purchased from Wuhan Yuangu Biotechnology Co., Ltd.) were paired to serve as T-line fixation antibodies and conjugates in fluorescent conjugates. Test strips were prepared according to the methods and conditions described in Example 3 (with the same reagent concentrations). Specific pairing methods are shown in Table 2. The preparation methods for the fluorescent conjugates corresponding to each monoclonal antibody are described in Example 2.

[0070] Using serially diluted p30 recombinant protein antigen (prepared in Example 1) as a positive sample and pure water as a blank control, a sensitivity test was performed. The specific detection method is described in Example 4.

[0071] The results (Table 2) show that when monoclonal antibody 027B is used as the conjugate antibody in the fluorescent conjugate and 2-12B is used as the T-line fixation antibody, the limit of detection of the test strip is 0.01 ng / mL; while the limits of detection of other combinations are between 0.04-10 ng / mL, and the sensitivity is significantly lower than that of the pairing of 027B as the conjugate antibody in the fluorescent conjugate and 2-12B as the T-line fixation antibody (i.e., the test strip of the present invention, see Table 2).

[0072] Table 2. Sensitivity tests of test strips prepared with four different monoclonal antibody pairings.

[0073] Note: In Table 2, the antibodies above the "+" are the fluorescent conjugates, and the antibodies below the "+" are the T-line fixation antibodies.

[0074] Example 6: Performance of the test strip of the present invention Samples prepared from African swine fever virus positive tissues by gradient dilution were tested using the test strip of this invention and the commercially available Jinno African swine fever virus fluorescent microsphere test strip (Beijing Jinno Biotech Co., Ltd.). The OIE-qPCR method (Xiao Qi, Jiang Caixia, Shi Hui, et al. Establishment of quantitative PCR for African swine fever virus and comparison of four detection kits [J]. Jiangsu Agricultural Sciences, 2021, 49(1):119-124.) was used as a reference method. The Jinno African swine fever virus fluorescent microsphere test strip (hereinafter referred to as the Jinno test strip) was operated according to the instructions. 40 μL of the test sample was added to the detection port, followed by 60 μL of working solution after 45 seconds. The results were observed after 15 minutes under UV light. The Jinno test strip showed a weak fluorescent line in the 1:10 positive sample detection line, therefore the limit of detection was 1:10. The limit of detection of the test strip of this invention was 1:30, showing significantly higher sensitivity than the Jinno test strip. The comparison results are shown in Table 3.

[0075] Table 3 Comparison of the test strip of the present invention with existing test strips and detection methods

Claims

1. A near-infrared fluorescent immunochromatographic test strip for detecting African swine fever virus, comprising a sample pad, a conjugate pad, an antibody-coated NC membrane, and an absorbent pad sequentially disposed on a support pad, characterized in that: The binding pad is coated with a fluorescent conjugate, which is obtained by conjugating African swine fever virus p30 protein monoclonal antibody 027B with a near-infrared fluorescent dye using biotin and streptavidin. The antibody-coated NC membrane has a detection line and a control line, and the detection line is coated with African swine fever virus p30 protein monoclonal antibody 2-12B. The monoclonal antibody 2-12B is secreted by hybridoma cell line 2-12B, with accession number CCTCC NO: C2022116. The monoclonal antibody 027B was purchased from Wuhan Yuangu Biotechnology Co., Ltd., product number 027B.

2. The test strip according to claim 1, characterized in that, The fluorescent conjugate was prepared by the following method: the African swine fever virus p30 protein monoclonal antibody 027B was labeled with biotin, streptavidin was labeled with DyLight 800 dye, and the biotin-labeled monoclonal antibody 027B was conjugated with the DyLight 800 dye-labeled streptavidin to obtain the fluorescent conjugate.

3. The test strip according to claim 1 or 2, characterized in that, The molar ratio of biotin-labeled monoclonal antibody 027B to streptavidin labeled with DyLight 800 dye is 1 to 4:1, preferably 4:

1.

4. The test strip according to claim 3, characterized in that... The quality control line is coated with rabbit anti-streptavidin polyclonal antibody.

5. The test strip according to claim 4, characterized in that, A binding pad was obtained by coating a nitrocellulose membrane with a fluorescent conjugate at a concentration of 0.05-0.2 mg / mL; a detection line was obtained by spraying a 0.8-2 mg / mL monoclonal antibody 2-12B against the p30 protein onto the nitrocellulose membrane; and a control line was obtained by spraying a 0.02-0.04 mg / mL rabbit anti-streptavidin polyclonal antibody onto the membrane.

6. The test strip according to claim 5, characterized in that, The nitrocellulose membrane is treated with a pretreatment blocking solution to obtain a sample pad. The pretreatment blocking solution is an aqueous solution containing 0.1-0.2 g / L boric acid, 45-55 g / L trehalose, 1.8-2.2 g / L polyethylene glycol, 2.3-2.8 g / L bovine serum albumin and 4-6 g / L Tween 20.

7. A method for detecting African swine fever virus using the test strip described in claim 1 for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Add the sample to be tested to the lysis buffer. After lysis, take a sample and drop it into the sample pad of the test strip for chromatography. (2) The fluorescence signal intensity of the C line (C) and the fluorescence signal intensity of the T line (T) were detected using a dry fluorescence immunoassay analyzer at a wavelength of 800 nm after chromatography. (3) Result determination: If the ratio of the T / C value of the test sample to the negative control T / C value is greater than or equal to 2, then the test sample contains African swine fever virus; if only the C line of the test sample shows an obvious fluorescence signal peak and the T line does not show a fluorescence signal peak, then the test sample does not contain African swine fever virus; if only the C line of the test sample shows an obvious fluorescence signal peak and the ratio of the T / C value of the test sample to the negative control T / C value is less than 2, then the test sample does not contain African swine fever virus.

8. The method according to claim 7, characterized in that, The lysis buffer is a phosphate buffer containing 2.3-2.8 g / L Tween 20.

9. The method according to claim 8, characterized in that, The negative control is African swine fever virus-negative pig serum.

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