Anti-swine IgA monoclonal antibody and application thereof in detection of PEDV IgA antibody

Abstract

The invention belongs to the technical field of detection, and particularly relates to an anti-swine IgA monoclonal antibody and application thereof in PEDV IgA antibody detection. The invention provides an anti-swine IgA monoclonal antibody and a sequence thereof, the monoclonal antibody is effective to swine IgA, but is ineffective to swine immune globulins IgG, IgM, IgD and IgE, and has good reactivity and specificity. According to the invention, an anti-swine IgA monoclonal antibody is labeled on lanthanide EU3 + fluorescent microspheres, and a time-resolved immunofluorescence test strip detection method is established and is used for qualitative detection of an IgA antibody. The detection method is wide in detection range, high in sensitivity and rapid in detection; good repeatability, accuracy and stability are achieved; in addition, the monoclonal antibody has no cross reaction with porcine immune globulins IgG, IgM, IgD and IgE, is high in specificity, has a clinical detection coincidence rate higher than 97.3%, and has strong clinical application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular and immunological diagnosis, and particularly relates to an anti-swine IgA monoclonal antibody and an application thereof in PEDV IgA antibody detection. Background Art

[0002] Porcine epidemic diarrhea (PED) is an intestinal disease of pigs caused by porcine epidemic diarrhea virus (PEDV), which can cause severe vomiting and acute diarrhea in pigs. Clinical symptoms vary with age, and piglets are particularly vulnerable, with a mortality rate of up to 100%. PEDV is a type of porcine enterovirus. After infection, it mainly stimulates mucosal immunity, and IgA is the most abundant immunoglobulin in animals. It plays an important role in intestinal mucosal immunity and effectively prevents the invasion of the virus. Studies have shown that piglets mainly obtain passive immune protection against PEDV through IgA (sIgA) antibodies secreted by the mother in sow milk. Therefore, the level of IgA antibodies in sows is a key indicator for evaluating the immunogenicity of vaccines. SIgA is an important component of the human mucosal defense system and is present in mammalian secretions such as colostrum, tears and saliva. It is an indispensable mucosal barrier. When pregnant sows are vaccinated with PEDV vaccines, elevated IgA levels activate intestinal mucosal immunity. These IgA-secreting cells migrate and localize near the mammary glands of sows, releasing PEDV-specific IgA into colostrum and milk, which are then consumed by newborn piglets, thereby preventing PEDV from penetrating the intestinal epithelium. Therefore, IgA antibodies are essential for protecting newborn piglets from PEDV infection. Monitoring IgA levels in colostrum and saliva is essential for the health of sows and newborn piglets. Detection of IgA antibodies by saliva and mucosal swabs provides a simpler and faster alternative to detecting IgA in sow colostrum.

[0003] PEDV is an enveloped single-stranded RNA virus belonging to the alpha-coronavirus subgenus, the Coronaviridae family, and the Nidovirales order. Its virus particles are spherical, ranging in size from 95 to 190 nanometers, and are similar in appearance and structure to other coronaviruses, but can be easily distinguished from other viruses under an electron microscope. Mature PEDV virus particles are composed of four major structural proteins: spike glycoprotein, nucleoprotein, glycosylated membrane protein, and glycosylated envelope protein. The S protein is the largest of these structural proteins and interacts with cell receptors during viral invasion. It is the main immunogenic protein and plays a key role in initiating immune responses and inducing neutralizing antibodies, leading to a strong host response. Therefore, the PEDV S protein has become a major target for virus detection and vaccine research. Most of the kits currently available on the market for porcine serum IgA antibody detection use indirect ELISA methods with purified viruses as coating antigens. These tests usually use sow colostrum as a test sample to assess vaccine immunity levels. However, this method has limitations, including long assay times and the need for highly purified antigens and enzyme-labeled secondary antibodies, which makes the kits cumbersome to use and high production costs.

[0004] Time-resolved fluorescence immunochromatography (TRFIA) test strips are an ultrasensitive detection method that combines immunoadsorption technology and time-resolved fluorescence technology. They have been widely used in fields such as food safety and clinical testing. Compared with traditional detection methods, they have the advantages of low cost, easy use, rapid detection, higher sensitivity and specificity. As a new type of material, fluorescent nanoparticles enable fluorescent immunochromatography to retain the convenience of traditional colloidal gold test strips in on-site detection, while emitting brighter fluorescent signals and significantly improving sensitivity. This makes them ideal for on-site detection. At the same time, rare earth chelates, as unique functional microspheres, can encapsulate hundreds of fluorescent molecules in each microsphere, greatly improving the efficiency and stability of fluorescent labeling. This makes them excellent luminescent materials for the detection process. Compared with traditional colloidal gold labeling, lanthanide metal labeling significantly improves the efficiency and durability of fluorescent labeling. It also provides lower background fluorescence interference, higher intensity fluorescence signals and greater labeling stability. These characteristics of lanthanide fluorescent microspheres make TRFIA more sensitive and specific, becoming a detection tool with great potential.

[0005] The present invention utilizes TRFIA technology, combined with anti-swine IgA monoclonal antibodies and PEDV S protein, to establish a rapid and qualitative PEDV IgA antibody detection method, which aims to effectively prevent PEDV and provide a new way to accurately monitor the immune status of pigs. Unlike traditional immunochromatography methods, this method uses Eu (III) chelate particles as a fluorescent reporter system, which effectively solves the problems of poor stability, easy photobleaching, and low sensitivity of traditional fluorescent labels. Compared with the traditional nanogold method, it also has higher sensitivity and accuracy. In addition, the fluorescent microsphere test strip is easy to use, and the entire detection process only takes 10-15 minutes, which can achieve rapid and convenient on-site detection without the need for complex laboratory equipment or technology. The performance of the Eu (III) chelate fluorescent microsphere test strip was further evaluated, and its sensitivity, specificity, accuracy and stability were evaluated, and its practicality was verified by clinical sample analysis. Summary of the invention

[0006] In view of the above technical problems, the purpose of the present invention is to provide an anti-swine IgA monoclonal antibody and its application in PEDV IgA antibody detection. Specifically, it includes the following contents:

[0007] In a first aspect, the present invention provides an anti-porcine IgA monoclonal antibody, wherein the heavy chain variable region CDR of the anti-porcine IgA monoclonal antibody comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO.1, a CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.3;

[0008] The light chain variable region CDR of the anti-porcine IgA monoclonal antibody comprises CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5 and CDR3 with an amino acid sequence as shown in SEQ ID NO.6.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the anti-porcine IgA monoclonal antibody is as shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region of the anti-porcine IgA monoclonal antibody is as shown in SEQ ID NO.8.

[0010] In a second aspect, the present invention provides the use of the anti-porcine IgA monoclonal antibody described in the first aspect in the preparation of a PEDVIgA antibody detection reagent, a test strip, or a kit.

[0011] In a third aspect, the present invention provides a TRFIA test strip for detecting PEDV IgA antibodies, the TRFIA test strip comprising a PVC base plate and a sample pad, a conjugation pad, a nitrocellulose membrane and absorbent paper sequentially mounted on the PVC base plate; the conjugation pad is coated with an anti-swine IgA monoclonal antibody labeled with fluorescent microspheres; a detection line and a quality control line are provided on the nitrocellulose membrane; the detection line is coated with PEDV S protein, and the quality control line is coated with IgA antibodies.

[0012] Preferably, the fluorescent microspheres are EU 3+ Chelating fluorescent microspheres.

[0013] Preferably, the volume-to-mass ratio of the fluorescent microspheres to the anti-porcine IgA monoclonal antibody is 1:1.

[0014] Preferably, the coating concentration of IgA antibody on the quality control line is 0.2 mg / ml.

[0015] In a fourth aspect, the present invention provides a method for preparing the TRFIA test strip according to the third aspect, the method comprising the following steps:

[0016] (1) preparing PEDV S recombinant protein and streaking on a nitrocellulose membrane to obtain a detection line;

[0017] (2) Streaking IgA antibody on a nitrocellulose membrane to obtain a quality control line;

[0018] (3) preparing an anti-porcine IgA monoclonal antibody labeled with fluorescent microspheres, and coating the monoclonal antibody labeled with fluorescent microspheres on a conjugate pad;

[0019] (4) Assemble the dried nitrocellulose membrane, conjugate pad, absorbent paper and sample pad on a PVC pad to obtain a TRFIA test strip.

[0020] Preferably, the preparation method of the fluorescent microsphere-labeled anti-porcine IgA monoclonal antibody is as follows: add 400 μL of fluorescent microsphere latex to boric acid buffer, add EDC and NHS solution, and activate the mixture at room temperature for 15 minutes; cool to 10°C, centrifuge to obtain the supernatant, and resuspend the particles with boric acid buffer; add 100 μL of IgA antibody, oscillate at room temperature, then add 10% BSA solution, seal or seal at room temperature and store in the dark at 4°C; centrifuge at 10°C for 10 minutes; finally, add a fluorescent microsphere-labeled protective agent, resuspend the microspheres, and prepare the fluorescent microsphere-labeled anti-porcine IgA monoclonal antibody.

[0021] Specifically: shake the container thoroughly, transfer 50 μL of fluorescent microspheres to a 2 mL centrifuge tube, add 400 μL of boric acid buffer to fully combine; add appropriate amounts of EDC and NHS solution, and activate the mixture at room temperature for 15 minutes; after activation, cool the sample to 10 ° C and centrifuge at 18800 rpm for 10 minutes; take the supernatant and resuspend the particles with 500 μL of boric acid buffer; repeat this process twice; if precipitation is observed, sonicate the sample for 1 minute (100W, 3S, 3S) to ensure complete dissolution. Add 100 μL of anti-porcine IgA monoclonal antibody to the centrifuge tube and oscillate at room temperature for 2 hours; then add 10% BSA solution and continue to oscillate at room temperature for 2 hours. Seal the test tube and store it in the dark at 4 ° C overnight. The next day, centrifuge the test tube at 10 ° C, 18800 rpm for 10 minutes, repeat 3 times. Finally, add 100 μL of fluorescent microsphere-labeled protective agent, resuspend the microspheres, and sonicate the sample if precipitation is observed. The prepared fluorescent microsphere-labeled anti-swine IgA monoclonal antibody solution was stored at 4°C.

[0022] In a fifth aspect, the present invention provides use of the TRFIA test strip described in the third aspect in detecting PEDV IgA antibodies for purposes other than disease diagnosis.

[0023] Preferably, the method for using the TRFIA test strip is:

[0024] Reaction: Dilute the sample with phosphate buffered saline at a ratio of 1:2 and mix thoroughly. Add 50 μL of the mixture to the sample pad of the TRFIA test strip and react at room temperature for 11-15 minutes.

[0025] Result determination: Under 365nm ultraviolet light, if both the quality control line and the test line show red fluorescent bands, it is positive, indicating the presence of IgA antibodies in the sample; if only the quality control line shows a red fluorescent band but the test line does not, it is negative, indicating that the sample does not contain IgA antibodies; if neither the quality control line nor the test line shows a red fluorescent band, or only the test line shows a red fluorescent band, the result is invalid.

[0026] The beneficial effects of the present invention are as follows: firstly, the present invention provides an anti-porcine IgA monoclonal antibody, which is effective against porcine IgA but ineffective against porcine immunoglobulins IgG, IgM, IgD and IgE, and has good reactivity and specificity; secondly, RNA is extracted from cells expressing the antibody, and its sequence is determined, and the characteristics of different CDR regions are analyzed; at the same time, the present invention labels the anti-porcine IgA monoclonal antibody on the lanthanide EU 3+A time-resolved immunofluorescence test strip detection method was established on fluorescent microspheres for the qualitative detection of IgA antibodies. The detection method has a wide detection range, high sensitivity, and rapid measurement; it has good repeatability, accuracy, and stability; and has no cross-reaction with porcine immunoglobulins IgG, IgM, IgD, and IgE, has high specificity, and can accurately reflect the IgA antibody level of clinical PEDV-immunized animals, with a compliance rate higher than 97.3%, and has strong clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of test strip assembly;

[0028] Figure 2 Based on EU 3+ Fluorescence reading analysis scheme of fluorescent microsphere test paper;

[0029] Figure 3 Expression and identification results of PEDV S protein; (A) SDS-PAGE analysis of purified PEDV S protein; (B) antigen analysis of S protein by western blotting using PEDV positive and PEDV negative sera as primary antibodies and HRP-labeled goat anti-swine antibody as secondary antibodies; M: protein marker; 1: control protein (PEDV positive serum); 2: PEDV S protein (PEDV positive serum); 3: control protein (PEDV negative serum); 4: PEDV S protein (PEDV negative serum).

[0030] Figure 4 Identification results of anti-porcine IgA monoclonal antibodies;

[0031] Figure 5 Optimization results of TRFIA reaction conditions; (A) is the antibody and EU 3+ Particle size change of fluorescent microspheres before and after coupling; (B) EU coupled with antibody 3+ The optimal amount of fluorescent microspheres; (C) is the optimal coating concentration of the QC line (C line);

[0032] Figure 6 Detection time optimization results;

[0033] Figure 7 Determination of test results;

[0034] Figure 8 Specific test results; 1: PEDV positive sample; 2: PRCV positive sample; 3: TEGV positive sample; 4: PDCOV positive sample; 5: PBS blank control;

[0035] Fig. 9Sensitivity test results; 1-6 are PEDV positive samples diluted 1:8 to 1:256;

[0036] Fig.10 Stability test results; samples collected at different time intervals (0, 7, 14, 21 and 28 days) were tested using test strips stored at room temperature. DETAILED DESCRIPTION

[0037] The technical scheme of the present invention is described in detail below by specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various equivalent changes and modifications according to the teachings of the present invention without departing from the spirit and scope of the present invention.

[0038] Materials and methods Ethical approval: This invention was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China. This invention was approved by the Animal Experiment Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Antigens, proteins, serum samples, PEDV S protein, and IgA antibodies were all commercially available or prepared according to conventional methods. Serum, saliva, and milk samples were provided by Zhengda Company, Qingyang, Gansu, China.

[0039] Main instruments and reagents: EU 3+ Time-resolved fluorescent microspheres were purchased from Nanjing Microtest Biological Co., Ltd. Fluorescence qualitative rapid detector was purchased from Shanghai Feiqi Biotechnology Co., Ltd. Nitrocellulose (NC) membrane, glass fiber, absorbent paper, sample pad, PVC base plate were purchased from Shanghai Jiening Biotechnology Co., Ltd., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and n-hydroxysulfosuccinimide (NHS) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Company, Shanghai, China.

[0040] Example 1 Expression and purification of PEDV S recombinant protein

[0041] The S sequence of PEDV is derived from the partial sequence of the S1 gene of PEDV HNCADA-2017 strain (GenBank: OP784565.1). The prokaryotic expression recombinant plasmid pET-28a-PEDV-S was synthesized and constructed by Wuhan GeneCreate Bioengineering Co., Ltd., transformed into Escherichia coli BL21 (DE3) competent cells (TransGen Biotech, China), and cultured in Luria-Bertani (LB) liquid medium containing ampicillin (100 μg / mL) at 37°C and shaken at 220 rpm. When OD 600nmWhen the value reached 0.6-0.8, IPTG with a final concentration of 1 mmol / L was added to induce expression for 6 hours. The S protein was purified by Ni Sepharose, analyzed by SDS-PAGE electrophoresis, and identified by western blotting.

[0042] PEDV S protein was expressed in E. coli and purified using a 6×his tag. SDS-PAGE analysis showed that the high-purity PEDV S protein was enriched at 50 kDa ( Figure 3 Western blotting analysis showed that the protein had strong reactivity and high specificity in reaction with PEDV positive serum ( Figure 3 (shown in B).

[0043] Example 2 Preparation and identification of anti-IgA monoclonal antibodies

[0044] Purified porcine IgA was used as an immunogen to immunize BALB / c mice. After immunization, mouse spleen cells were fused with SP2 / 0 myeloma cells to prepare hybridoma cells. Since IgA immunoglobulins usually contain some IgG impurities during the purification process, the indirect ELISA method was used to confirm the specificity of the monoclonal antibody. Polystyrene plates were coated with porcine IgA, IgG, IgM, IgD and IgE respectively. The supernatant of subcloned hybridoma cells was used as the primary antibody, and HRP-labeled goat anti-mouse antibody was used as the secondary antibody.

[0045] In order to identify the obtained monoclonal antibody cell line, the reactivity of the subcloned hybridoma cell supernatant with different immunoglobulins was evaluated. The ELISA results showed that the monoclonal antibody was effective against porcine IgA, showing strong specificity and reactivity; but it was ineffective against porcine IgG, IgM, IgD or IgE, indicating that the monoclonal antibody prepared by the present invention has good reactivity and specificity for porcine IgA ( Figure 4 shown).

[0046] The prepared monoclonal antibodies were sequenced for comparison and analysis. The heavy chain variable region and light chain variable region sequences of the anti-porcine IgA monoclonal antibodies prepared in this example are as follows:

[0047] Heavy chain variable region:

[0048] CDR1: GFSCFAFT (SEQ ID NO. 1)

[0049] CDR2: MGMINGGG (SEQ ID NO. 2)

[0050] CDR3:KIRERMVPIVACYGS(SEQ ID NO.3)

[0051] Light chain variable region:

[0052] CDR1:ESVDNYGISF(SEQ ID NO.4)

[0053] CDR2: AAS (SEQ ID NO. 5)

[0054] CDR3: QQSKEVPWT (SEQ ID NO. 6)

[0055] Anti-porcine IgA monoclonal antibody VH sequence:

[0056] SGSLLVKLGASMKILCKACGFSCFAFTMYWVKQIHRKTGECMECMGMNGGGSARSS RASPHLTSHPARVHGATVSHLRTMQYTGCKIRERMVPIVACYGSGVKGPQSP(SEQ ID NO.7)

[0057] Anti-porcine IgA monoclonal antibody VLκ sequence:

[0058] DIVMTQSPASLAVSLGQRAAISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEADDTAMYFCQQSKEVPWTFGGGTKL (SEQ ID NO. 8) Example 3 Preparation of TRFIA test strips

[0059] 1. Preparation of anti-swine IgA antibody fluorescent microspheres:

[0060] Add 50 μL of EU 3+ Transfer the time-resolved fluorescent microspheres to a 2 mL centrifuge tube and shake the container thoroughly to activate the EU 3+ For time-resolved fluorescent microspheres, add 400 μL of boric acid buffer to allow for full binding. Add appropriate amounts of EDC and NHS solution and activate the mixture at room temperature for 15 minutes. After activation, cool the sample to 10°C and centrifuge at 18,800 rpm for 10 minutes. Take the supernatant and resuspend the pellet with 500 μL of boric acid buffer. Repeat this process twice. If precipitation is observed, sonicate the sample for 1 minute (100 W, 3S, 3S) to ensure complete dissolution.

[0061] Add 100 μL of the prepared anti-swine IgA monoclonal antibody to the centrifuge tube and shake at room temperature for 2 hours. Then add 10% BSA solution and continue shaking at room temperature for 2 hours or seal the tube and store it at 4°C in the dark overnight.

[0062] The next day, the tube was centrifuged at 10°C, 18,800 rpm for 10 minutes, and repeated three times. Finally, the microspheres were resuspended in resuspension liquid. If precipitation was observed, the sample was sonicated to prepare EU 3+ Anti-porcine IgA monoclonal antibody labeled with chelated fluorescent microspheres, stored at 4℃.

[0063] 2. Preparation of TRFIA test strips:

[0064] Indirect method 3+ The carboxyl groups on the surface of the chelated fluorescent microspheres were activated and coupled with anti-porcine IgA monoclonal antibodies.

[0065] IgA antibody and PEDV S protein were immobilized on nitrocellulose (NC) membranes as the quality control line (C line) and detection line (T line), respectively.

[0066] After antibody conjugation, EU 3+ Anti-porcine IgA monoclonal antibody labeled with chelated fluorescent microspheres was sprayed onto the conjugate pad.

[0067] The NC membrane and conjugate pad were placed in a drying chamber at 37 °C for 30 min to dry.

[0068] Assemble the dried NC membrane, conjugate pad, absorbent paper and sample pad on the PVC pad at their designated positions ( Figure 1 As shown), ensure that there is 1 to 2 mm overlap between each pad. Then cut it into 4 mm wide strips with a strip cutter, assemble it into a cassette, and store it at room temperature.

[0069] Test method and determination of results: Dilute the sample to be tested with phosphate buffered saline (PBS) at a ratio of 1:2, mix thoroughly, and add 50 μL of the mixture to the sample well. After 13 ± 2 minutes at room temperature, evaluate the results ( Figure 2 shown).

[0070] Judgment criteria: Under 365nm ultraviolet light, if both the C line and the T line show red fluorescence bands, the result is considered positive, indicating that IgA antibodies exist in the sample being tested. If only the C line shows a red fluorescence band, but the T line does not, the result is considered negative, indicating that the sample being tested does not contain IgA antibodies; if neither the C line nor the T line shows a red fluorescence band, or only the T line shows a red fluorescence band, the result is invalid.

[0071] In order to accurately determine the PEDV IgA level, a rapid fluorescence qualitative detector should be used to simultaneously detect the fluorescence values ​​of the T line and C line.

[0072] 3. Test strip condition optimization

[0073] (1)EU 3+Optimization of Chelated Fluorescent Microsphere-Antibody Coupling Rate

[0074] Marking process EU 3+ The ratio of chelated fluorescent microspheres to antibodies significantly affects the performance of the test strip. 3+ If there are too few available groups on the surface of the chelated fluorescent microspheres, the coupling efficiency will be low. Conversely, using too much antibody will reduce the fluorescence intensity. 100μLEU was used in the experiment. 3+ The chelated fluorescent microspheres and antibody masses were 25, 50, 75, 100, and 125 ng, respectively. The corresponding fluorescent microsphere to antibody ratios were 2:1, 4:3, 1:1, and 1:2, respectively. Samples of the same concentration were tested and analyzed to determine the optimal microsphere-antibody coupling ratio.

[0075] By adjusting the EU 3+ The ratio of chelated fluorescent microspheres to monoclonal antibodies and other parameters were optimized and the reaction conditions were optimized. 3+ The size of the chelated fluorescent microspheres increased significantly from 132.8nm to about 757nm ( Figure 5 A), as confirmed by the particle size measurements before and after coupling, indicating successful coupling. In addition, the optimal reaction parameters of the test strip were determined, and the optimal coupling ratio of the monoclonal antibody was 100ng ( Figure 5 (shown in B).

[0076] (2) IgA antibody coating concentration on line C

[0077] Line C was coated with 0.2, 0.5, 0.8, and 1.0 mg / mL of IgA antibody, respectively. A fluorescence quantitative rapid detector was used to measure the control, and the optimal coating concentration of line C was determined based on the results.

[0078] The results showed that the optimal coating concentration of the quality control line (C line) was 0.2 mg / ml ( Figure 5 (as shown in C).

[0079] (3) Optimize detection time

[0080] If the detection time is too short, the accuracy of the result will be affected, while if the detection time is too long, the detection efficiency will be reduced. The present invention adopts the best process to prepare the test strips, and detects the sample concentrations of 0, 1, 2, and 5 ng / mL respectively. Each test is repeated three times. The sample is added, and the test is started after 3 minutes, and the measurement is performed every two minutes for up to 29 minutes. The fluorescence value of the T line is recorded, and a curve changing with time is drawn.

[0081] The reaction time of the TRFIA test strip was optimized and it was found that the maximum fluorescence value was reached at about 13 minutes. After 15 minutes, the fluorescence value began to decrease and then stabilized. Therefore, the optimal reaction time was determined to be 13±2 minutes ( Figure 6shown).

[0082] 4.Performance testing of TRFIA test strips

[0083] (1) Determine the detection limit

[0084] Fifty saliva samples negative for PEDV IgA antibodies were diluted 1:2 in PBS and tested as described previously. After the reaction was complete, the test strip was placed in the fluorescence immunoassay analyzer for 3 seconds. The mean and standard deviation of the Line T fluorescence values ​​were calculated, and the detection limit was determined using the formula X±3SD(2).

[0085] To evaluate the reactivity of the TRFIA test strips, 50 saliva samples negative for PEDV IgA antibodies were diluted 1:2 in PBS and tested using the method described previously. After the reaction was complete, the TRFIA test strips were placed in a fluorescent immunoassay analyzer for 3 seconds. The test line of the test strips was determined by calculating the mean (X) and standard deviation (SD) of the measured values. Figure 7 The fluorescence value was calculated as X±3SD. The average fluorescence value was 1466.38, the standard deviation was 1085.273, and the T-line detection limit was 4722.199. In summary, a T-line fluorescence value ≥ 4722 was positive, and < 4722 was negative.

[0086] (2) Specificity

[0087] The TRFIA test strip prepared by the present invention was used to detect PEDV IgA standard (100 ng / mL), and PRCV-positive, TEGV-positive and PDCOV-positive samples were simultaneously detected to evaluate the specificity of the method.

[0088] The specific samples were tested using the TRFIA test strips, and the PEDV ELISA antibody detection kit was used for control experiments. The results showed that only the PEDV-positive samples showed a positive reaction, while the PEDV-negative samples showed a negative reaction. In addition, all other positive samples produced negative results, proving that the TRFIA test strips prepared by the present invention have excellent specificity ( Figure 8 shown).

[0089] (3) Sensitivity:

[0090] The PEDV-positive sample was diluted gradually and applied to the sample application area of ​​each test strip. The results were analyzed to determine the maximum dilution ratio at which the TRFIA test strip could detect the substance.

[0091] The PEDV positive samples were diluted step by step and applied to the sample application area of ​​the TRFIA test strip. The results were analyzed to determine the maximum dilution ratio at which the TRFIA test strip could detect the substance. The positive samples of known concentration were tested and were still positive after being diluted from 1:8 to 1:64. The minimum detection limit was 1:64 ( Fig. 9 In contrast, the IDEXX antibody test kit had a limit of detection of 1:4 for the same positive samples (shown in Table 1).

[0092] Table 1 IDEXX Antibody Test Kit 1:2-1:256 dilution positive sample test results

[0093] Dilution gradient Test results S / P + / - 2× 0.858136 + 4× 0.449235 + 8× 0.246175 - 16× 0.109857 - 32× 0.048679 - 64× 0.018081 - 128× -0.00695 - 256× -0.00417 -

[0094] (4) Repeatability

[0095] The same batch of TRFIA test strips were used to test specific and clinical samples, and each test strip was tested three times. To test the repeatability of test strips between batches, three different batches of PEDV antibody test strips produced at different times were tested together with the PEDV ELISA (IgA) antibody test kit on the same specific and clinical samples.

[0096] The same batch of TRFIA test strips were used to detect specificity and clinical samples, and each test strip was tested 3 times. The results showed that all three test results were consistent with each other and with the IDEXX PEDV ELISA antibody detection kit, indicating that the TRFIA test strips described in the present invention have high repeatability within the batch. In order to verify the repeatability between different batches, three different batches of PEDV antibody test strips produced at different times were tested on the same specificity and clinical samples together with the PEDV ELISA (IgA) antibody detection kit. The results are consistent with the results of the IDEXX PEDV ELISA (IgA) antibody detection kit, indicating that the TRFIA test strips prepared by the present invention have high repeatability.

[0097] (5) Stability:

[0098] The prepared TRFIA test strips were stored at 4°C and 37°C, respectively, and the same positive samples were tested. The fluorescence values ​​of the T line were recorded on days 0, 7, 14, 21, and 28.

[0099] The test results show that the specificity and sensitivity of the TRFIA test strip prepared by the present invention remain consistent throughout the storage period ( Fig.10 This indicates that the TRFIA test strip prepared by the present invention has higher stability.

[0100] (6) Accuracy

[0101] Comparative study of clinical samples To evaluate the effectiveness and accuracy of the fluorescent test strips, 76 samples collected were tested using the same batch of test strips. The results were compared with those of commercially available ELISA kits, and the accuracy of the fluorescent test strips was evaluated on this basis.

[0102] 76 clinical samples were tested simultaneously using the TRFIA test strips prepared by the present invention and the IDEXX PEDV IgA antibody detection kit. The results showed that the TRFIA test strips prepared by the present invention had extremely high accuracy, with 31 positive results and 45 negative results, matching the results of the IDEXX test kit as a control. Of the 76 samples, 74 samples showed consistent results from the two methods, with an overall consistency rate of 97.3% (as shown in Table 2), indicating that the TRFIA test strips prepared by the present invention have potential clinical relevance.

[0103] Table 2 TRFIA test strip clinical sample test results

[0104]

[0105] PEDV is an intestinal infectious disease that mainly affects suckling piglets, with a mortality rate of 100%. At present, vaccination is the main effective means to prevent and control the disease, which makes the monitoring of antibody levels after immunization particularly important. In theory, serum IgG levels after vaccination are positively correlated with the protection rate. However, clinical PEDV antibody testing in pigs showed that pigs with higher IgG levels still had diarrhea, indicating that there are defects in evaluating the immune effect of vaccines using only IgG levels in serum. As the unique role of IgA in fighting PEDV was explored, researchers found that specific sIgA antibodies can fight PEDV, prevent PEDV from adhering to and replicating in the intestinal epithelium, and thus protect piglets from infection. Mammary immunity can be induced by vaccinating sows, and sows pass PEDV-specific IgA antibodies to suckling piglets, thereby inhibiting the vertical transmission of PEDV through breast milk. Therefore, IgA levels have gradually become the most important indicator for evaluating PEDV vaccines. Real-time monitoring of PEDV IgA antibody levels in pig herds can detect the IgA level of pregnant sows at an early stage, thereby replenishing immunity in time to reduce the morbidity of piglets, which puts forward more requirements for the detection of PEDV IgA antibody levels.

[0106] Currently, ELISA is the mainstream method for detecting PEDV-specific antibodies, and most methods focus on using fragments of neutralizing epitope proteins as antigens or developing methods for detecting IgA antibodies. Studies have shown that serum IgA antibody levels are most strongly correlated with colostrum IgA antibodies and remain high both before and after delivery. These findings suggest that serum IgA levels can be used as a reliable immune predictor of immune efficacy and that colostrum IgA antibody levels can be assessed 21 days in advance. Currently, most ELISA techniques are designed to detect IgG levels, and only a few mature methods can be used to detect IgA. However, ELISA tests have several disadvantages, including long detection time, complex procedures, inability to perform on-site testing, and strict operational requirements. The purpose of the present invention is to develop a simple, on-site rapid method for detecting PEDV antibodies that does not require equipment and can monitor PEDV IgA antibody levels in pig herds.

[0107] TRFIA is a fairly mature detection technology that has been widely used in various research fields. It has several advantages, including no radioactive contamination, easy to use, and clear fluorescence readings. The design principle of the experiment is as follows: when the sample containing PEDV IgA antibodies is applied to the sample pad, capillary action causes the sample to pass through the chromatogram forward and bind to the anti-porcine IgA monoclonal antibody fluorescent microsphere conjugate fixed on the conjugate pad. As the chromatography proceeds, the PEDV IgA antibodies in the sample bind to the PEDV S protein in the detection zone to form antigen-antibody conjugate microspheres, which are then captured by the detection line. The unbound fluorescent microsphere conjugates continue to migrate and bind to the IgA protein in the quality control zone, and are finally fixed on the control line. After incubation for 15 minutes under light-proof conditions, the fluorescent immunoassay analyzer detects the fluorescent signals (T and C values) of the detection line (T line) and the control line (C line). The T / C values ​​are plotted on the standard curve, and PEDV IgA antibodies can be detected in real time.

[0108] The present invention uses porcine IgA antibodies as immunogens to prepare anti-porcine IgA specific monoclonal antibodies. The established TRFIA test strip can identify porcine IgA antibodies, and the detection limit after multiple repetitions is 1:64. The optimal detection time is 13±2 minutes, and there is no cross-reaction with the positive specimens of the laboratory pig test (PRCV positive, TGRV positive, PDCoV positive). The present invention introduces a new method for on-site and sensitive detection of PEDV IgA antibodies in pigs using highly specific antigen-antibody immunochromatography technology combined with carboxylated fluorescent microspheres as fluorescent probes. Although ELISA is more sensitive and has a lower detection limit (only a small amount of antibody is required for detection), fluorescent test strips have the advantages of being fast and easy to use. Unlike traditional detection methods, this test strip does not require strict operator requirements and does not require specialized equipment. In fact, it is easy to use and relies only on visual analysis. More importantly, fluorescent test strips provide comparable specificity to other methods. They also have the additional advantages of requiring no preparation before use and being easy to store, and can be obtained as needed, making them particularly suitable for small ranches and grassroots frontlines where laboratories cannot be established. In addition, the method can be applied to various sample types, including saliva and colostrum, with the advantage of not being limited to colostrum. Used across multiple sample types, the test strip provides greater applicability and flexibility.

[0109] The innovative PEDV IgA TRFIA test strip developed by the present invention has an extremely low detection limit (1:64), a wide detection range, high sensitivity, fast and convenient detection, and the detection completion time is only 13±2 minutes. In the stability test, it showed good repeatability, accuracy and stability. In addition, the test strip had no cross-reaction with the other four positive sera, confirming its high specificity. In the clinical sample test, the compliance rate reached 97.3%, which has strong clinical application potential.

Claims

1. An anti-porcine IgA monoclonal antibody, characterized in that: The heavy chain variable region CDR of the anti-porcine IgA monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; the light chain variable region CDR of the anti-porcine IgA monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

6.

2. The anti-porcine IgA monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the anti-porcine IgA monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region of the anti-porcine IgA monoclonal antibody is shown in SEQ ID NO.

8.

3. Use of the anti-porcine IgA monoclonal antibody as claimed in claim 1 or 2 in the preparation of a PEDVIgA antibody detection reagent, a test strip, or a kit.

4. A TRFIA test strip for detecting PEDVIgA antibodies, the TRFIA test strip comprising a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and a blotting paper sequentially mounted on the PVC base plate; characterized in that: The conjugate pad is coated with the anti-porcine IgA monoclonal antibody of claim 1 labeled with fluorescent microspheres; the nitrocellulose membrane is provided with a detection line and a quality control line; the detection line is coated with PEDV S protein, and the quality control line is coated with IgA antibody.

5. The TRFIA test strip according to claim 4, wherein The fluorescent microspheres are EU 3+ Chelated fluorescent microspheres.

6. The TRFIA test strip according to claim 5, wherein: The volume-to-mass ratio of the fluorescent microspheres to the anti-porcine IgA monoclonal antibody is 1:

1.

7. The TRFIA test strip according to claim 6, wherein The coating concentration of IgA antibody on the quality control line is 0.2 mg / ml.

8. The method for preparing a TRFIA test strip according to any one of claims 4 to 7, wherein: The method comprises the following steps: (1) preparing PEDV S recombinant protein and streaking on a nitrocellulose membrane to obtain a detection line; (2) streaking the IgA antibody on a nitrocellulose membrane to obtain a test line; (3) preparing an anti-porcine IgA monoclonal antibody labeled with fluorescent microspheres, and coating the anti-porcine IgA monoclonal antibody labeled with fluorescent microspheres on a conjugate pad; (4) Assemble the dried nitrocellulose membrane, binding site, absorbent paper and sample pad on a PVC pad to obtain a TRFIA test strip.

9. The method according to claim 8, characterized in that The preparation method of the fluorescent microsphere-labeled anti-porcine IgA monoclonal antibody is as follows: adding 400 μL of fluorescent microsphere latex to a boric acid buffer, adding EDC and NHS solutions, and activating the mixture at room temperature for 15 minutes; cooling to 10°C, centrifuging to obtain a supernatant, and resuspending the particles with a boric acid buffer; adding 100 μL of IgA antibody, oscillating at room temperature, and then adding a 10% BSA solution, sealing or sealing at room temperature and storing at 4°C in the dark; centrifuging at 10°C for 10 minutes; and finally, adding a fluorescent microsphere-labeled protective agent, resuspending the microspheres, and preparing the fluorescent microsphere-labeled anti-porcine IgA monoclonal antibody.

10. Use of the TRFIA test strip according to any one of claims 4 to 7 in the detection of PEDVIgA antibodies for purposes other than disease diagnosis.

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