A double colloidal gold test strip for simultaneously detecting porcine circovirus type 2 and type 3 antigens and a preparation method and application thereof

Abstract

The application discloses a double colloidal gold test strip for simultaneously detecting porcine circovirus type 2 (PCV2) and type 3 (PCV3) antigens and a preparation method and application thereof, and belongs to the technical field of biological detection. The application creates a PCV2-PCV3 antigen rapid detection double colloidal gold test strip, which can detect 0.04 mu g of PCV2-capsid protein (Cap) and 0.02 mu g of PCV3-Cap at the minimum, and has no cross reaction with PEDV, PoRV, PRRSV, PRV and CSFV antigens. The test strip can be used for identifying and simultaneously detecting PCV2 and PCV3 antigens, can rapidly detect PCV2 or PCV3 infected pigs on site, and provides important tools and technical support for rapid clinical diagnosis of PCV2 and PCV3 at the grassroots level.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a dual colloidal gold test strip for simultaneously detecting porcine circovirus type 2 and type 3 antigens, its preparation method, and its application. Background Technology

[0002] Porcine circovirus type 2 (PCV2) is the main pathogen causing porcine circovirus disease. It is highly contagious and immunosuppressive, and is prevalent globally. Infected pigs exhibit a variety of clinical symptoms, including weaned piglet multisystemic wasting syndrome, finishing pig dermatitis and nephropathy syndrome, and reproductive disorders in sows, severely impacting growth, development, and reproductive performance, causing significant economic losses to the pig industry. Porcine circovirus type 3 (PCV3), first discovered in the United States in 2015, can cause reproductive disorders, dermatitis and nephropathy syndrome, and other diseases in pigs. Infection is widespread and often co-infects with other pathogens. Both PCV2 and PCV3 have high infection rates in pig herds, and co-infection is common. The similar clinical symptoms and pathological changes caused by both make clinical differential diagnosis difficult.

[0003] Currently, the main detection methods for PCV2 and PCV3 include PCR and its derivative technologies (such as dual quantitative PCR, dual nano-PCR, etc.) and serological methods such as ELISA. While PCR methods offer high sensitivity and specificity, they require specialized equipment and operators, and are time-consuming, making them unsuitable for rapid on-site testing at the grassroots level. Existing ELISA methods are mostly indirect methods, with specificity needing improvement, and typically only detect a single virus. Therefore, developing a detection method and rapid on-site testing tool capable of simultaneously, rapidly, and accurately detecting PCV2 and PCV3 antigens is of great significance for the epidemiological surveillance, clinical diagnosis, and epidemic prevention and control of PCV2 and PCV3. Summary of the Invention

[0004] The purpose of this invention is to provide a dual colloidal gold test strip for simultaneously detecting porcine circovirus type 2 and type 3 antigens, its preparation method, and its application, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is a dual colloidal gold test strip for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 antigens, comprising a PVC base plate and a sample pad, a gold label pad, a nitrocellulose membrane and an absorbent pad sequentially overlapped on the PVC base plate. The gold-labeled pad is adsorbed with colloidal gold-labeled PCV2 monoclonal antibody 1C10 and colloidal gold-labeled PCV3 monoclonal antibody 1D8. The nitrocellulose membrane is provided with a control line C, a detection line T1, and a detection line T2; the C line is coated with goat anti-mouse IgG, the T1 line is coated with PCV2 monoclonal antibody 1C6, and the T2 line is coated with PCV3 monoclonal antibody 1D1. The PCV2 monoclonal antibodies 1C10 and 1C6 recognize different antigenic epitopes of PCV2-Cap; the PCV3 monoclonal antibodies 1D8 and 1D1 recognize different antigenic epitopes of PCV3-Cap.

[0006] The second technical solution of the present invention is a method for preparing the aforementioned dual colloidal gold test strip, comprising the following steps: (1) Preparation of gold-labeled antibodies: The colloidal gold suspension was adjusted to pH 7.3, and PCV2 monoclonal antibody 1C10 and PCV3 monoclonal antibody 1D8 were added to a final concentration of 10 μg / mL, respectively. The mixture was labeled at room temperature for 45 min, and BSA was added to a final concentration of 1% for blocking for 45 min. After centrifugation, the precipitate was resuspended in colloidal gold resuspension. (2) Preparation of gold-labeled pad: The gold-labeled antibody prepared in step (2) was adsorbed onto a glass cellulose membrane and dried at 37 °C for 1 h; (3) Preparation of nitrocellulose membrane: 0.5 mg / mL goat anti-mouse IgG was scratched at the C line, 2 mg / mL PCV2 monoclonal antibody 1C6 was scratched at the T1 line, and 3 mg / mL PCV3 monoclonal antibody 1D1 was scratched at the T2 line. (4) Assembly: The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are sequentially overlapped and pasted onto the PVC base plate, and then cut into test strips.

[0007] The third technical solution of the present invention is the application of the aforementioned dual colloidal gold test strip in the preparation of a detection reagent for detecting porcine circovirus type 2 and / or porcine circovirus type 3 antigen.

[0008] The fourth technical solution of the present invention is a method for detecting porcine circovirus type 2 and / or type 3 antigens for non-disease diagnosis or treatment purposes, which utilizes the aforementioned double colloidal gold test strip to detect the sample to be tested.

[0009] Based on the above technical solution, the present invention has the following technical effects: This invention presents a dual colloidal gold test strip for rapid detection of PCV2 and PCV3 antigens, capable of detecting minimum levels of 0.04 μg PCV2-Cap and 0.02 μg PCV3-, respectively, and exhibiting no cross-reactivity with PEDV, PoRV, PRRSV, PRV, and CSFV antigens. This test strip can be used to identify and simultaneously detect PCV2 and PCV3 antigens, enabling rapid on-site detection of PCV2 or PCV3-infected pigs, providing crucial tools and technical support for the rapid clinical diagnosis of PCV2 and PCV3 at the basal cap layer. Attached Figure Description

[0010] Figure 1 The expression and purification of Cap are shown below. A. Expression and purification of PCV2-Cap. B. Expression and purification of PCV3-Cap. M. Protein molecular size standard; 1 and 2. Recombinant strains before and after induction, respectively; 3 and 4. Supernatant and precipitate after ultrasonic lysis of the induced expression cells, respectively; 5. Purified Cap.

[0011] Figure 2 The results are from Western blot analysis of the proteins. A. Western blotting of PCV2-Cap; B. Western blotting of PCV3-Cap; M. Protein size standard. 1. Expressed Cap; 2. Induced product of pET-28a empty plasmid transformation into E. coli.

[0012] Figure 3 This section describes the Western blot analysis of monoclonal antibodies. Specifically, it includes: A. Identification of PCV2 monoclonal antibodies; B. Identification of PCV3 monoclonal antibodies.

[0013] Figure 4 The results show the purification of PCV2 and PCV3 monoclonal antibodies in ascites fluid. A. SDS-PAGE of purified PCV2 monoclonal antibody; B. SDS-PAGE of purified PCV3 monoclonal antibody.

[0014] Figure 5 This is for the quality determination of colloidal gold. The results include: A. Color of the colloidal gold suspension; B. Absorbance value of the colloidal gold particles; C. Electron micrograph of the colloidal gold particles.

[0015] Figure 6 This is a pairing of monoclonal antibodies. Specifically, A. PCV2 monoclonal antibody; B. PCV3 monoclonal antibody.

[0016] Figure 7 This study aims to determine the optimal pH for colloidal gold-labeled monoclonal antibodies. The assay includes: A. Mey's stabilization test (PCV2); B. OD... 530nmC. Value determination (PCV2); D. Mey's stabilization test (PCV3); E. OD 530nm Value determination (PCV3).

[0017] Figure 8 This study aims to determine the optimal stability level of colloidal gold-labeled monoclonal antibodies. The determination includes: A. Mey's stabilization assay (PCV2); B. OD... 530nm C. Value determination (PCV2); D. Mey's stabilization test (PCV3); E. OD 530nm PCV3 value determination.

[0018] Figure 9 Determination of the optimal antibody concentration for the PCV2 and PCV3 dual colloidal gold control line. Wherein, 1 represents 1 mg / mL; 2 represents 0.5 mg / mL; and 3 represents 0.25 mg / mL.

[0019] Figure 10 This study determined the optimal antibody concentration for the detection line of the PCV2 and PCV3 dual test strips. Where 1 represents T1: 0.5 mg / mL, T2: 1 mg / mL; 2 represents T1: 1 mg / mL, T2: 2 mg / mL; and 3 represents T1: 2 mg / mL, T2: 3 mg / mL.

[0020] Figure 11 Screening for the optimal diluent for PCV2 and PCV3 dual test strips. Where 1 represents Tris-HCl buffer; 2 represents PBS; 3 represents PB solution; and 4 represents ddH2O.

[0021] Figure 12 The concentration of Triton X-100 was determined. Where 1 represents 2%; 2 represents 1%; 3 represents 0.5%; 4 represents 0.25%; and 5 represents 0.1%.

[0022] Figure 13 Specificity analysis of the PCV2 and PCV3 dual test strips. Wherein, 1 represents PEDV; 2 represents PoRV; 3 represents PRRSV; 4 represents PRV; 5 represents CSFV; 6 represents PCV2 Cap; 7 represents PCV2; 8 represents PCV3 Cap; 9 represents PCV3 infectious clone; 10 represents (PCV2+PCV3) Cap; 11 represents PCV2+PCV3; 12 represents negative control. Detailed Implementation

[0023] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0024] This invention provides a dual colloidal gold test strip for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 antigens, comprising a PVC base plate and a sample pad, a gold label pad, a nitrocellulose membrane, and an absorbent pad sequentially overlapped on the PVC base plate; The gold-labeled pad is adsorbed with colloidal gold-labeled PCV2 monoclonal antibody 1C10 and colloidal gold-labeled PCV3 monoclonal antibody 1D8. The nitrocellulose membrane is provided with a control line C, a detection line T1, and a detection line T2; the C line is coated with goat anti-mouse IgG, the T1 line is coated with PCV2 monoclonal antibody 1C6, and the T2 line is coated with PCV3 monoclonal antibody 1D1. The PCV2 monoclonal antibodies 1C10 and 1C6 recognize different antigenic epitopes of PCV2-Cap; the PCV3 monoclonal antibodies 1D8 and 1D1 recognize different antigenic epitopes of PCV3-Cap.

[0025] In some specific embodiments, the colloidal gold has a particle size of 20 nm; the optimal pH for colloidal gold-labeled PCV2 monoclonal antibody 1C10 and PCV3 monoclonal antibody 1D8 is 7.3; and the optimal stable amount of PCV2 monoclonal antibody 1C10 and PCV3 monoclonal antibody 1D8 per milliliter of colloidal gold suspension is 10 μg.

[0026] In some specific embodiments, the concentration of the C-line goat anti-mouse IgG is 0.5 mg / mL; the concentration of the T1-line PCV2 monoclonal antibody 1C6 is 2 mg / mL; and the concentration of the T2-line PCV3 monoclonal antibody 1D1 is 3 mg / mL.

[0027] In some specific implementations, the sample diluent for the sample pad is a Tris-HCl buffer containing 0.1% Triton X-100; the optimal color development time is 11 min.

[0028] In some specific implementation plans, the criteria for determining the results are as follows: When both the T1 line and the C line show red bands, it is determined to be PCV2 antigen positive; When both the T2 line and the C line show red bands, it is determined to be PCV3 antigen positive; When red bands appear on T1, T2 and C lines, it is determined that PCV2 and PCV3 are co-infected. When only line C shows a red band, it is determined that the PCV2 and PCV3 antigens are negative; If there is no red band on line C, the test strip is considered invalid.

[0029] This invention also provides a method for preparing the aforementioned dual colloidal gold test strip, comprising the following steps: (1) Preparation of gold-labeled antibodies: The colloidal gold suspension was adjusted to pH 7.3, and PCV2 monoclonal antibody 1C10 and PCV3 monoclonal antibody 1D8 were added to a final concentration of 10 μg / mL, respectively. The mixture was labeled at room temperature for 45 min, and BSA was added to a final concentration of 1% for blocking for 45 min. After centrifugation, the precipitate was resuspended in colloidal gold resuspension. (2) Preparation of gold-labeled pad: The gold-labeled antibody prepared in step (1) was adsorbed onto a glass cellulose membrane and dried at 37 °C for 1 h; (3) Preparation of nitrocellulose membrane: 0.5 mg / mL goat anti-mouse IgG was scratched at the C line, 2 mg / mL PCV2 monoclonal antibody 1C6 was scratched at the T1 line, and 3 mg / mL PCV3 monoclonal antibody 1D1 was scratched at the T2 line. (4) Assembly: The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are sequentially overlapped and pasted onto the PVC base plate, and then cut into test strips.

[0030] This invention also provides an application of the aforementioned dual colloidal gold test strip in the preparation of a detection reagent for detecting porcine circovirus type 2 and / or type 3 antigens.

[0031] This invention also provides a method for detecting porcine circovirus type 2 and / or type 3 antigens for non-disease diagnosis or treatment purposes, using the aforementioned dual colloidal gold test strip to detect the sample to be tested.

[0032] Example 1 1. Materials and Methods 1.1 Materials Porcine circovirus (PCV2) hybridoma cells 1D2, 1C10, and 1C6, and porcine circovirus (PCV3) hybridoma cell lines 1D8, 3B5, 1D1, 1G10, and 2H3 were all prepared and identified by the Animal Infectious Disease Laboratory of the College of Veterinary Medicine, Hebei Agricultural University. The monoclonal antibodies secreted by these cells were all IgG1-type κ chains. *Escherichia coli* (E. coli) Escherichia coli , E. coli Rosetta (DE3) competent cells and the prokaryotic recombinant expression plasmids pET28a(+)-Cap (GenBank ID: MK585076) and PCV2-Cap (GenBank ID: MF593110) were preserved by the Animal Infectious Diseases Laboratory of the College of Veterinary Medicine, Hebei Agricultural University.

[0033] Porcine serum samples that were positive and negative for PCV2 and PCV3 antibodies were provided by the Baoding Municipal Animal Disease Prevention and Control Center.

[0034] SPF-grade BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0035] 1.2 Methods 1.2.1 Induction, purification, and identification of proteins 1.2.1.1 Induction and purification of protein Take PCV2-Cap recombinant expression bacterial culture (OD) 600 nm (0.7) was inoculated into Kan at a volume ratio of 1:100. + In resistant liquid LB medium, culture at 37 ℃ and 200 r / min until the logarithmic growth phase (OD) is reached. 600nm (Values ​​were between 0.6 and 0.8). One mL of bacterial culture was extracted as a control sample before induction. Protein expression was then induced for 5 h at 32 ℃ and 200 r / min using 0.25 mmol / L isopropyl-β-D-thiogalactoside (IPTG). The bacterial cell pellet was collected by centrifugation; it was resuspended in 20 volumes of PBS and lysed by sonication at 4 ℃. After centrifugation, the supernatant and pellet, along with previously extracted samples before and after induction, were subjected to SDS-PAGE to detect protein expression. PCV2-Cap was purified using a His-tagged protein purification kit, and the purification results were analyzed by SDS-PAGE.

[0036] The induction and purification process of PCV3-Cap was the same as that of PCV2-Cap, but the final concentration of IPTG was 0.5 mmol / L, the induction temperature was 31 ℃, and the induction time was 6 h.

[0037] 1.2.1.2 Western blotting of proteins The purified PCV2-Cap, PCV3-Cap and the induction products of the transformed empty plasmid pET28a(+) were used as controls for SDS-PAGE analysis and Western blot identification.

[0038] 1.2.2 Preparation and purification of monoclonal antibodies PCV2 hybridoma cell lines 1C10, 1C6, and 1D2, and PCV3 hybridoma cell lines 1D8, 3B5, 1D1, 1G10, and 2H3 were revived, and the cell culture supernatant was used for Western blot identification.

[0039] 1.2.2.1 Large-scale preparation of monoclonal antibodies Monoclonal antibodies were prepared in large quantities using an in vivo ascites in mouse method. SPF-grade BALB / c multiparous female mice were selected, and autoclaved liquid paraffin was injected intraperitoneally at a rate of 0.5 mL per mouse. One week later, rapidly growing hybridoma cells were injected intraperitoneally into the mice. When the mice's abdomens were fully inflated, the ascites was aspirated, centrifuged, and the supernatant was collected and stored at -80 °C.

[0040] All animal experiments were conducted with the approval of the Experimental Animal Ethics Committee of Hebei Agricultural University.

[0041] 1.2.2.2 Monoclonal antibody titer detection The titer of monoclonal antibodies was detected using an indirect ELISA method.

[0042] 1.2.2.3 Purification of Monoclonal Antibodies The antibody was purified using the octanoic acid-ammonium sulfate method.

[0043] 1.2.2.4 Affinity determination of monoclonal antibodies The equilibrium dissociation constant KD was determined using an indirect ELISA method. PCV2-Cap and PCV3-Cap were diluted to 5 μg / mL and 1 μg / mL, respectively, with coating buffer (0.05 mol / L carbonate buffer, pH 9.6). 100 μL of each buffer was used to coat the microplate, and the plates were incubated at 37 ℃ for 1 h, followed by overnight incubation at 4 ℃. The liquid in the wells was discarded, and blocking buffer was added, followed by blocking at 37 ℃ for 1 h. The blocking buffer was then discarded, and serially diluted PCV2 or PCV3 monoclonal antibodies (400 pmol / L, 200 pmol / L, 100 pmol / L, 50 pmol / L, 25 pmol / L, 12.5 pmol / L, 6.25 pmol / L, 3.125 pmol / L) were added to each well at 100 μL / well, and the reaction was carried out at 37 ℃ for 1 h. After washing, 100 μL of the target PCV2 or PCV3 monoclonal antibody was added to each well. Incubate μL of HRP-goat anti-mouse IgG (1:10000 dilution) at 37 °C for 45 min; wash, add 100 μL of TMB single-component chromogenic solution to each well and react in the dark for 15 min; stop the reaction by adding 50 μL of 2 mol / L H2SO4 to each well; and detect OD using a microplate reader. 450nm The measured data were analyzed using nonlinear fitting plots with OriginPro software, and a custom equation was applied: Y = BmaxX / (KD + X) (where Y represents the measured OD). 450nm Value, Bmax is the maximum OD 450nm The KD value of the monoclonal antibody is calculated by taking the KD value (where KD is the dissociation constant to be determined, and X is the concentration of the monoclonal antibody).

[0044] 1.2.2.5 HRP labeling and titer detection of monoclonal antibodies Following the HRP labeling kit instructions, high-affinity monoclonal antibodies selected through HRP labeling were used for labeling. The enzyme-labeled antibodies were named PCV2-HRP-1C6 and PCV3-HRP-1D8, respectively. Microplates were coated with 5 μg / mL PCV2-Cap and 1 μg / mL PCV3-Cap. The titers of PCV2-HRP-1C6 and PCV3-HRP-1D8, serially diluted 1:100 to 1:12800, were detected by ELISA. Unlabeled monoclonal antibodies were used as negative controls. When the OD of the test samples... 450nm Value > OD of negative sample 450nm A value multiplied by 2.1 is considered positive.

[0045] 1.2.3 Preparation of gold nanoparticles Add 99 mL of ultrapure water and 1 mL of 1% chloroauric acid solution to a 250 mL Erlenmeyer flask and heat to boiling for 2 min. Quickly transfer the Erlenmeyer flask to a magnetic stirrer (temperature 100 ℃, speed 200 r / min), add 1.8 mL of 1% trisodium citrate solution, and continue heating and stirring for 8 min after the solution turns wine red. After cooling, filter to sterilize.

[0046] The colloidal gold suspension was detected by ultraviolet spectrophotometer at OD. 400-700nm The absorption spectra within the specified range were analyzed, and the morphology of the absorption peaks was observed. Smaller peak widths indicate more uniform gold particle size and morphology, while larger peak widths indicate less uniform gold particle size and morphology. Finally, the prepared colloidal gold suspension was dropped onto a copper grid and dried. The shape, size, uniformity, and presence of aggregation of the particles were observed using a transmission electron microscope.

[0047] 1.2.3.1 Monoclonal antibody pairing First, the saturated dilutions of different monoclonal antibodies against PCV2 and PCV3 strains were determined using an indirect ELISA method. For each well coated with PCV2-Cap or PCV3-Cap, 100 μL of saturated dilution of PCV2 or PCV3 monoclonal antibody was added and incubated at 37 °C for 30 min. After washing, another monoclonal antibody from each pair was added (100 μL / well) and incubated at 37 °C for 30 min. After washing, 100 μL of HRP-goat anti-mouse IgG (1:10000 dilution) was added and incubated at 37 °C for 1 h. After washing, TMB substrate chromogenic solution was added and incubated in the dark for 15 min. The reaction was stopped by adding 50 μL of 2 mol / L H2SO4 to each well, and the OD was measured. 450nm The AI ​​value of the two monoclonal antibodies was calculated using the additivity index (AI) method, according to the following formula: AI = (A1.2 - A1) / A2 × 100% (A1 represents the superposition of OD of a single monoclonal antibody) 450nm Value, A2 represents the OD superposition of another monoclonal antibody. 450nm Value, A1.2 represents the OD of the two monoclonal antibodies superimposed. 450nm (Value). When the absolute value of AI is >10%, the two monoclonal antibodies recognize different antigenic sites; when the absolute value of AI is <10%, the two monoclonal antibodies recognize the same antigenic site.

[0048] The monoclonal antibodies with high AI values ​​selected by the above method were paired (Table 1): purified PCV2 monoclonal antibodies 1D2 and 1C10 were mixed with colloidal gold suspension and blocked with bovine serum albumin (BSA); the prepared gold-labeled antibodies were adsorbed onto a glass fiber membrane to prepare gold-labeled pads. 0.25 mg / mL goat anti-mouse IgG was blotted at the control line, and PCV2 monoclonal antibody 1C6 was blotted at a certain concentration at the test line (T line), and the strips were assembled. Similarly, four PCV3 monoclonal antibodies, 3B5, 1D8, 1G10 and 2H3, were labeled with colloidal gold and prepared as gold-labeled pads. 0.25 mg / mL goat anti-mouse IgG was streaked at the control line (C line) on the NC membrane, and PCV3 monoclonal antibody 1D1 was streaked at the test line (T line) on the NC membrane, and the strips were assembled. The pre-assembled test strips were used to test PCV2 and PCV3 positive samples respectively. The two antibodies that showed bands at both the test line and the control line were considered paired antibodies.

[0049] Table 1. Monoclonal antibody pairing

[0050] 1.2.4 Determination of the optimal pH for gold-labeled antibodies Add 1 mL of the prepared colloidal gold suspension to each of the nine 1.5 mL centrifuge tubes (see Table 2 for the specific amounts of each solution added). Add 0 μL, 1 μL, 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of 0.2 mol / L K2CO3 solution to tubes 1-8, respectively. Tube 9 is the control tube (pH 7.2). After inverting and mixing, add 10 μg of PCV2 monoclonal antibody 1C10 or PCV3 monoclonal antibody 1D8 to each tube, mix well, and let stand for 20 min. Add 100 μL of 10% NaCl to tubes 1-8, and add an equal amount of water to tube 9 as a control. Mix slowly and let stand for 2 h, then observe the color change.

[0051] Simultaneously, 200 μL of each tube was added to an ELISA plate, and the OD of the colloidal gold solution in each tube was measured using a Syrergy HTX ELISA reader. 530nm The values ​​are calculated, and a line graph is plotted.

[0052] The tube with the same solution color as the control tube, no sediment at the bottom, and whose broken line is closest to the X-axis is determined to be the optimal addition amount of 0.2 mol / L K2CO3 solution, and the pH of the solution in this tube is determined to be the optimal pH.

[0053] Table 2. Determination of Optimal pH for Gold-Labeled Antibodies

[0054] 1.2.5 Determination of the optimal stable amount of gold-labeled antibody Add 1 mL of colloidal gold suspension to each of eight 1.5 mL centrifuge tubes (see Table 3 for specific amounts of each solution). Add the optimal amount of 0.2 mol / L K2CO3 solution determined in the previous step to each tube and adjust it to the optimal pH. Add 0 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, and 30 μg of PCV2 monoclonal antibody 1C10 or PCV3 monoclonal antibody 1D8 to tubes 1-7 respectively. Mix gently and let stand for 20 min. Add 100 μL of 10% NaCl to each tube. Add an equal amount of water to tube 8 as a control. Mix well and let stand for 2 h. Observe the color change with the naked eye.

[0055] Simultaneously, 200 μL of each tube was added to an ELISA plate, and the OD of the colloidal gold solution in each tube was measured using a Syrergy HTX ELISA reader. 530nm The values ​​are calculated, and a line graph is plotted.

[0056] The tube with the same solution color as the control tube, no sedimentation at the bottom, and whose fold line is closest to the X-axis is determined to be the optimal stable amount of the gold-labeled antibody.

[0057] Table 3 Determination of the optimal stable amount of gold-labeled antibody

[0058] 1.2.6 Colloidal gold labeling of monoclonal antibodies Labeling was performed based on the established optimal pH and stable antibody levels for colloidal gold labeling. The colloidal gold suspension was adjusted to the optimal pH, and half a volume of stable PCV2 and PCV3 monoclonal antibodies (1 mg / mL) were added respectively. The mixture was then labeled at room temperature for 45 min on a shaking platform. 1% BSA was added, and the mixture was blocked for 45 min. The mixture was then incubated at 4 °C for 2 h. The labeled antibody solution was centrifuged at 3000 r / min for 15 min. The supernatant was collected and centrifuged at 12000 r / min for 10 min. The precipitate was collected, mixed with 1 / 20 volume of labeled PBS, and stored at 4 °C for later use.

[0059] 1.2.7 Preparation of gold-labeled pads The prepared gold-labeled antibodies were uniformly adsorbed onto glass cellulose membranes, dried in a 37 ℃ constant temperature oven for 1 h, sealed, and stored at 4 ℃.

[0060] 1.2.8 Assembly of test strips The sample pad, gold label pad, NC membrane, and absorbent pad are pasted onto the PVC base plate in the following order: sample pad overlaps the gold label pad by 2 mm and rests on top of the gold label pad; the gold label pad and absorbent pad overlap the NC membrane by 2 mm and rest on top of the NC membrane. The assembled materials are then cut into 3 mm wide test strips and sealed and dried at 4 ℃.

[0061] 1.2.9 Screening of working concentration for quality control lines Goat anti-mouse IgG was diluted to 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL and imprinted onto the control line (C line) on a nitrocellulose membrane. Test strips were then assembled with PCV2 and PCV3 gold-labeled pads, respectively. 50 μL of PCV2 and PCV3 negative samples were added to each sample well, and the results were observed within 15 min to determine the optimal working concentration of the control line.

[0062] 1.2.10 Screening of working concentration for the detection line The monoclonal antibody PCV2-1C6 was blotted onto the nitrocellulose membrane at the optimal working concentration selected for the quality control line. The monoclonal antibody PCV3-1D1 was diluted to 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, and the monoclonal antibody PCV3-1D1 was diluted to 1 mg / mL, 2 mg / mL, and 3 mg / mL. The two antibodies were then blotted onto the detection lines (T1 and T2) of the nitrocellulose membrane and assembled with their corresponding gold-labeled pads to form a test strip.

[0063] Dilute PCV2-Cap and PCV3-Cap to a final concentration of 10 μg / mL, add 50 μL to the sample well, and observe and record the experimental results within 15 min.

[0064] 1.2.11 Screening of Sample Diluents Triton X-100 was added to Tris-HCl, PBS, PB and deionized water to a final concentration of 0.1% to prepare sample dilution solutions. PCV2-Cap and PCV3-Cap were diluted with different sample dilution solutions, and 50 μL of each solution was added to the corresponding sample well. The experimental results were observed and recorded within 15 min.

[0065] 1.2.12 Exploring the Concentration of Triton X-100 Add different amounts of Triton X-100 to the buffer solution determined in the previous step to final concentrations of 2%, 1%, 0.5%, 0.25%, and 0.1%, respectively. Take 50 μL of each sample dilution and add it to the sample well. Let it stand for 15 min and observe and record the experimental results.

[0066] 1.2.13 Determination of Optimal Color Development Time Positive samples were prepared by simultaneously diluting PCV2-Cap and PCV3-Cap with a predetermined sample diluent. The color development of the test strips was observed and recorded every 1 minute to determine the optimal color development time.

[0067] 1.2.14 Specificity Detection Porcine rotavirus (PoRV), PEDV, PRRSV (JXA1-R strain), PRV (Bartha-K16 strain), and CSFV (cell-derived attenuated strain) were diluted 1:10 and 50 μL of each was added to the sample wells. At the same time, PCV2 or / and PCV3 Cap, PCV2 culture medium or / and PCV3 infectious clone (F5) culture medium were used as positive controls, and cell nutrition medium was used as a negative control. 50 μL of each sample was loaded, and the experimental results were observed and recorded.

[0068] 1.2.15 Sensitivity Testing PCV2-Cap with an initial concentration of 1 mg / mL and PCV3-Cap with an initial concentration of 2 mg / mL were extracted and added to the same sample dilution buffer until the concentration of both proteins was 3.125 μg / mL. They were then diluted to 1:16 at ratios of 1:2 and 1:4, respectively. 50 μL of each protein was added to the sample, and the reaction was allowed to proceed for 15 min. The experimental results were observed and recorded.

[0069] 1.2.16 Compliance Rate and Clinical Application Seven pig spleens were taken, and the tissue samples were mixed with physiological saline in a certain proportion and ground on ice. After grinding, the mixture was repeatedly frozen and thawed three times, centrifuged at 8000 r / min for 8 min, and 50 μL of supernatant was added to the sample well. After the reaction was completed, the experimental results were observed and recorded.

[0070] 2 Results 2.1 Prepared Protein 2.1.1 Expression and Purification of Proteins Transformed PCV2-Cap and PCV3-Cap prokaryotic recombinant expression plasmids pET28a(+)-Cap respectively E. coli After IPTG induction, the results were analyzed by SDS-PAGE as follows: Figure 1As shown, the target protein band with the expected molecular weight (25 kDa) is visible in the corresponding lane. After purification, the recombinant Cap protein exhibits a single band on electrophoresis with almost no impurities.

[0071] 2.1.2 Western blotting of proteins Western blotting showed that the expressed recombinant PCV2-Cap and PCV3-Cap could specifically bind to porcine PCV2 and PCV3 antiserum, respectively. Figure 2 As shown, a brown band appeared at 25 kDa in the corresponding lane on the PVDF membrane, and no reaction band of the corresponding molecular weight was observed in the lane of the induced product of E. coli transformed with empty plasmid, indicating that the expressed recombinant PCV2-Cap and PCV3-Cap have good reactivity.

[0072] 2.2 Preparation of Monoclonal Antibodies 2.2.1 Western blotting of monoclonal antibodies Western blotting results showed ( Figure 3 After the supernatant of revived PCV2 and PCV3 hybridoma cells reacted with their corresponding proteins, a clear brown band of the expected size appeared in the corresponding lane on the PVDF membrane, while the supernatant of SP2 / 0 cells did not react with either protein. This demonstrates that both PCV2 and PCV3 monoclonal antibodies can specifically react with their corresponding proteins.

[0073] 2.2.2 Monoclonal antibody titer Ascites fluid titers were detected by indirect ELISA after coating PCV2-Cap and PCV3-Cap, respectively. The results showed that the titers of the three PCV2 ascites antibodies and the five PCV3 ascites antibodies could reach up to 1:10. 7 .

[0074] 2.2.3 Purified monoclonal antibodies The harvested ascites monoclonal antibodies, after purification using the caprylic acid-ammonium sulfate method, showed clear heavy and light chains upon SDS-PAGE analysis, with few impurity bands. Figure 4 ), which can be used for subsequent experiments.

[0075] 2.2.4 Affinity of Monoclonal Antibodies The OD obtained by measuring the binding of different concentrations of monoclonal antibodies to their corresponding proteins 450nmThe KD values ​​of the monoclonal antibodies were obtained by nonlinear fitting analysis using Origin Pro 2021 software. The KD values ​​of the two monoclonal antibodies 1C10 and 1C6 for PCV2 were 48.08 nmol / L and 33.64 nmol / L, respectively. The KD values ​​of the two monoclonal antibodies 1D8 and 3B5 for PCV3 were 33.91 pmol / L and 60.89 pmol / L, respectively. It can be seen that monoclonal antibody 1C6 has a higher binding affinity to PCV2-Cap than 1C10, and monoclonal antibody 1D8 has a higher binding affinity to PCV3-Cap than 3B5. Therefore, monoclonal antibodies 1C6 and 1D8 were selected for HRP labeling.

[0076] 2.2.5 Titer of HRP-labeled monoclonal antibodies The titer of enzyme-labeled antibodies was determined using a direct ELISA method. The results showed that the negative control OD of PCV2 direct ELISA was [missing value]. 450nm The value is 0.036, therefore when the sample OD 450nm A value > 0.076 is considered positive. When HRP-PCV2-1C6 is diluted 12800 times, the OD... 450nm The value was 0.188 > 0.076, and further dilution resulted in a negative result; therefore, the titer of HRP-PCV2-1C6 reached 1:12800. PCV3 direct ELISA negative control OD 450nm The value is 0.042, therefore when the sample OD... 450nm A value > 0.088 is considered positive. When HRP-PCV3-1D8 is diluted 12800 times, the OD... 450nm The value was 0.151 > 0.088, and further dilution resulted in a negative result. Therefore, the titer of HRP-PCV3-1D8 reached 1:12800.

[0077] 2.3 PCV2-PCV3 Antigen Rapid Detection Dual Colloidal Gold Test Strip 2.3.1 Nanogold quality The prepared colloidal gold suspension, when observed with the naked eye, is clear, transparent, and free of sediment, with a wine-red color. Figure 5 (A)

[0078] The absorbance values ​​at 400–600 nm were measured using a UV spectrophotometer, and the curve was plotted as follows. Figure 5 As shown in Figure B, an absorption peak appears at 520 nm, with a maximum absorbance value of 0.9215.

[0079] The colloidal gold particles were further observed using transmission electron microscopy, such as... Figure 5 As shown in Figure C, the colloidal gold particles are uniform in morphology and size, with little aggregation, and an average particle size of about 20 nm, proving that the colloidal gold is of good quality and can be used for subsequent experiments.

[0080] 2.3.2 Characterization of monoclonal antibodies Indirect ELISA showed that the saturation dilutions of 1D2 and 1C10 in the three PCV2 hybridoma cells were 1:160, and the saturation dilution of 1C6 was 1:80. In the five PCV3 hybridoma cells, the saturation dilutions of 1G10 and 2H3 were 1:80, and the saturation dilutions of 1D8, 1D1, and 3B5 were 1:160, 1:320, and 1:640, respectively.

[0081] Superimposed ELISA assays revealed that in PCV2 hybridoma cells, the AI ​​value of superimposed 1C10 and 1C6 was greater than 10%, demonstrating that these two monoclonal antibodies could recognize different antigenic epitopes. In PCV3 hybridoma cells, the AI ​​value of superimposed 1D1 with 1D8, 3B5, and 1G10 was greater than 10%, and the AI ​​value of superimposed 1D8 with 1G10 was also greater than 10%, demonstrating that 1D1 with 1D8, 3B5, and 1G10 recognized different antigenic epitopes, and 1D8 with 1G10 recognized different antigenic epitopes.

[0082] The PCV2 monoclonal antibody pairing results showed that when the gold-labeled antibody was 1C10 and the streaking antibody was 1C6, both the test line and the control line of the test strip showed bands. Figure 6 (A) indicates that PCV2 monoclonal antibodies 1C6 and 1C10 can recognize different antigenic epitopes and can be used for subsequent experiments.

[0083] The PCV3 monoclonal antibody pairing results showed that when the gold-labeled antibody was 1D8 and the streak antibody was 1D1, both the test line and the control line of the test strip showed bands. Figure 6 (B) Therefore, it was determined that PCV3 monoclonal antibodies 1D8 and 1D1 can recognize different antigenic epitopes and can be used for subsequent experiments.

[0084] 2.3.3 Optimal pH for Colloidal Gold-Labeled Monoclonal Antibodies The optimal pH for colloidal gold-labeled PCV2 monoclonal antibody can be visually displayed based on the results of the Mey stabilization assay. Compared to the control tubes, tubes 1 and 2 showed significant color changes at pH 6.0-6.2, while tubes 3-8 showed no significant color difference at pH 6.5-7.6. Figure 7 Therefore, the optimal pH range for PCV2 gold-labeled antibody is 6.5-8.0. Simultaneously, the OD values ​​of the colloidal gold solutions in each tube were measured. 530nm The values ​​were plotted as a line graph, with pH between 6.5 and 8.0 and OD... 530nm The pH value closest to the X-axis is the optimal pH for colloidal gold, i.e., the optimal pH is 7.3. Figure 7 (B)

[0085] The optimal pH for colloidal gold-labeled PCV3 monoclonal antibody was determined in the same way as for PCV2. According to the Mey stabilization assay results, compared to the control tubes, tubes 1-3 showed significant color changes at pH 6.0-6.5, while tubes 4-7 showed virtually no color change at pH 6.8-7.6. Figure 7 (C), therefore the optimal pH range for PCV3 gold-labeled antibody is 6.8~7.6, and the OD of the colloidal gold solution is measured. 530nm The values ​​were plotted as a line graph, with pH between 6.8 and 7.6 and OD... 530nm The value closest to the X-axis is the optimal pH for colloidal gold, i.e., the optimal pH is 7.3. Figure 7 (D).

[0086] Therefore, the optimal pH for colloidal gold-labeled PCV2 and PCV3 monoclonal antibodies is 7.3.

[0087] 2.3.4 Optimal Stable Amount of Colloidal Gold-Labeled Monoclonal Antibodies The optimal stabilization amount of colloidal gold-labeled PCV2 monoclonal antibody can be visually displayed based on the results of the Mey stabilization experiment. Compared with the control tubes, tubes 1 and 2 show slight color changes, while tubes 3-7 show no color difference when 10-30 μg is added. Figure 8 Therefore, the optimal stable amount range for adding 10-30 μg of antibody per mL of colloidal gold suspension is (A).

[0088] Simultaneously, 200 μL of each tube was added to an ELISA plate, and the OD of the colloidal gold solution in each tube was measured using a Syrergy HTX ELISA reader. 530nm The values ​​were calculated and a line graph was plotted, as shown in the figure. When the amount of monoclonal antibody added was 15 μg, the OD value was... 530nm The value closest to the X-axis ( Figure 8 Therefore, the optimal stable amount of PCV2 1C10 monoclonal antibody is 10 μg added to 1 mL of colloidal gold suspension (B).

[0089] The optimal stabilization level of colloidal gold-labeled PCV3 monoclonal antibody was determined using the same method as for PCV2. Based on the results of the Mey stabilization experiment, tubes 1 and 2 showed slight color changes compared to the control tubes, while tubes 3-7 showed no color difference when 10-30 μg was added. Figure 8 Therefore, the optimal stable amount range for adding 10-30 μg of antibody per mL of colloidal gold suspension is (C).

[0090] Simultaneously, 200 μL of each tube was added to an ELISA plate, and the OD of the colloidal gold solution in each tube was measured using a Syrergy HTX ELISA reader. 530nm The values ​​were calculated and a line graph was plotted. As shown in the figure, when the amount of monoclonal antibody added was 15 μg, the OD value was... 530nm The value closest to the X-axis ( Figure 8 Therefore, the optimal stable amount of PCV3 1D8 monoclonal antibody is 10 μg added to 1 mL of colloidal gold suspension (D).

[0091] 2.3.5 Working concentration of the quality control line Different concentrations of goat anti-mouse IgG were streaked onto the control line (C line) on a nitrocellulose membrane. 50 μL of negative sample was added, and the results were as follows: Figure 9 As shown, when the control line concentration is 0.5 mg / mL, the band is both clear and not too dark. Therefore, the optimal control line concentration for the test strip is determined to be 0.5 mg / mL.

[0092] 2.3.6 Working concentration of the detection line Different concentrations of PCV2 and PCV3 monoclonal antibodies were blotted onto the detection lines T1 and T2 on a nitrocellulose membrane. Positive samples were then added, and the results were as follows: Figure 10 As shown, bands of varying thicknesses can be seen at the test lines of test strips with different antibody concentrations. When the concentration of PCV2 monoclonal antibody is 2 mg / mL and the concentration of PCV3 monoclonal antibody is 3 mg / mL, the test lines of the test strips are the most obvious and clear. Therefore, the optimal working concentration of PCV2 and PCV3 dual colloidal gold T1 is determined to be 2 mg / mL and the optimal working concentration of T2 is determined to be 3 mg / mL.

[0093] 2.3.7 Optimal Sample Diluent The positive samples were diluted with the four prepared test strip diluents: Tris-HCl containing 0.1% Triton X-100, PBS, PB buffer, and deionized water, respectively. The results after sample addition are as follows: Figure 11 As shown, the buffer solution that produces distinct bands at both the control line (C line) and the test lines (T1, T2) is a Tris-HCl solution containing 0.1% Triton X-100. Therefore, this solution is determined to be the optimal sample dilution.

[0094] Further, Tris-HCl buffer solutions containing different concentrations of Triton X-100 were added to the sample wells, and the results showed that ( Figure 12 When Triton X-100 is present, no false positives will be produced. However, when Triton X-100 is present at other concentrations, weak bands will appear on the test line. Therefore, Tris-HCl buffer containing 0.1% Triton X-100 is the best sample dilution.

[0095] 2.3.8 Optimal color development time A clear band appeared 11 minutes after the positive sample was added to the sample well, and the background was clean. No significant changes were observed in the band after 13 and 15 minutes of further reaction. Therefore, 11 minutes was determined to be the optimal color development time for the test strip.

[0096] 2.3.9 Result Judgment Criteria When the sample is positive for PCV2 or PCV3 antigen, a red band will appear at the test line (T1) or (T2) and the control line (C) of the colloidal gold test strip. When the sample is co-infected with PCV2 and PCV3, a red band will appear at the test line (T1), (T2), and control line (C) of the colloidal gold test strip. If the sample is negative for both PCV2 and PCV3 antigen, only a red band will appear at the control line (C) of the test strip. If there is no color at the control line (C), it is invalid.

[0097] 2.3.10 Specificity The assembled PCV2 and PCV3 dual test strips were used to detect other pathogens, with positive and negative controls. The results showed that the T1 test line of the test strip could only specifically bind to PCV2-Cap or viral fluid, and the T2 test line could only specifically bind to PCV3-Cap or the supernatant of infectious clones. Figure 13 This demonstrates that the method has good specificity.

[0098] 2.3.11 Sensitivity Different concentrations of PCV2 and PCV3-Cap were added to the sample wells to determine the sensitivity of the test strips. The results showed that when the content of the two proteins in the sample was 0.16 μg, the bands were clear; as the protein content decreased, the two detection lines gradually became fainter. When the sample contained 0.02 μg of PCV2-Cap, there was no band at the T1 detection line, while when the PCV2-Cap content in the sample was 0.04 μg, there was a faint band at T1. Therefore, the detection limit for PCV2 was determined to be 0.04 μg. When the PCV3-Cap content was 0.02 μg, there was a weak band at the T2 detection line, and no band was observed when the content decreased further. Therefore, the detection limit for PCV3 was determined to be 0.02 μg of protein.

[0099] 2.3.12 Compliance Rate and Clinical Application Seven porcine spleen homogenates were simultaneously tested using assembled PCV2 and PCV3 dual colloidal gold test strips and PCR technology. The results showed that the detection rates of PCV2 and PCV3 using the dual test strips and PCR were 85.71% (6 / 7) and 100% (7 / 7), respectively, with a positive concordance rate of 85.71%. The detection rates of PCV3 using both methods were 100% (7 / 7), with a positive concordance rate of 100%. These results indicate that the dual colloidal gold test strips for PCV2 and PCV3 have high concordance rates with PCR, enabling rapid and simultaneous detection of both PCV2 and PCV3, and can be used for clinical diagnosis.

[0100] In summary, this embodiment aims to detect antigens by screening PCV2 and PCV3 monoclonal antibodies capable of recognizing different antigenic epitopes. One of these antibodies was used as a colloidal gold-labeled antibody to prepare a PCV2-PCV3 dual gold-labeled pad. Goat anti-mouse IgG was used as the control line (C line) of the colloidal gold test strip, and another monoclonal antibody of PCV2 and PCV3 was used as the detection line (T line) of the colloidal gold test strip. The colloidal gold antigen-antibody complex that passed through the detection line was captured and colored. This method can detect as little as 0.04 μg of PCV2-Cap and 0.02 μg of PCV3-Cap, exhibiting high sensitivity and no non-specific reactions with antigens such as ASFV, PRRSV, and PoRV. Seven clinical samples were simultaneously tested using this dual test strip and PCR method. The detection rates for PCV2 using the dual test strip and PCR method were 85.71% (6 / 7) and 100% (7 / 7), respectively, with a positive concordance rate of 85.71%. The detection rates for PCV3 using both methods were 100% (7 / 7), with a positive concordance rate of 100%. The results indicate that the dual colloidal gold test strip, constructed using monoclonal antibodies prepared from anti-PCV2 and PCV3 proteins, can simultaneously and rapidly detect both PCV2 and PCV3, and can be used for clinical diagnosis.

[0101] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A dual colloidal gold test strip for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 antigens, characterized in that, Includes a PVC base plate and a sample pad, a gold label pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped on the PVC base plate; The gold-labeled pad is adsorbed with colloidal gold-labeled PCV2 monoclonal antibody 1C10 and colloidal gold-labeled PCV3 monoclonal antibody 1D8. The nitrocellulose membrane is provided with a control line C, a detection line T1, and a detection line T2; the C line is coated with goat anti-mouse IgG, the T1 line is coated with PCV2 monoclonal antibody 1C6, and the T2 line is coated with PCV3 monoclonal antibody 1D1. The PCV2 monoclonal antibodies 1C10 and 1C6 recognize different antigenic epitopes of PCV2-Cap; the PCV3 monoclonal antibodies 1D8 and 1D1 recognize different antigenic epitopes of PCV3-Cap.

2. The test strip according to claim 1, characterized in that, The colloidal gold has a particle size of 20 nm; the optimal pH for colloidal gold-labeled PCV2 monoclonal antibody 1C10 and PCV3 monoclonal antibody 1D8 is 7.3; the optimal stable amount of PCV2 monoclonal antibody 1C10 and PCV3 monoclonal antibody 1D8 per milliliter of colloidal gold suspension is 10 μg.

3. The test strip according to claim 1, characterized in that, The concentration of the C-line goat anti-mouse IgG was 0.5 mg / mL; the concentration of the T1-line PCV2 monoclonal antibody 1C6 was 2 mg / mL; and the concentration of the T2-line PCV3 monoclonal antibody 1D1 was 3 mg / mL.

4. The test strip according to claim 1, characterized in that, The sample diluent for the sample pad is Tris-HCl buffer containing 0.1% Triton X-100; the optimal color development time is 11 min.

5. The test strip according to claim 1, characterized in that, The criteria for determining the results are as follows: When both the T1 line and the C line show red bands, it is determined to be PCV2 antigen positive; When both the T2 line and the C line show red bands, it is determined to be PCV3 antigen positive; When red bands appear on T1, T2 and C lines, it is determined that PCV2 and PCV3 are co-infected. When only line C shows a red band, it is determined that the PCV2 and PCV3 antigens are negative; If there is no red band on line C, the test strip is considered invalid.

6. A method for preparing a dual colloidal gold test strip as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Preparation of gold-labeled antibodies: The colloidal gold suspension was adjusted to pH 7.3, and PCV2 monoclonal antibody 1C10 and PCV3 monoclonal antibody 1D8 were added to a final concentration of 10 μg / mL, respectively. The mixture was labeled at room temperature for 45 min, and BSA was added to a final concentration of 1% for blocking for 45 min. After centrifugation, the precipitate was resuspended in colloidal gold resuspension. (2) Preparation of gold-labeled pad: The gold-labeled antibody prepared in step (2) was adsorbed onto a glass cellulose membrane and dried at 37 °C for 1 h; (3) Preparation of nitrocellulose membrane: 0.5 mg / mL goat anti-mouse IgG was scratched at the C line, 2 mg / mL PCV2 monoclonal antibody 1C6 was scratched at the T1 line, and 3 mg / mL PCV3 monoclonal antibody 1D1 was scratched at the T2 line. (4) Assembly: The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are sequentially overlapped and pasted onto the PVC base plate, and then cut into test strips.

7. The use of a dual colloidal gold test strip as described in any one of claims 1-5 in the preparation of a detection reagent for detecting porcine circovirus type 2 and / or porcine circovirus type 3 antigen.

8. A method for detecting porcine circovirus type 2 and / or type 3 antigens for purposes other than disease diagnosis or treatment, characterized in that, The sample to be tested was tested using the dual colloidal gold test strip as described in any one of claims 1-5.

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