A time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus and its preparation method

Abstract

This invention belongs to the field of biotechnology and specifically relates to a time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus (TGEV) and its preparation method. The test strip comprises a PVC base, a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad, which are sequentially overlapped on the PVC base. A test line T and a quality control line C are provided on the nitrocellulose membrane, with the test line T positioned near one end of the conjugation pad and the quality control line C positioned near the other end of the water-absorbing pad. The conjugation pad is coated with monoclonal antibody A against the TGEV N protein and rabbit anti-IgG labeled with time-resolved fluorescent microspheres. The test line T is coated with monoclonal antibody B against the TGEV N protein, and the quality control line C is coated with goat anti-rabbit IgG. This test strip is simple to operate and can be used for timely, rapid, and sensitive detection of TGEV clinical samples, providing technical support for precise prevention and control of the disease on pig farms.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus and a preparation method thereof. Background Art

[0002] Transmissible gastroenteritis (TGE) is an acute, highly contagious disease caused by the transmissible gastroenteritis virus (TGEV). Rapid and accurate diagnosis is crucial for the prevention and control of the disease, as there are currently no safe and effective drugs or vaccines.

[0003] Commonly used pathogen detection methods include virus isolation and identification, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) and real-time quantitative PCR.

[0004] However, these methods are time-consuming, complex, and require specialized technicians and specialized instruments, making them difficult to meet the requirements for rapid, cost-effective, and efficient monitoring of clinical samples. Compared to these methods, immunochromatographic detection technology can maintain high sensitivity and strong specificity while strictly controlling the overall reaction time to within 10-15 minutes, significantly shortening the operation time and improving detection efficiency.

[0005] Immunochromatographic rapid detection technology is a new diagnostic test that effectively combines immunochromatographic techniques, monoclonal antibody technology, and novel material labeling. A time-resolved fluorescence immunochromatographic assay (TRFIA) was developed based on the luminescence characteristics of lanthanide chelates. TRFIA is reportedly 10 to 100 times more sensitive than colloidal gold and 2 to 4 times more sensitive than latex. Summary of the Invention

[0006] The present invention provides a convenient, rapid, and highly sensitive time-resolved fluorescent immunochromatographic test strip for detecting porcine transmissible gastroenteritis virus (TGEV), and a method for preparing the same. This method employs a double-antibody sandwich antigen detection approach, using a lanthanide element europium-coupled monoclonal antibody specific for the TGEV N protein as a marker. This method develops a time-resolved fluorescent immunochromatographic test strip for rapid TGEV detection. The test strip is simple to operate and can be used for timely, rapid, and sensitive detection of TGEV clinical samples, providing technical support for precise prevention and control measures on pig farms.

[0007] The present invention specifically includes the following contents:

[0008] The invention provides a time-resolved fluorescent immunochromatographic test strip for porcine transmissible gastroenteritis virus. The time-resolved fluorescent immunochromatographic test strip comprises a PVC base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane and the water-absorbing pad are sequentially overlapped on the PVC base plate; a detection line T and a quality control line C are provided on the nitrocellulose membrane, wherein the detection line T is arranged at one end close to the conjugation pad, and the quality control line C is arranged at one end close to the water-absorbing pad; the conjugation pad is coated with anti-TGEV N protein monoclonal antibody 1B7 and rabbit anti-IgG (purchased from abcam, article number: ab6702) labeled with time-resolved fluorescent microspheres; the detection line T is coated with anti-TGEV N protein monoclonal antibody 2A9; and the quality control line C is coated with goat anti-rabbit IgG.

[0009] The heavy chain variable region of the monoclonal antibody 1B7 of the present invention includes a CDR1 with an amino acid sequence of GFNIKDTY, a CDR2 with an amino acid sequence of IDPADGYT, and a CDR3 with an amino acid sequence of ARPGTLDY. The heavy chain variable region of the monoclonal antibody 2A9 of the present invention includes a CDR1 with an amino acid sequence of GYTFTDFN, a CDR2 with an amino acid sequence of INPNNGRS, and a CDR3 with an amino acid sequence of ARRHWDWYFDV.

[0010] The light chain variable region of the monoclonal antibody 1B7 of the present invention includes a CDR1 with an amino acid sequence of KSVSTSGYSY, a CDR2 with an amino acid sequence of LVS, and a CDR3 with an amino acid sequence of QHIRELTR. The light chain variable region of the monoclonal antibody 2A9 of the present invention includes a CDR1 with an amino acid sequence of KSLLHSN, a CDR2 with an amino acid sequence of QMS, and a CDR3 with an amino acid sequence of AQNLEFPWT.

[0011] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B7 of the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2A9 of the present invention is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0012] The DNA sequence of the heavy chain variable region of the monoclonal antibody 1B7 of the present invention is shown in SEQ ID NO: 5, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 6; the DNA sequence of the heavy chain variable region of the monoclonal antibody 2A9 of the present invention is shown in SEQ ID NO: 7, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0013] The heavy chain constant region of the monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus of the present invention is of IgG1 or IgG2b type, and the light chain constant region of the monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus of the present invention is of Kappa type.

[0014] The sample pad is immersed in a sample pad blocking solution; the sample pad blocking solution is a boric acid solution containing 2% BSA, 1% S17, 2% PEG20000, 0.3% sodium caseinate and 0.05% Krovin 300, wherein S17 is a surfactant.

[0015] The preparation method of the conjugate pad is as follows:

[0016] The conjugate pad was immersed in a conjugate pad blocking solution; the conjugate pad blocking solution was a boric acid solution containing 5% m / v trehalose, 1% m / v PEG20000, 1% m / v Tween-20, 1% m / v TritonX-100 and 0.05% Krovin 300.

[0017] Time-resolved fluorescent microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), centrifuged, resuspended in borate buffer, and ultrasonicated to fully disperse the fluorescent microspheres. Anti-TGEV N protein monoclonal antibody 1B7 or rabbit anti-IgG was then added, incubated at room temperature, and fluorescent microsphere marker blocking solution was added. The mixture was incubated at room temperature and centrifuged. The precipitate was resuspended in a fluorescent microsphere marker protective agent to obtain a time-resolved fluorescent microsphere labeling solution of anti-TGEV N protein monoclonal antibody 1B7 or rabbit anti-IgG.

[0018] The time-resolved fluorescent microsphere labeling solution of the anti-TGEV N protein monoclonal antibody 1B7 and the time-resolved fluorescent microsphere labeling solution of rabbit anti-IgG prepared above were mixed in a 1:1 volume ratio and evenly sprayed on the treated conjugate pad.

[0019] The fluorescent microsphere marker blocking solution is an ultrapure aqueous solution containing 10% m / v BSA;

[0020] The fluorescent microsphere marker protective agent is a boric acid solution containing 2% BSA, 1% m / v PVP-40, 0.5% v / v Tween-20 and 0.05% Krovin 300.

[0021] The time-resolved fluorescent microsphere labeling solution of the anti-TGEV N protein monoclonal antibody 1B7 is composed of: 120 μg of TGEV N protein monoclonal antibody 1B7 per 1 ml of fluorescent microsphere solution.

[0022] The rabbit anti-IgG time-resolved fluorescent microsphere labeling solution is composed of: 50 μg rabbit anti-IgG per 1 ml of fluorescent microsphere solution.

[0023] Preferably, the detection line T is coated with 1 mg / ml of TGEV N protein monoclonal antibody 2A9.

[0024] Preferably, the quality control line C is coated with 0.5 mg / ml goat anti-rabbit IgG.

[0025] In a second aspect, the present invention provides a method for preparing a time-resolved fluorescent immunochromatographic test strip for porcine transmissible gastroenteritis virus, the method comprising the following steps:

[0026] (1) preparing a sample pad and a conjugate pad sprayed with a time-resolved fluorescent microsphere-labeled antibody complex;

[0027] (2) Anti-TGEV N protein monoclonal antibody 2A9 (1 mg / ml) and goat anti-rabbit IgG antibody (0.5 mg / ml) were sprayed onto nitrocellulose membrane at intervals of 5–8 mm to serve as the test line T and quality control line C, respectively;

[0028] (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are overlapped on the PVC base plate in sequence to obtain a time-resolved fluorescent immunochromatographic test strip for detecting porcine transmissible gastroenteritis virus.

[0029] In a third aspect, a method for using the test strip of the present invention comprises the following steps:

[0030] (1) Prepare samples to be tested.

[0031] (2) Sample testing

[0032] Take the sample to be tested (determine whether to dilute it according to actual needs) and drop it into the sample addition area of ​​the sample pad. After 13 minutes, put it into the portable dry immunoassay analyzer to observe the results. The sample diluent is: PBS solution containing 0.5% v / v Tween-20.

[0033] (3) Result determination

[0034] Within 30 seconds after the reaction is completed, insert the test strip into the detector and detect by reading the size of the fluorescence signal.

[0035] The beneficial effects of the present invention are:

[0036] The time-resolved fluorescence test strip for TGEV provided by the present invention can be used for specific detection of TGEV pathogens. When used in conjunction with a dry immunofluorescence analyzer, the fluorescence intensity values ​​of specific T lines and C lines can be read. The digitalization of the test results avoids human subjective bias. The minimum limit of the virus that the test strip can detect in the culture is 103.2 TCID 50 The test strip prepared by the present invention has the advantages of good specificity, sensitivity, simple operation, and short detection time, and provides a rapid detection method for on-site or grassroots detection of porcine transmissible gastroenteritis virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 PCR amplification results of the TGEV N gene. M: DL2000 relative molecular mass standard; 1: control water; 2: N gene, approximately 1000 bp in size.

[0038] Figure 2 The expression and purification results of the pET32a-N recombinant protein are shown. (A) M: Protein marker; 1: pET32a-N before induction; 2: pET32a-N after induction; 3: pET32a-N lysis supernatant; 4: pET32a-N lysis precipitate. (B) M: Protein marker; 1: Purified pET32a-N protein.

[0039] Figure 3 ELISA test of mouse antibody titers after N protein immunization. 1-3: immunized mice 1, 2, and 3, NC: negative control.

[0040] Figure 4 Identification of monoclonal antibody reactivity for IFA. (A) 1B7 monoclonal antibody; (B) 2A9 monoclonal antibody; (C) SP2 / 0 cell culture medium.

[0041] Figure 5 Western blot analysis of monoclonal antibody reactivity. (A) 1B7 monoclonal antibody; (B) 2A9 monoclonal antibody; (C) internal control. M: protein marker, Mock: PK cell control; TGEV: TGEV-infected PK cells.

[0042] Figure 6 The results of subclass identification.

[0043] Figure 7 The results of antibody purification SDS-PAGE identification are shown. M: protein marker, 1: 1B7 monoclonal antibody; 2: 2A9 monoclonal antibody.

[0044] Figure 8Variable region PCR amplification results. (A) Heavy chain variable region PCR amplification results. M: DL2000 marker; 1: Water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody; (B) Light chain kappa variable region PCR amplification results. M: DL2000 marker; 1: Water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody.

[0045] Figure 9 Determination of the optimal amount of fluorescent microsphere-labeled antibody.

[0046] Figure 10 Determination of the optimal antibody labeling concentration at the T line.

[0047] Figure 11 Schematic diagram of test strip assembly.

[0048] Figure 12 Determination of the optimal reaction time of the test strip.

[0049] Figure 13 Determination of the critical value of the test strip.

[0050] Figure 14 Test strip sensitivity test results chart.

[0051] Figure 15 Graph of test strip specificity detection results.

[0052] Figure 16 Test strip stability test results. DETAILED DESCRIPTION

[0053] The present invention will be described in more detail below through specific implementation methods to facilitate understanding of the technical solution of the present invention, but it is not intended to limit the scope of protection of the present invention.

[0054] Example 1 Preparation of monoclonal antibodies against the N protein of porcine infectious diarrhea coronavirus

[0055] 1. Construction, expression, and purification of TGEV N protein recombinant plasmid

[0056] 1.1 Construction of pET32a-N recombinant plasmid

[0057] The TGEV strain was HLJ-17 (Genbank accession number: MT522161.1). The N protein of this strain was used as a reference to design specific primers. The upstream P1:5 ′ -CGC GGATCC ATGGCCAACCAGGGACAACG-3 ′ , downstream P2: 5 ′ -CCG CTCGAGTTAGTTCGTTACCTCATCAATTATC-3 ′ , upstream and downstream primers 5 ′ BamHI and XhoI restriction sites (underlined parts) were introduced at the end. After primer synthesis, the supernatant RNA of TGEV cells infected with PK15 cells was extracted and reverse transcribed into cDNA, which was then used as a template for PCR amplification. The PCR reaction system was: PrimeSTAR Max Premix (2×) 25μL, 1μL each of P1 and P2, 1μL of cDNA, and ddH2O was added to 50μL. The reaction procedure was: pre-denaturation at 98℃ for 2min; denaturation at 98℃ for 10s, annealing at 55℃ for 30s, extension at 72℃ for 2min, 30 cycles; extension at 72℃ for 10min. The PCR product was detected by 1% agarose gel electrophoresis, and the results are as follows. Figure 1 As shown, the size of the TGEV N fragment is approximately 1000 bp. Gel extraction was then performed according to the instructions of the EZNAGel Extraction Kit from Omega.

[0058] The TGEV N gene PCR gel-recovered product was digested with the pET32a vector using BamHI and XhoI, ligated, and transformed into DH5α competent cells. The constructed recombinant plasmid was sequenced and identified, and the positive recombinant plasmid was designated pET32a-N.

[0059] 1.2N recombinant protein induction and purification

[0060] The recombinant plasmid pET32a-N was transformed into Escherichia coli BL21 (DE3), and a single colony was picked and placed in 5 mL of ampicillin-resistant LB medium. 600 When the concentration was about 0.8, IPTG was added to a final concentration of 1 mmol / L to induce expression. 5 hours after induction, the precipitate was collected by centrifugation at 12000 rpm for 2 minutes. The precipitate was resuspended in an appropriate amount of PBS and sonicated for 5 minutes (supersonic for 3 seconds, stop for 3 seconds). After sonication, the supernatant and precipitate were centrifuged at 4°C for 10 minutes, and the supernatant and precipitate were collected separately. The supernatant and precipitate were added to 5× loading buffer, boiled in boiling water for 10 minutes, and identified by SDS-PAGE. The results showed that pET32a-N was expressed in both the supernatant and the precipitate ( Figure 2 A), indicating that part of the N protein is soluble.

[0061] The pET32a-N protein was induced to express in amplified according to the above method. The supernatant was collected after sonication and purified by nickel column. The specific purification steps are as follows:

[0062] (1) Resin loading: Take an empty column and add 2 mL of nickel column NTA resin. When the preservation solution drops to the surface of the resin, wash the column once with 5 column volumes of distilled water, and then equilibrate the column with 5 column volumes of equilibration solution (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.4);

[0063] (2) Sample loading: When the equilibrium solution reaches the resin surface, add 3 mL of lysate containing the recombinant protein. Repeat the sample loading 2 to 3 times, each time for 2 minutes, and collect the sample flow-through. Rinse the column once with 5 column volumes of equilibrium solution.

[0064] (3) Protein elution: The protein sample was eluted with different concentrations of imidazole (25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM and 300 mM) using the equilibrium solution, 1 mL each time, and each concentration was repeated twice. The purpose was to determine the optimal concentration of imidazole for washing and protein elution;

[0065] (4) Column cleaning and storage: Rinse the column once with 5 volumes of 0.5M NaOH, then rinse once with distilled water, then add an appropriate amount of 70% anhydrous ethanol and store in a refrigerator at 4°C. Each collected sample was identified by SDS-PAGE. Figure 2 As shown in B, a high-purity N protein was obtained.

[0066] 2. Animal immunization

[0067] Three 6-week-old female BALB / c mice were immunized with 30 μg of purified N recombinant protein per mouse. For the first immunization, equal volumes of N protein were mixed with complete Freund's adjuvant, emulsified, and injected subcutaneously at multiple sites (one on the back and two on the abdomen). Booster immunizations were performed every two weeks for a total of four immunizations. Booster immunizations consisted of equal volumes of N recombinant protein mixed with incomplete Freund's adjuvant, emulsified, using the same method as the first immunization. Seven days after the fourth immunization, tail blood was collected to determine antibody titers.

[0068] 3. ELISA method to detect polyclonal antibody titer

[0069] The TGEV virus solution and coating solution were diluted 1:1 and coated on the ELISA plate, 50 μL / well, coated overnight at 4°C, washed four times with PBST, and blocked with 2% trehalose at 4°C for 10 hours. The positive serum and negative serum were diluted with PBST in a series ratio, with a dilution gradient from 1:100 to 1:12800, with 8 dilution gradients, at 37°C for 1 hour, washed four times with PBST, and HRP-labeled goat anti-mouse IgG (1:20000 dilution) was added. After washing four times with PBST, TMB was used for color development and the OD was measured on a microplate reader. 450 .

[0070] The results are as follows Figure 3 As shown, the serum of non-immunized mice was used as negative control (NC). When the serum was diluted 1:102400, the OD values ​​of immunized mice No. 1, 2 and 3 were 450 It was still greater than that of the NC group, indicating that the antibody titer could reach above 1:102400.

[0071] 4. Preparation of Monoclonal Antibodies

[0072] The specific steps are as follows:

[0073] (1) Preparation of feeder layer cells: Inject HAT culture medium into the peritoneal cavity of female BALB / c mice, slowly and repeatedly aspirate the liquid, and then plate it into a 96-well plate.

[0074] (2) Cell fusion: Splenocytes and an appropriate amount of SP20 cells were fused with the fusion agent PEG. The fused cells were plated in a 96-well plate containing feeder cells.

[0075] (3) Screening of positive clones:

[0076] a) Indirect ELISA detection method: the purified N protein is coated on an ELISA plate to detect the secretion of antibodies by the fused cells.

[0077] b) Indirect immunofluorescence (IFA) detection method: the supernatant from the ELISA antibody-positive cell wells was added to TGEV-infected PK15 cells, and then FITC-labeled goat anti-mouse IgG was added. After the reaction was completed, the results were observed under a fluorescence microscope.

[0078] (4) Subcloning of positive hybridoma cells: Select ELISA and IFA positive wells and subclone the screened positive hybridoma cells using the limiting dilution method. Observe under an inverted microscope, mark the wells where only a single clone has grown, collect the supernatant, and perform antibody detection using the above ELISA and IFA methods. Positive cells will enter the next round of subcloning, and this will be repeated three times.

[0079] (5) Preparation of ascites: 10-12 week old Balb / c mice were taken and 0.5 mL of Freund's incomplete adjuvant was injected intraperitoneally into each mouse. 1 week later, 5 × 10 5 After 7 to 10 days, the abdominal cavity of the mouse was obviously bulging, and the ascites was collected. The titer was detected by ELISA, and the cells were aliquoted and stored at -80℃.

[0080] (6) Purification of ascites: Affinity chromatography was performed using the PIERCE NAb™ Protein G Spin Purification Kit, and the purity was determined by SDS-PAGE electrophoresis. The heavy and light chains of the purified 1B7 and 2A9 mAbs were approximately 55 kDa and 25 kDa, respectively. Figure 7 ) This indicates that the antibody bands after purification by this method are correct and the purity is high.

[0081] 5. Identification of Monoclonal Antibodies

[0082] (1) Specificity identification: including IFA and Western blot verification.

[0083] a) IFA verification: PK15 cells were infected with TGEV. 36 hours after virus infection, the cells were fixed with paraformaldehyde. After cell fixation, ascites was diluted with PBS (500 times) and added to the virus-infected cells. Then, FITC-anti-mouse secondary antibody (100 times dilution) was incubated and the cells were observed under a fluorescence microscope after the reaction was completed. The results are shown in Figure 2. Figure 4 As shown in A and 4B, monoclonal antibodies 1B7 and 2A9 can detect specific green fluorescence signals when used on TGEV-infected PK15 cells, while no fluorescence signals were observed when the supernatant of SP2 / 0 cells was used on TGEV-infected cells ( Figure 4 C).

[0084] b) Western blot verification: Purified N protein or virus-infected and uninfected PK15 cells were collected for SDS-PAGE, and then the protein gel was transferred to NC membrane for western blot verification. The primary antibody was monoclonal antibody 1B7 or 2A9, and the secondary antibody was HRP-anti-mouse IgG. The results are shown in Figure 2. Figure 5 As shown in A and 5B, monoclonal antibodies 1B7 and 2A9 can both recognize the N protein in virus-infected cells. Figure 5 C is the detection of the intracellular reference β-actin.

[0085] (2) Subclass identification: The obtained monoclonal antibodies were identified according to the instructions of the Southern Biotech SBA Clonotyping™ System / HRP Antibody Subclass Identification Kit. The results showed that Figure 6 The heavy chain constant regions of 1B7 monoclonal antibody are of IgG1 type, and those of 2A9 are of IgG2b type, and their light chain constant regions are of Kappa type.

[0086] 6. PCR amplification and sequence determination of monoclonal antibody variable region genes

[0087] First, RNA of monoclonal antibody hybridoma cells was extracted and reverse transcribed into cDNA using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A).

[0088] The antibody variable region genes were amplified using nested PCR. First, the variable region genes were amplified using the aforementioned cDNA as a template using the first round of mouse IgG1 and κ light chain primers. Then, the first round product was used as a template for the second round of mouse IgG1 and κ light chain primers. The PCR reaction system consisted of 25 μL of PrimeSTAR Max Premix (2×), 1 μL each of P1 and P2, 1 μL of cDNA, and ddH2O to 50 μL. The reaction procedure was as follows: pre-denaturation at 98°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and extension at 72°C for 10 min. Primers for antibody variable region gene amplification were referenced from the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.).

[0089] After amplification, 1% agarose gel electrophoresis was performed and the gene sizes of the heavy chain and κ light chain variable regions were approximately 300 bp ( Figure 8 A and 8B), the target fragment was recovered by gel excision and inserted into the pMD-19T vector for sequence determination.

[0090] The DNA sequence of the heavy chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO: 5, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 6; the DNA sequence of the heavy chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO: 7, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0091] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0092] The heavy chain variable region of monoclonal antibody 1B7 includes CDR1 with the amino acid sequence of GFNIKDTY, CDR2 with the amino acid sequence of IDPADGYT, and CDR3 with the amino acid sequence of ARPGTLDY; the heavy chain variable region of monoclonal antibody 2A9 includes CDR1 with the amino acid sequence of GYTFTDFN, CDR2 with the amino acid sequence of INPNNGRS, and CDR3 with the amino acid sequence of ARRHWDWYFDV.

[0093] The light chain variable region of monoclonal antibody 1B7 includes CDR1 with the amino acid sequence of KSVSTSGYSY, CDR2 with the amino acid sequence of LVS, and CDR3 with the amino acid sequence of QHIRELTR; the light chain variable region of monoclonal antibody 2A9 includes CDR1 with the amino acid sequence of KSLLHSN, CDR2 with the amino acid sequence of QMS, and CDR3 with the amino acid sequence of AQNLEFPWT.

[0094] Example 2 Preparation of Time-Resolved Fluorescent Immunochromatographic Test Strips for Detecting Porcine Transmissible Gastroenteritis Virus

[0095] 1. Preparation of the Conjugate Pad

[0096] 1.1 Determination of the optimal amount of fluorescent microsphere-labeled TGEV N protein monoclonal antibody 1B7

[0097] The prepared TGEV N protein monoclonal antibody 1B7 was coupled with time-resolved Eu fluorescent microspheres (purchased from Nanjing Microtest Biotechnology Co., Ltd.) to a final concentration of 1B7 of 160 μg / mL, 140 μg / mL, 120 μg / mL, 100 μg / mL, and 50 μg / mL, respectively. After successful coupling, the conjugate was stored at 4°C overnight and found that when the final concentration was 50 μg / mL, almost all of the conjugates precipitated, indicating that the coupling efficiency was too low and unsuccessful. The other four successfully labeled conjugates were used to test the positive and negative controls, such as Figure 9 As shown in the figure, when the final antibody concentration is 120 μg / mL, the T1 / T2 value is the largest, so this concentration is selected as the optimal fluorescent microsphere-labeled antibody amount.

[0098] 1.2 Preparation of TGEV N protein monoclonal antibody 1B7 and rabbit anti-IgG time-resolved fluorescent microsphere markers

[0099] Take two 800μl portions of 0.05M pH8.2 boric acid buffer and add them to 2mL centrifuge tubes respectively, add 200μL of time-resolved Eu fluorescent microspheres with a solid content of 1% (Nanjing WeiCe Biotechnology Co., Ltd.) to each and mix gently; then add 20μL of 10mg / mL EDC (purchased from Sigma, product number: 03449) and 50μL of 10mg / mL NHS (purchased from Sigma, product number: 8045180025), mix thoroughly and activate at room temperature for 15min, centrifuge at 14000r for 30min; remove the supernatant solution and resuspend with 0.05mol / LpH8.2 boric acid buffer; if there is a precipitate that is obviously difficult to mix, sonicate at 100W for 3s, stop for 3s, and sonicate for a total of 1min to completely disperse the time-resolved Eu fluorescent microspheres; add 120μg TGEV to the solution respectively Incubate the N protein monoclonal antibody 1B7 and 50 μg rabbit anti-IgG at room temperature for 2 h; add 110 μl of 10% BSA solution for blocking, incubate at room temperature for 2 h, centrifuge at 14,000 rpm for 30 min, and discard the supernatant; finally, add 1 mL of fluorescent microsphere marker protective agent and resuspend; if there is a precipitate that is obviously difficult to mix, sonicate at 100 W for 3 seconds, then 3 seconds, for a total of 1 minute, and store at 4°C until use.

[0100] 1.3 Preparation of Time-Resolved Fluorescent Microsphere-Antibody Conjugate Pad

[0101] Soak the conjugate pad in a blocking solution (boric acid solution containing 5% m / v trehalose, 1% m / v PEG 20000, 1% m / v Tween-20, 1% m / v Triton X-100, and 0.05% Krovin 300) at 37°C–38°C for 2 hours. Then, remove and dry in an oven for 2–3 hours. Mix the prepared complex of fluorescent microsphere-labeled anti-TGEV N protein monoclonal antibody 1B7 and rabbit anti-IgG in a 1:1 volume ratio and evenly spray the conjugate pad using a film sprayer. Dry in a 37°C oven for 2–3 hours. Remove and store at 4°C in a sealed container.

[0102] 2. Preparation of Sample Pad

[0103] At 37°C to 38°C, soak the sample pad in sample pad blocking solution (boric acid solution containing 2% BSA, 1% S17, 2% PEG20000, 0.3% sodium caseinate and 0.05% Krovin 300) for 2 hours, take it out and place it in an oven to dry for 2 to 3 hours, and then place it in an aluminum foil bag to dry for later use.

[0104] 3. Preparation of Nitrocellulose Membrane

[0105] In order to determine the optimal coating concentration of the antibody at the T line, the monoclonal antibody 2A9 against TGEV N protein was diluted to 0.5, 1, 1.5, and 2 mg / ml, sprayed on the T line, and TGEV cytotoxicity (positive control) and uninfected healthy cell supernatant (negative control) were added. After reacting for 13 minutes, the values ​​were read on a dry fluorescent immunoassay. The fluorescence value T1 at the positive control T line was recorded; the fluorescence value T2 at the negative control T line was recorded, and the parameters when T1 / T2 was the largest were selected as the optimal conditions. The test results are as follows: Figure 10 As shown, when the concentration of the anti-TGEVN protein monoclonal antibody 2A9 was 1 mg / ml, the T1 / T2 value was the largest.

[0106] 1 mg / ml anti-TGEV N protein monoclonal antibody 2A9 and 0.5 mg / ml goat anti-rabbit IgG antibody were sprayed on the nitrocellulose membrane at intervals of 5 to 8 mm to serve as the test line (T) and quality control line (C), respectively. After the lines were drawn, they were placed at 37°C to 38°C to dry for 2 to 3 hours.

[0107] 4. Assembly of test strips

[0108] The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are connected on the PVC base plate in sequence to obtain a test strip. The test strip is cut into strips according to the size of the test strip card, and then the cut test strips are loaded into the test strip card. The structural diagram of the test strip is shown in FIG. Figure 11 shown.

[0109] Example 3 Application of Time-Resolved Fluorescent Immunochromatographic Test Strips for Detecting Porcine Transmissible Gastroenteritis Virus

[0110] 1. How to use the test strips

[0111] (1) Detection: Take out the test strip and equilibrate it to room temperature. Add the sample to be tested to the sample adding area of ​​the sample pad, let it stand at room temperature, and determine the result.

[0112] (2) Result determination: Insert the test strip into the dry immunoassay analyzer and determine the result by reading the size of the fluorescent signal.

[0113] 2. Determination of the optimal reaction time of the test strip

[0114] For test strips, the determination of detection time greatly affects the overall reaction process. If the time is too short, it may lead to insufficient overall reaction. On the contrary, if the time is too long, it will lead to a waste of detection time. After adding TGEV positive control and negative control, the detection was carried out at intervals of 0, 1, 3, 5, 7, 9, 11, 13, 15, and 17 minutes, and the fluorescence values ​​T1 and T2 were recorded in time. The T1 / T2 value was calculated and a trend chart of the change of T1 / T2 value over time was drawn. The results are as follows Figure 12As shown in the figure, when the exposure time is 13 minutes, the T1 / T2 value is the largest, indicating that the optimal reaction time of the test strip is 13 minutes.

[0115] 3. Determination of the critical value of the test strip

[0116] Use the assembled test strips to test 30 TGEV-negative anal swab samples, record the T value after the test, and calculate the corresponding average value and standard deviation (SD). According to the formula The critical value for judging positive or negative. Figure 13 As shown, the average T value of 30 negative samples The value is 1254 and the SD value is 610, so the critical value of this test strip is 3084.

[0117] 4. Sensitivity of the test strip

[0118] The TGEV TCID50 was 10 7.8 After 5-fold dilution of the virus / 0.1mL, the sensitivity of the established test strip was tested by 2-fold dilution. 1 to 1:2 13 When diluted, the T value of the test line is greater than the critical value; and 1:2 14 When the test strip is released, the T value is less than the critical value. Therefore, the sensitivity of the test strip reaches 1:2 13 , that is, the minimum virus detection amount of the present invention is 10 3.2 TCID 50 / 0.1mL, such as Figure 14 shown.

[0119] 5. Specificity of the test strip

[0120] TGEV, SADS-CoV, PEDV and PDCoV were detected by test strips, and it was found that only TGEV was positive, and the others had no cross reaction, indicating that the method established by the present invention can specifically identify TGEV. Figure 15 shown.

[0121] 6. Test strip stability test

[0122] Stability is an important factor for future applications. The test strips were stored at room temperature (18-25°C) and 4°C in the dark. Stability tests were performed on the test strips at 0, 1, 2, 3, 6, 9, 12, and 15 months. Figure 16 It can be seen that the test strips can be stably stored for 9 months at room temperature and for 12 months at 4°C.

[0123] Table 1 Test strip stability test results

[0124]

[0125] The embodiments described above are only preferred embodiments of the present invention and do not limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus, comprising a PVC base, a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad, wherein the sample pad, conjugation pad, nitrocellulose membrane, and water-absorbing pad are sequentially overlapped on the PVC base; a detection line T and a quality control line C are provided on the nitrocellulose membrane, wherein the detection line T is provided at one end near the conjugation pad, and the quality control line C is provided at one end near the water-absorbing pad; and characterized in that: The conjugate pad is coated with time-resolved fluorescent microspheres-labeled anti-TGEV N protein monoclonal antibody 1B7 and rabbit anti-IgG; the detection line T is coated with anti-TGEV N protein monoclonal antibody 2A9; the quality control line C is coated with goat anti-rabbit IgG; The heavy chain variable region of monoclonal antibody 1B7 includes a CDR1 with an amino acid sequence of GFNIKDTY, a CDR2 with an amino acid sequence of IDPADGYT, and a CDR3 with an amino acid sequence of ARPGTLDY; the light chain variable region of monoclonal antibody 1B7 includes a CDR1 with an amino acid sequence of KSVSTSGYSY, a CDR2 with an amino acid sequence of LVS, and a CDR3 with an amino acid sequence of QHIRELTR; The heavy chain variable region of monoclonal antibody 2A9 includes CDR1 with the amino acid sequence of GYTFTDFN, CDR2 with the amino acid sequence of INPNNGRS, and CDR3 with the amino acid sequence of ARRHWDWYFDV; the light chain variable region of monoclonal antibody 2A9 includes CDR1 with the amino acid sequence of KSLLHSN, CDR2 with the amino acid sequence of QMS, and CDR3 with the amino acid sequence of AQNLEFPWT.

2. The time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; The amino acid sequence of the heavy chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

3. The time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus according to claim 1, characterized in that: The DNA sequence of the heavy chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO: 5, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 6; The DNA sequence of the heavy chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO: 7, and the DNA sequence of the light chain variable region is shown in SEQ ID NO:

8.

4. The time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus according to claim 1, characterized in that: The sample pad was immersed in a sample pad blocking solution; the sample pad blocking solution was a boric acid solution containing 2% BSA, 1% S17, 2% PEG20000, 0.3% sodium caseinate and 0.05% Krovin 300.

5. The time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus according to claim 1, characterized in that: The preparation method of the conjugate pad is as follows: Soak the conjugate pad in a conjugate pad blocking solution; The time-resolved fluorescent microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, centrifuged, resuspended in boric acid buffer, and ultrasonicated to fully disperse the fluorescent microspheres. Anti-TGEV N protein monoclonal antibody 1B7 or rabbit anti-IgG was then added, incubated at room temperature, and fluorescent microsphere marker blocking solution was added. The mixture was incubated at room temperature and centrifuged. The precipitate was resuspended in a fluorescent microsphere marker protective agent to obtain a time-resolved fluorescent microsphere labeling solution of anti-TGEV N protein monoclonal antibody 1B7 or rabbit anti-IgG. The time-resolved fluorescent microsphere labeling solution of the anti-TGEV N protein monoclonal antibody 1B7 and the time-resolved fluorescent microsphere labeling solution of rabbit anti-IgG were mixed in a 1:1 volume ratio and evenly sprayed on the treated conjugate pad.

6. The time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus according to claim 5, characterized in that: The conjugate pad blocking solution is a boric acid solution containing 5% m / v trehalose, 1% m / v PEG20000, 1% m / v Tween-20, 1% m / v TritonX-100 and 0.05% Krovin 300; The fluorescent microsphere marker blocking solution is an ultrapure aqueous solution containing 10% m / v BSA; The fluorescent microsphere marker protective agent is a boric acid solution containing 2% BSA, 1% m / v PVP-40, 0.5% v / v Tween-20 and 0.05% Krovin 300.

7. The time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus according to claim 5, characterized in that: The time-resolved fluorescent microsphere labeling solution of the anti-TGEV N protein monoclonal antibody 1B7 is composed of: 120 μg of TGEV N protein monoclonal antibody 1B7 per 1 ml of fluorescent microsphere labeling solution; The composition of the rabbit anti-IgG time-resolved fluorescent microsphere labeling solution is as follows: 50 μg rabbit anti-IgG per 1 ml of fluorescent microsphere labeling solution.

8. The time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus according to claim 1, characterized in that: The test line T is coated with 1 mg / ml of TGEV N protein monoclonal antibody 2A9; The quality control line C was coated with 0.5 mg / ml goat anti-rabbit IgG.

9. The method for preparing a time-resolved fluorescent immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus according to claim 1, characterized in that: The following steps are involved: (1) Prepare a sample pad and a binding pad coated with time-resolved fluorescent microspheres labeled with anti-TGEV N protein monoclonal antibody 1B7 and rabbit anti-IgG; (2) Anti-TGEV N protein monoclonal antibody 2A9 and goat anti-rabbit IgG antibody were sprayed on the nitrocellulose membrane alternately, serving as the test line T and the quality control line C, respectively; (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are overlapped on the PVC base plate in sequence to obtain a time-resolved fluorescent immunochromatographic test strip for detecting porcine transmissible gastroenteritis virus.

10. The preparation method according to claim 9, characterized in that The distance between the test line T and the quality control line C is 5 to 8 mm.

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