Porcine Lawsonia intracellularis antibody detection test paper and application thereof

Abstract

The invention provides swine Lawsonia intracellularis antibody detection test paper and application thereof, and belongs to the technical field of biological detection. According to the swine Lawsonia intracellularis antibody detection test paper developed on the basis of the immunochromatography technology, by optimizing antigen labeling and signal amplification strategies, rapid and high-sensitivity detection of antibodies in serum / whole blood samples is achieved. Compared with traditional ELISA and IFA methods, the technology does not need to depend on large instruments, the detection time is shortened to be within 10 minutes, high specificity and stability are achieved, and cross reaction and complex environment interference can be effectively avoided. The test paper supports portable fluorescent quantitative interpretation, is suitable for scenes such as grassroots farms and veterinary clinics, provides a reliable tool for real-time monitoring of infection states of swinery, assists accurate prevention and control and epidemiological investigation, remarkably reduces the detection cost, improves the prevention and control efficiency, and has important application value for promoting sustainable development of the pig breeding industry.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a test paper for detecting antibodies against Lawsonia intracellularis of pigs and an application thereof. Background Art

[0002] Lawsonia intracellularis is an obligate intracellular Gram-negative bacterium that primarily invades the intestinal epithelium of pigs, causing porcine proliferative enteropathy (PPE). This disease, characterized by adenomatous hyperplasia of the intestinal mucosa, diarrhea, and growth retardation, causes economic losses to the swine industry. Because the bacterium is difficult to culture in vitro and has a high latent infection rate, serological testing has become a key tool in epidemiological surveys and vaccine efficacy assessment.

[0003] Currently, the detection methods for antibodies to Lawsonia intracellularis suis are mainly divided into two categories: 1. Serological tests based on antigen-antibody reactions, such as the enzyme-linked immunosorbent assay (ELISA), are available. Patent CN201710821996 proposes an ELISA method using a recombinant LsaA protein as the coating antigen. While this method avoids the difficulty of preparing whole-bacterial antigens, it relies on a microplate reader for interpretation, requiring a 2-hour or longer procedure. It also exhibits low sensitivity (serum dilutions of only 1:800), and the antigen is susceptible to cross-reactions with other pathogens. Another approach is immunofluorescence assay (IFA), such as patent CN202110855204, which utilizes an Hsp60 monoclonal antibody for immunofluorescence detection. While highly specific, it requires fluorescence microscopy for interpretation, resulting in high costs and the inability to achieve quantitative analysis, limiting its applicability in primary farms.

[0004] 2. Molecular biological testing, such as PCR and fluorescent quantitative PCR, is sensitive and specific, but it can only detect pathogen nucleic acids and cannot reflect antibody levels. It also relies on specialized equipment and is difficult to meet the needs of rapid on-site screening.

[0005] Existing methods generally suffer from strong equipment dependence, complex operation, insufficient sensitivity, or high cost. Therefore, developing a rapid, highly sensitive, and quantitative antibody detection technology that does not require complex instrumentation is of great significance for achieving real-time monitoring of pig infection status and precise prevention and control. The immunochromatographic test strip technology of the present invention aims to overcome these limitations by optimizing antigen selection and labeling strategies, providing pig farms with an efficient and economical detection tool. Summary of the Invention

[0006] The purpose of the present invention is to provide a test strip for detecting antibodies to Lawsonia intracellularis in pigs and its application. The detection time is shortened to 10 minutes and no complex equipment is required. The signal intensity of the fluorescent microspheres is 10 times that of colloidal gold, and the sensitivity is increased to 1:3200. In addition, the OMP antigen used in the present invention is highly conservative and specific, and can cover the main prevalent strains of Lawsonia intracellularis in pigs, avoiding missed detection due to strain variation. In addition, the test strip has no cross-reaction with common pathogens such as porcine circovirus and classical swine fever virus.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a fluorescent test strip for detecting antibodies to the outer membrane protein of intracellular Lawsonia intracellularis, comprising a base plate, a sample pad, a conjugation pad, a chromatography membrane, and an absorption pad; the sample pad, the conjugation pad, the chromatography membrane, and the absorption pad are sequentially arranged on the base plate in the order of liquid flow; a detection line and a quality control line are sequentially arranged on the chromatography membrane; The test line contains anti-swine IgG monoclonal antibodies; The quality control line contains goat anti-rabbit IgG; The conjugate pad contains a fluorescent microsphere-antigen complex; The fluorescent microsphere-antigen complex is obtained by coupling polyethyleneimine-modified microspheres and intracellular Lawsonia outer membrane protein antigen; The amino acid sequence of the Lawsonia intracellularis outer membrane protein antigen is shown in SEQ ID NO.1.

[0008] Preferably, the Lawsonia intracellularis outer membrane protein antigen is produced by recombinantly expressing a gene encoding the Lawsonia intracellularis outer membrane protein, and the nucleotide sequence of the gene encoding the outer membrane protein is shown in SEQ ID NO.3.

[0009] Preferably, the polyethyleneimine modified microspheres are obtained by a preparation method comprising the following steps: Will Eu 3+ The doped polystyrene microspheres are mixed with polyethyleneimine to react and obtain polyethyleneimine modified microspheres.

[0010] Preferably, the Eu 3+ The particle size of the doped polystyrene microspheres is 100–300 nm; The Eu 3+ The mass ratio of the doped polystyrene microspheres to polyethyleneimine is 1:0.05~0.15.

[0011] Preferably, the reaction time is 1.5 to 2.5 hours.

[0012] Preferably, the fluorescent microsphere-antigen complex is obtained by a preparation method comprising the following steps: Polyethyleneimine-modified microspheres and intracellular Lawsonia outer membrane protein antigen were mixed at a mass ratio of 1:4-6, and EDC and NHS were added for coupling reaction to obtain fluorescent microsphere-antigen complexes.

[0013] Preferably, the coupling reaction time is 1.5 to 2.5 hours; The coupling reaction was carried out under light-protection conditions.

[0014] The present invention also provides the use of the above-mentioned Lawsonia intracellularis outer membrane protein antibody fluorescent detection test paper in preparing a porcine Lawsonia intracellularis antibody detection product.

[0015] The present invention also provides a system for detecting antibodies to intracellular Lawsoniae, comprising the above-mentioned fluorescent detection test paper for outer membrane protein antibodies to intracellular Lawsoniae and a fluorescent reading detection device.

[0016] Preferably, the excitation wavelength of the fluorescence reading detection device is 365 nm, and the emission wavelength is 615 nm.

[0017] Beneficial effects of the present invention: The porcine intracellular Lawsoniae antibody detection test strip developed by the present invention based on immunochromatographic technology realizes rapid and highly sensitive detection of antibodies in serum / whole blood samples by optimizing antigen labeling and signal amplification strategies. Compared with traditional ELISA and IFA methods, this technology does not need to rely on large instruments, the detection time is shortened to less than 10 minutes, and it has both high specificity and stability, which can effectively avoid cross-reactions and complex environmental interference. The test strip supports portable fluorescence quantitative interpretation and is suitable for scenarios such as grassroots farms and veterinary clinics. It provides a reliable tool for real-time monitoring of the infection status of pig herds, assists in precise prevention and control and epidemiological surveys, significantly reduces detection costs and improves prevention and control efficiency, and has important application value in promoting the sustainable development of the pig farming industry. DETAILED DESCRIPTION

[0018] The present invention provides a fluorescent test strip for detecting antibodies to an intracellular Lawsonia spp. outer membrane protein, comprising a base plate, a sample pad, a conjugation pad, a chromatography membrane, and an absorption pad; the sample pad, the conjugation pad, the chromatography membrane, and the absorption pad are sequentially arranged on the base plate according to the order of liquid flow; a detection line and a quality control line are sequentially arranged on the chromatography membrane; the detection line contains an anti-pig IgG monoclonal antibody; the quality control line contains a goat anti-rabbit IgG; the conjugation pad contains a fluorescent microsphere-antigen complex; the fluorescent microsphere-antigen complex is obtained by coupling polyethyleneimine-modified microspheres and an intracellular Lawsonia spp. outer membrane protein antigen; the amino acid sequence of the intracellular Lawsonia spp. outer membrane protein antigen is shown in SEQ ID NO.1.

[0019] In the present invention, preferably, the outer membrane protein antigen of Lawsonia intracellularis is obtained by recombinantly expressing the coding gene of the outer membrane protein of Lawsonia intracellularis, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.3. Preferably, the polyethyleneimine modified microspheres are obtained by a preparation method comprising the following steps: 3+ Doped polystyrene microspheres are mixed with polyethyleneimine to obtain polyethyleneimine modified microspheres. 3+ The particle size of the doped polystyrene microspheres is 100-300 nm; the Eu 3+ The mass ratio of the doped polystyrene microspheres to the polyethyleneimine is 1:0.05~0.15. Preferably, the reaction time is 1.5~2.5h. Preferably, the fluorescent microsphere-antigen complex is obtained by a preparation method comprising the following steps: mixing polyethyleneimine-modified microspheres and intracellular Lawsonia outer membrane protein antigens in a mass ratio of 1:4~6, adding EDC and NHS, and performing a coupling reaction to obtain a fluorescent microsphere-antigen complex. Preferably, the coupling reaction time is 1.5~2.5h; and the coupling reaction is carried out under light-proof conditions.

[0020] The present invention also provides the use of the above-mentioned Lawsonia intracellularis outer membrane protein antibody fluorescent detection test paper in preparing a porcine Lawsonia intracellularis antibody detection product.

[0021] The present invention also provides a system for detecting antibodies to Lawsonia intracellularis, comprising the above-mentioned fluorescent test strips for detecting antibodies to Lawsonia intracellularis outer membrane protein and a fluorescence reading detection device. Preferably, the fluorescence reading detection device has an excitation wavelength of 365 nm and an emission wavelength of 615 nm.

[0022] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Sequence information involved in the embodiment: Lawsonia intracellularis OMP protein amino acid sequence: >ACC77464.1 outer membrane protein [Lawsonia intracellularis] MKIIHSAIFAVTLLTAWSTVCFAAEVTASCTKRVESYNYLVDYSGSMMMKHVAVREPKIELAKEAILKINAAMPKMSYQGGLYTFAPYSVIIPQGSWNSCVAECAVNTIKSDLEIFGRLTPMGDGIKMHETVINQMPPQAAVILLTDGHNNLGMNPVEEVKSIYQTNPNVCFHVASFADDAEGKAIIDQIVALNSGSVLVDGLQLLQNPAVCQEFVNSVFCQGQILVTEEVVVLRGVNFAFDSFALDDTAKAISEETVRLIRANPDFNVRLLGWTDSTGPDAYNLRLSQERADAVKNFLVKMGIPSNRLFAKGMGKSYQYNNATKEGRYMNRRTELVFFD, as shown in SEQ ID NO.1; Base sequence of the OMP gene of Lawsonia intracellularis >EU621796.1 Lawsonia intracellularis clone CG123453 outer membraneprotein gene, complete cds Base sequence after codon optimization: Example 1 OMP antigen preparation: Step 1: Gene synthesis and cloning We commissioned Sangon Biotechnology to synthesize the codon-optimized OMP gene (SEQ ID NO. 3), and introduced EcoRI and SalI restriction sites at both ends; The double-enzyme-digested pET-28a vector and the OMP gene fragment were ligated using T4 ligase at 16°C for 12 h; The ligation product was transformed into Escherichia coli DH5α competent cells, coated on LB plates containing kanamycin (50 μg / mL), cultured at 37°C for 16 hours, and single colonies were picked for sequencing verification.

[0024] Step 2: Recombinant protein expression The verified correct recombinant plasmid was transformed into Escherichia coli BL21 (DE3); Inoculate a single colony into LB liquid medium containing kanamycin and culture at 37°C with shaking until OD 600 =0.6; IPTG (final concentration 0.5 mM) was added and expression was induced at 20°C for 16 h, and the bacteria were collected by centrifugation.

[0025] Step 3: Protein purification After ultrasonic disruption, the cells were centrifuged (12000 rpm, 30 min), and the supernatant was passed through a Ni-NTA affinity chromatography column; Contaminants were eluted with PBS buffer containing 20 mM, 50 mM, and 100 mM imidazole, and finally the target protein was eluted with a buffer containing 250 mM imidazole. The eluate was dialyzed into PBS (pH 7.4) and further purified through Superdex 200 molecular sieves to obtain OMP antigen with a purity of ≥95% (concentration 2.5 mg / mL), the amino acid sequence of which is shown in SEQ ID NO.1.

[0026] Fluorescent microsphere labeling and test strip assembly: Step 1: Microsphere surface modification Take commercially available Eu 3+ 1 mg of doped polystyrene microspheres (particle size 200 nm) were dispersed in 1 mL of PBS (pH 7.4); Polyethyleneimine (PEI, molecular weight 10 kDa) was added to a final concentration of 0.1 mg / mL and shaken at 25°C for 2 h; Free PEI was removed by centrifugation (10,000 rpm, 15 min), and the pellet was resuspended in PBS to obtain PEI-modified microspheres.

[0027] Step 2: Antigen conjugation PEI-modified microspheres were mixed with OMP antigen at a mass ratio of 1:5 (1 mg microspheres, 5 mg antigen), and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added to the final concentrations of 0.4 mM and 0.1 mM, respectively; The reaction was carried out at 25°C in the dark for 2 hours, and the unbound antigen was removed by centrifugation (10,000 rpm, 15 minutes). The precipitate was resuspended in PBS containing 0.1% BSA to obtain the fluorescent microsphere-antigen complex (concentration 1 mg / mL).

[0028] Step 3: Test Strip Assembly Sample pad: Glass fiber membrane soaked in PBS containing 0.1% Triton X-100 and 5 mM EDTA and dried at 37 °C; Conjugate pad: The fluorescent microsphere-antigen complex was evenly sprayed onto a glass fiber membrane (purchased from Sartorius, product number CN140) at a spray volume of 0.5 μL / cm, freeze-dried, and sealed. Detection line (T line): Anti-swine IgG monoclonal antibody (concentration 1.2 mg / mL, purchased from Abcam, catalog number ab112767) was sprayed on a membrane streaker (purchased from Golden Biotechnology); Quality control line (line C): sprayed with goat anti-rabbit IgG (concentration 1.5 mg / mL, purchased from Sigma, product number R7139); Absorbent pad: absorbent filter paper (purchased from Whatman, product number 3030-691) cut into 5 mm wide strips; Chromatographic membrane: T-line and C-line were fixed on nitrocellulose membrane (purchased from Millipore, product number HF13504). Each component was affixed to a PVC base plate in the order (sample pad → binding pad → chromatography membrane → absorption pad), cut into 3 mm wide test strips, and stored in sealed aluminum foil bags (with built-in desiccant).

[0029] Example 2 OMP antigen preparation: Step 1: Gene synthesis and cloning We commissioned Sangon Biotechnology to synthesize the codon-optimized OMP gene (SEQ ID NO. 3), and introduced EcoRI and SalI restriction sites at both ends; The double-enzyme-digested pET-28a vector and the OMP gene fragment were ligated using T4 ligase at 16°C for 12 h; The ligation product was transformed into Escherichia coli DH5α competent cells, coated on LB plates containing kanamycin (50 μg / mL), cultured at 37°C for 16 hours, and single colonies were picked for sequencing verification.

[0030] Step 2: Recombinant protein expression The verified correct recombinant plasmid was transformed into Escherichia coli BL21 (DE3); Inoculate a single colony into LB liquid medium containing kanamycin and culture at 37°C with shaking until OD 600 =0.6; IPTG (final concentration 0.5 mM) was added and expression was induced at 20°C for 16 h, and the bacteria were collected by centrifugation.

[0031] Step 3: Protein purification After ultrasonic disruption, the cells were centrifuged (12000 rpm, 30 min), and the supernatant was passed through a Ni-NTA affinity chromatography column; Contaminants were eluted with PBS buffer containing 20 mM, 50 mM, and 100 mM imidazole, and finally the target protein was eluted with a buffer containing 250 mM imidazole. The eluate was dialyzed into PBS (pH 7.4) and further purified through Superdex 200 molecular sieves to obtain OMP antigen with a purity of ≥95% (concentration 2.5 mg / mL), the amino acid sequence of which is shown in SEQ ID NO.1.

[0032] Fluorescent microsphere labeling and test strip assembly: Step 1: Microsphere surface modification Take commercially available Eu 3+ 1 mg of doped polystyrene microspheres (particle size 200 nm) were dispersed in 1 mL of PBS (pH 7.4); Polyethyleneimine (PEI, molecular weight 10 kDa) was added to a final concentration of 0.05 mg / mL and shaken at 25°C for 2 h; Free PEI was removed by centrifugation (10,000 rpm, 15 min), and the pellet was resuspended in PBS to obtain PEI-modified microspheres.

[0033] Step 2: Antigen conjugation PEI-modified microspheres were mixed with OMP antigen at a mass ratio of 1:5 (1 mg microspheres, 5 mg antigen), and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added to the final concentrations of 0.4 mM and 0.1 mM, respectively; The reaction was carried out at 25°C in the dark for 2 hours, and the unbound antigen was removed by centrifugation (10,000 rpm, 15 minutes). The precipitate was resuspended in PBS containing 0.1% BSA to obtain the fluorescent microsphere-antigen complex (concentration 1 mg / mL).

[0034] Step 3: Test Strip Assembly Sample pad: Glass fiber membrane soaked in PBS containing 0.1% Triton X-100 and 5 mM EDTA and dried at 37 °C; Conjugate pad: The fluorescent microsphere-antigen complex was evenly sprayed onto a glass fiber membrane (purchased from Sartorius, product number CN140) at a spray volume of 0.5 μL / cm, freeze-dried, and sealed. Detection line (T line): Anti-swine IgG monoclonal antibody (concentration 1.2 mg / mL, purchased from Abcam, catalog number ab112767) was sprayed on a membrane streaker (purchased from Golden Biotechnology); Quality control line (line C): sprayed with goat anti-rabbit IgG (concentration 1 mg / mL, purchased from Sigma, product number R7139); Absorbent pad: absorbent filter paper (purchased from Whatman, product number 3030-691) cut into 5 mm wide strips; Chromatographic membrane: T-line and C-line were fixed on nitrocellulose membrane (purchased from Millipore, product number HF13504). Each component was affixed to a PVC base plate in the order (sample pad → binding pad → chromatography membrane → absorption pad), cut into 3 mm wide test strips, and stored in sealed aluminum foil bags (with built-in desiccant).

[0035] Example 3 OMP antigen preparation: Step 1: Gene synthesis and cloning We commissioned Sangon Biotechnology to synthesize the codon-optimized OMP gene (SEQ ID NO. 3), and introduced EcoRI and SalI restriction sites at both ends; The double-enzyme-digested pET-28a vector and the OMP gene fragment were ligated using T4 ligase at 16°C for 12 h; The ligation product was transformed into Escherichia coli DH5α competent cells, coated on LB plates containing kanamycin (50 μg / mL), cultured at 37°C for 16 hours, and single colonies were picked for sequencing verification.

[0036] Step 2: Recombinant protein expression The verified correct recombinant plasmid was transformed into Escherichia coli BL21 (DE3); Inoculate a single colony into LB liquid medium containing kanamycin and culture at 37°C with shaking until OD 600 =0.6; IPTG (final concentration 0.5 mM) was added and expression was induced at 20°C for 16 h, and the bacteria were collected by centrifugation.

[0037] Step 3: Protein purification After ultrasonic disruption, the cells were centrifuged (12000 rpm, 30 min), and the supernatant was passed through a Ni-NTA affinity chromatography column; Contaminants were eluted with PBS buffer containing 20 mM, 50 mM, and 100 mM imidazole, and finally the target protein was eluted with a buffer containing 250 mM imidazole. The eluate was dialyzed into PBS (pH 7.4) and further purified through Superdex 200 molecular sieves to obtain OMP antigen with a purity of ≥95% (concentration 2.5 mg / mL), the amino acid sequence of which is shown in SEQ ID NO.1.

[0038] Fluorescent microsphere labeling and test strip assembly: Step 1: Microsphere surface modification Take commercially available Eu 3+ 1 mg of doped polystyrene microspheres (particle size 200 nm) were dispersed in 1 mL of PBS (pH 7.4); Polyethyleneimine (PEI, molecular weight 10 kDa) was added to a final concentration of 0.15 mg / mL and shaken at 25°C for 2 h; Free PEI was removed by centrifugation (10,000 rpm, 15 min), and the pellet was resuspended in PBS to obtain PEI-modified microspheres.

[0039] Step 2: Antigen conjugation PEI-modified microspheres were mixed with OMP antigen at a mass ratio of 1:4 (1 mg microspheres, 4 mg antigen), and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added to the final concentrations of 0.4 mM and 0.1 mM, respectively; The reaction was carried out at 25°C in the dark for 2 hours, and the unbound antigen was removed by centrifugation (10,000 rpm, 15 minutes). The precipitate was resuspended in PBS containing 0.1% BSA to obtain the fluorescent microsphere-antigen complex (concentration 1 mg / mL).

[0040] Step 3: Test Strip Assembly Sample pad: Glass fiber membrane soaked in PBS containing 0.1% Triton X-100 and 5 mM EDTA and dried at 37 °C; Conjugate pad: The fluorescent microsphere-antigen complex was evenly sprayed onto a glass fiber membrane (purchased from Sartorius, product number CN140) at a spray volume of 0.6 μL / cm, freeze-dried, and sealed. Detection line (T line): Anti-swine IgG monoclonal antibody (concentration 1.2 mg / mL, purchased from Abcam, catalog number ab112767) was sprayed on a membrane streaker (purchased from Golden Biotechnology); Quality control line (line C): sprayed with goat anti-rabbit IgG (concentration 1.5 mg / mL, purchased from Sigma, product number R7139); Absorbent pad: absorbent filter paper (purchased from Whatman, product number 3030-691) cut into 5 mm wide strips; Chromatographic membrane: T-line and C-line were fixed on nitrocellulose membrane (purchased from Millipore, product number HF13504). Each component was affixed to a PVC base plate in the order (sample pad → binding pad → chromatography membrane → absorption pad), cut into 3 mm wide test strips, and stored in sealed aluminum foil bags (with built-in desiccant).

[0041] Experimental example The following experiments were carried out using the product prepared in Example 1: Step 1: Sample testing Serum sample: directly take 50 μL and drop it onto the sample pad, then add 50 μL of sample diluent; Whole blood sample: Take 50 μL of fresh pig whole blood and add 50 μL of sample diluent; Chromatographic reaction: Incubate at room temperature for 10 minutes and read the T-line signal intensity using a portable fluorescence reader (excitation wavelength 365 nm, emission wavelength 615 nm). The threshold is set at 3 times the standard deviation of the negative control signal mean.

[0042] Step 2: Sensitivity test Gradual dilution of positive pig serum (1:100 to 1:6400), the lowest dilution that the test strip can detect is 1:3200 Step 3: Specificity testing In the detection of positive samples of porcine circovirus (PCV2), classical swine fever virus (CSFV), and Escherichia coli (E. coli), the T line showed no signal (cross-reaction rate 0%).

[0043] Step 4: Clinical Validation 50 clinical serum samples were tested and compared with commercially available ELISA kits, with a coincidence rate of 94% (47 / 50) and a Kappa value of 0.89.

[0044] Step 5: Stability Test After the test strips were stored at 4°C, 25°C, and 40°C for 6 months, the signal intensity of positive samples attenuated by 2.3%, 4.7%, and 4.9%, respectively (all <5%).

[0045] The results showed that the detection scheme provided by the present invention can shorten the detection time to 10 minutes without the need for complex equipment; the signal intensity of the fluorescent microspheres is 10 times that of colloidal gold, and the sensitivity is improved to 1:3200; and the OMP antigen used in the present invention is highly conservative and highly specific, and can cover the main prevalent strains of porcine intracellular Lawsonia intracellularis, avoiding missed detection due to strain variation; and has no cross-reaction with common pathogens such as porcine circovirus and classical swine fever virus.

[0046] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fluorescent test strip for outer membrane protein antibodies of intracellular Lawsonia spp., characterized in that: The method comprises a bottom plate, a sample pad, a conjugation pad, a chromatography membrane and an absorption pad; the sample pad, the conjugation pad, the chromatography membrane and the absorption pad are sequentially arranged on the bottom plate according to the order of liquid flow; a detection line and a quality control line are sequentially arranged on the chromatography membrane; The test line contains anti-swine IgG monoclonal antibodies; The quality control line contains goat anti-rabbit IgG; The conjugate pad contains a fluorescent microsphere-antigen complex; The fluorescent microsphere-antigen complex is obtained by coupling polyethyleneimine-modified microspheres and intracellular Lawsonia outer membrane protein antigen; The amino acid sequence of the Lawsonia intracellularis outer membrane protein antigen is shown in SEQ ID NO.

1.

2. The fluorescent test strip for the outer membrane protein antibody of Lawsonia intracellularis according to claim 1, characterized in that: The Lawsonia intracellularis outer membrane protein antigen is prepared by recombinantly expressing the coding gene of the Lawsonia intracellularis outer membrane protein, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.

3.

3. The fluorescent test strip for the outer membrane protein antibody of Lawsonia intracellularis according to claim 1, wherein The polyethyleneimine modified microspheres are obtained by a preparation method comprising the following steps: Will Eu 3+ The doped polystyrene microspheres are mixed with polyethyleneimine to react and obtain polyethyleneimine modified microspheres.

4. The fluorescent test strip for the outer membrane protein antibody of Lawsonia intracellularis according to claim 3, wherein The Eu 3 + The particle size of the doped polystyrene microspheres is 100–300 nm; The Eu 3+ The mass ratio of the doped polystyrene microspheres to polyethyleneimine is 1:0.05~0.

15.

5. The fluorescent test strip for the outer membrane protein antibody of Lawsonia intracellularis according to claim 3, characterized in that: The reaction time is 1.5 to 2.5 hours.

6. The fluorescent test strip for the outer membrane protein antibody of Lawsonia intracellularis according to claim 1, wherein The fluorescent microsphere-antigen complex is obtained by a preparation method comprising the following steps: Polyethyleneimine-modified microspheres and intracellular Lawsonia outer membrane protein antigen were mixed at a mass ratio of 1:4-6, and EDC and NHS were added for coupling reaction to obtain fluorescent microsphere-antigen complexes.

7. The fluorescent test strip for detecting antibodies to the outer membrane protein of intracellular Lawsonia spp. according to claim 6, wherein: The coupling reaction time is 1.5 to 2.5 hours; The coupling reaction was carried out under light-protection conditions.

8. Use of the Lawsonia intracellularis outer membrane protein antibody fluorescent detection test paper according to any one of claims 1 to 7 in the preparation of a porcine Lawsonia intracellularis antibody detection product.

9. A system for detecting antibodies to Lawsonia intracellularis, characterized in that: The invention comprises the Lawsonia intracellularis outer membrane protein antibody fluorescence detection test paper and fluorescence reading detection equipment according to any one of claims 1 to 7.

10. The Lawsonia intracellularis antibody detection system according to claim 9, characterized in that: The excitation wavelength of the fluorescence reading detection device is 365 nm, and the emission wavelength is 615 nm.