A kit and method for simultaneously screening antibodies against PCV2, Mhp, and PRRSV.

Abstract

This invention discloses a kit and method for simultaneously screening antibodies against PCV2, Mhp, and PRRSV, belonging to the field of veterinary product technology. The kit comprises a mixed antigen composed of porcine circovirus type 2a, 2b, and 2d virus-like particles, recombinant protein of Mycoplasma hyopneumoniae, and inactivated virus solution of porcine reproductive and respiratory syndrome virus (PRRSV) strain SCqq. By jointly coating an enzyme-linked immunosorbent assay (ELISA) plate with antigens from the three pathogens PCV2, Mhp, and PRRSV, this invention overcomes the steric hindrance effect of the three antigens, establishing a kit capable of simultaneously detecting PCV2, Mhp, and PRRSV antibodies in a single detection operation. This kit exhibits high specificity and sensitivity against these three antibodies, facilitating the screening of animals that are triple-negative for PCV2, Mhp, and PRRSV antibodies and the purification of the three pathogens, saving detection time and cost, reducing sample consumption, and shortening the animal enrollment cycle.

Description

Technical Field

[0001] This invention belongs to the field of veterinary product technology, specifically relating to a kit and method for simultaneously screening PCV2, Mhp and PRRSV antibodies. Background Technology

[0002] Porcine circovirus type 2 (PCV2), porcine mycoplasmal pneumonia (MPP), and porcine reproductive and respiratory syndrome (PRRS) are all significant diseases threatening the pig industry. The proportion of mixed infections of diseases in pig farms is increasing. In response to this situation, more and more research institutions are focusing on developing a trivalent vaccine combining PCV2, Mycoplasmal pneumonia (Mhp), and PRRSV. For example, patent CN104271153B discloses a "PCV / Mycoplasma hyopneumoniae / PRRS combined vaccine," and patent CN120531864A discloses a "trivalent inactivated vaccine for porcine circovirus type 2, porcine mycoplasma pneumoniae, and porcine reproductive and respiratory syndrome, its preparation method, and application." Shandong Binzhou Wohua Bioengineering Co., Ltd. and Shandong Bolaiwei Biotechnology Research Institute have applied for a "clinical trial of a trivalent inactivated vaccine for porcine circovirus type 2, porcine mycoplasma pneumoniae, and porcine reproductive and respiratory syndrome (Cap protein + LY1 strain + LF25 strain)." Qingdao National Engineering Research Center for Animal Health Co., Ltd., Qingdao Weilan Biological Products Co., Ltd., Jiangsu Nannong High-Tech Co., Ltd., and Sinopharm Yangzhou Weike Bioengineering Co., Ltd. have applied for a "trivalent inactivated vaccine for porcine circovirus type 2, porcine mycoplasma pneumoniae, and porcine reproductive and respiratory syndrome (rE. coli)." Huapai Biotechnology (Group) Co., Ltd. and Juxing Agriculture and Animal Husbandry Co., Ltd. have applied for a clinical trial of a "trivalent inactivated vaccine against porcine circovirus type 2, porcine mycoplasma pneumoniae, and porcine reproductive and respiratory syndrome virus (recombinant baculovirus HP162 strain + HP-G strain + HP-NADC30 strain)". Correspondingly, the safety and efficacy testing of this PCV2, Mhp, and PRRSV trivalent vaccine requires a large number of pigs that are negative for PCV2, Mhp, and PRRSV triple antibodies. Therefore, there is a significant need for screening pigs that are negative for PCV2, Mhp, and PRRSV triple antibodies.

[0003] In non-immunized pig farms, antibody monitoring followed by culling of antibody-positive animals is a common method for disease eradication. However, testing for multiple antibodies individually faces many difficulties. Considering labor costs, financial costs, and time costs, the market hopes for a kit that can detect PCV2, Mhp, and PRRSV antibodies in a single operation.

[0004] However, there is currently no kit that can simultaneously detect PCV2, Mhp, and PRRSV antibodies. Therefore, there is an urgent need and a broad market prospect for developing a kit that can simultaneously detect these three antibodies. However, coating multiple antigens simultaneously on an ELISA plate can easily cause steric hindrance, a common challenge in ELISA kit development. Numerous studies have shown that steric hindrance exists between molecules, and this steric hindrance can severely affect the sensitivity of ELISA detection.

[0005] For example, the 1985 paper by TE Koertge et al. (The relationship between the binding of primary antibody to solid-phase antigen in microtiter plates and its detection by the ELISA, J Immunol Methods. 1985) discussed the steric hindrance effect caused by the obstruction of spatial conformation between molecules, and is a classic paper that clearly pointed out the steric hindrance problem in ELISA. The paper by DW Brown et al. (The influence of immune complexes, steric effects, and antigen-antigen interactions on the sensitivity of enzyme-linked immunosorbent assays, J Immunol Methods. 1989) showed that the steric hindrance effect may have a significant impact on the sensitivity of ELISA. The paper by Dinesh Kumar et al. in recent years (Determining factor of enzyme conjugates, bridge heterology and analytical variables of immunogens in prednisolone ELISA, Sensing and Bio-Sensing Research. 2024) elaborated on the steric hindrance and then discussed the use of linkers to reduce and overcome the steric hindrance effect between large protein molecules. This illustrates that the steric hindrance between molecules is a significant factor affecting detection sensitivity during the development of ELISA kits, and it is also an unavoidable challenge. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a kit and method for simultaneously screening antibodies against PCV2, Mhp, and PRRSV. By coating an ELISA plate with antigens from three pathogens—porcine circovirus type 2a, 2b, and 2d virus-like particles, recombinant protein of Mycoplasma hyopneumoniae, and inactivated virus solution of porcine reproductive and respiratory syndrome virus (PRRSV) strain SCqq—the steric hindrance effect of the three antigens is overcome by utilizing the difference in molecular weight of the coated antigens. This establishes an indirect ELISA kit capable of simultaneously detecting antibodies against porcine circovirus type 2, Mycoplasma hyopneumoniae, and PRRSV in a single detection operation. This kit is simple to operate, highly specific and sensitive against the three antibodies, facilitating the screening of animals that are triple-negative for these three pathogens and the purification of the three pathogens. It saves detection time and cost, reduces sample consumption, and shortens the animal enrollment cycle.

[0007] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide a kit for simultaneously screening antibodies against porcine circovirus type 2, Mycoplasma hyopneumoniae, and porcine reproductive and respiratory syndrome virus (PRRSV). The kit comprises a mixed antigen consisting of porcine circovirus type 2a, 2b, and 2d virus-like particles, recombinant Mycoplasma hyopneumoniae protein, and inactivated virus solution of porcine PRRSV strain SCqq.

[0008] Furthermore, the concentrations of porcine circovirus type 2a, 2b, and 2d virus-like particles, recombinant protein of Mycoplasma hyopneumoniae, and inactivated virus solution of porcine reproductive and respiratory syndrome virus SCqq strain in the mixed antigen were 0.5-8 μg / mL, 1-16 μg / mL, and 10 μg / mL, respectively. 6.0 -10 8.0 TCID 50 / mL.

[0009] Furthermore, the concentrations of porcine circovirus type 2a, 2b, and 2d virus-like particles, recombinant protein of Mycoplasma hyopneumoniae, and inactivated virus solution of porcine reproductive and respiratory syndrome virus SCqq strain in the mixed antigen were 1.5-3 μg / mL, 2-5 μg / mL, and 10 μg / mL, respectively. 7.0 -10 8.0 TCID 50 / mL.

[0010] Furthermore, the kit also includes an antigen-coated plate, a PCV2 positive control, a Mhp positive control, a PRRSV positive control, a PCV2, Mhp, and PRRSV triple antibody negative control, sample diluent, washing buffer, enzyme-labeled secondary antibody solution, substrate chromogenic solution, and stop solution.

[0011] Furthermore, after the antigen-coated plate is blocked with 2-3% bovine serum albumin at 35-37°C for 1-3 hours, it is incubated overnight at 4°C using ELISA plate stabilizer I.

[0012] Furthermore, the enzyme-labeled secondary antibody solution is an HRP-labeled goat anti-pig IgG enzyme-labeled secondary antibody solution.

[0013] Furthermore, the serum control was a serum control diluted with protein stabilizer II.

[0014] Another object of the present invention is to provide a method for screening antibodies against porcine circovirus type 2, Mycoplasma hyopneumoniae, and porcine reproductive and respiratory syndrome virus, comprising the following steps: The serum sample to be tested is detected using the kit described in any one of claims 1 to 7, and the presence of the corresponding antibody is determined based on the P / N value.

[0015] Furthermore, the P / N value represents the ratio of the sample or positive control to the negative control, the P value represents the OD value of the sample or positive control, and the N value represents the OD value of the negative control; A sample is considered positive when the P / N value is ≥2.1, indicating that at least one of the three antibodies, PCV2, Mhp, and PRRSV, is suspected. A sample is considered negative when the P / N value is <2.1, indicating that all three antibodies, PCV2, Mhp, and PRRSV, are negative.

[0016] Another object of the present invention is to provide the use of the above-described kit in the preparation of formulations for screening triple-negative animals for PCV2, Mhp, and PRRSV antibodies and for the purification of PCV2, Mhp, and PRRSV diseases.

[0017] The beneficial effects of this invention are: When multiple antigens are co-coated onto an ELISA plate, steric hindrance often occurs due to the interactions and spatial arrangement of antigen groups, affecting sample activity. This invention uses medium-molecular-weight virus-like particle antigens (Polyovirus 2a, 2b, and 2d), relatively small-molecular-weight protein antigens (Mycoplasma hyopneumoniae recombinant protein), and relatively large-molecular-weight whole-virus antigens (Porcine reproductive and respiratory syndrome virus SCqq strain inactivated virus solution) to coat the ELISA plate, forming a combination of large and small molecular weights, effectively overcoming the steric hindrance effect between the three antigens. Specifically, the Porcine circovirus type 2 coating antigen uses trivalent Porcine circovirus 2a, 2b, and 2d virus-like particles instead of a single subtype antigen, effectively detecting antibodies produced by infections with multiple currently prevalent subtypes. The Mycoplasma hyopneumoniae coating antigen uses a recombinant protein, a conserved protein of Mycoplasma hyopneumoniae, effectively detecting antibodies produced by Mycoplasma hyopneumoniae. The Porcine reproductive and respiratory syndrome virus coating antigen uses a whole-virus inactivated antigen, capable of detecting antibodies produced by multiple antigenic epitopes, making the detection results more sensitive.

[0018] This invention utilizes the combined coating of antigens from three pathogens—porcine circovirus type 2a, 2b, and 2d virus-like particles, recombinant protein of Mycoplasma hyopneumoniae, and porcine reproductive and respiratory syndrome virus (PRRSV) strain SCqq—on an enzyme-linked immunosorbent assay (ELISA) plate. By optimizing reaction conditions and overcoming the steric hindrance effect of the three antigens through differences in molecular weight, this invention establishes an indirect ELISA kit capable of simultaneously detecting antibodies against porcine circovirus type 2, Mycoplasma hyopneumoniae, and PRSV in a single assay. This kit is simple to operate, highly specific and sensitive against the three antibodies, and facilitates the screening of animals that are triple-negative for porcine circovirus type 2, Mycoplasma hyopneumoniae, and PRSV antibodies, as well as the purification of the three pathogens. It saves testing time and costs, reduces sample consumption, and shortens the animal enrollment cycle. Detailed Implementation

[0019] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0020] The reagents and related detection methods involved in this invention are as follows: 1. Reagents Washing solution: NaCl 8.0 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4 0.24 g, 0.5 ml Tween-20, dissolved in purified water and brought to a final volume of 1000 mL.

[0021] 0.05M pH 9.6 carbonate buffer: Na2CO3 1.59g, NaHCO3 2.94g, dissolved in purified water and brought to a final volume of 1000mL, filtered through a 0.22μm filter and stored at 4℃.

[0022] Sample dilution solution: Add 5g BSA to 1000mL of washing solution.

[0023] PCV2 positive control, Mhp positive control, PRRSV positive control, PCV2, Mhp, and PRRSV triple antibody negative control: porcine serum diluted with protein stabilizer II.

[0024] HRP-labeled goat anti-pig IgG enzyme-labeled secondary antibody solution: HRP-labeled goat anti-pig IgG diluted with HRP conjugate stabilizer I.

[0025] The ELISA plate stabilizer I, protein stabilizer II, and HRP conjugate stabilizer I were all purchased from Huzhou Yingchuang Biotechnology Co., Ltd.

[0026] Substrate chromogenic solution: single-component TMB solution (3,3',5,5'-tetramethylbenzidine).

[0027] Termination solution: 2M H2SO4 solution.

[0028] 2. Test methods First, a general experimental method is established, and then optimizations are made based on this method: S1. Antigen coating: Dilute the antigen to a certain concentration with carbonate buffer, add 100 μL / well to a 96-well microplate, and coat overnight at 4°C.

[0029] S2. Washing: Add 300 μL of washing solution to each well, wash 3 times, and pat dry.

[0030] S3. Sealing: Add 100 μL of sealing solution to each well and seal at 37°C for 2 hours.

[0031] S4. Washing: Repeat S2.

[0032] S5. Serum incubation: Add 100 μL of diluted serum sample to each well and incubate at 37°C for 1 hour.

[0033] S6. Washing: Repeat S2.

[0034] S7. Secondary antibody incubation: Add 100 μL of secondary antibody solution to each well and incubate at 37°C for 1 hour.

[0035] S8. Washing: Repeat S2.

[0036] S9. Color development: Add 100 μL of substrate color development solution to each well and incubate at room temperature for 15 minutes.

[0037] S10, Termination: Add 50 μL of termination solution to each well and read the OD. 450nm Absorbance value.

[0038] Example 1 Preparation of mixed coated antigens 1. The antigen-coated plate is coated with a mixture of antigens consisting of porcine circovirus type 2a, 2b and 2d virus-like particles, porcine mycoplasma pneumoniae recombinant protein and porcine reproductive and respiratory syndrome virus SCqq strain inactivated virus solution.

[0039] (1) Porcine circovirus type 2a, 2b and 2d virus-like particle antigens were prepared by the method disclosed in CN117143888A.

[0040] (2) The recombinant protein of Mycoplasma hyopneumoniae was prepared by the method disclosed in CN105524148B.

[0041] (3) Preparation of inactivated virus solution of porcine reproductive and respiratory syndrome virus SCqq strain (which has been disclosed in invention patent application number 202411424270.5): S1. Virus propagation: Discard the nutrient solution of monolayer PAMs cells, inoculate with virus at a ratio of 5% of the nutrient solution, adsorb at 37°C for 30-60 minutes, then replenish the maintenance medium, and incubate at 37°C for 48-72 hours. Harvest when more than 80% of the cells show cytopathic effects, and centrifuge to harvest the supernatant after three freeze-thaw cycles.

[0042] S2, Virus content detection: Disperse PAMs cells into 1×10⁻⁶ particles. 6 Cells were seeded at 100 μL / well in a 96-well plate. After cell attachment, the culture medium was discarded, and 100 μL of a 10-fold serially diluted virus solution was added to each well. -1 ~10 -8 Each dilution was replicated in 4 wells, with normal PAMs cells serving as a control. Cells were incubated at 37°C with 5% CO2 for 72 h. The number of wells showing cytopathic effects and the number of wells without cytopathic effects were observed and recorded under a microscope. The Reed-Muench method was used to calculate the viral TCID. 50 .

[0043] S3. Virus inactivation: Add 0.2% formaldehyde to the harvested virus solution and inactivate at 37°C for 48 hours.

[0044] 2. Preparation of the mixed coated antigen: Porcine circovirus type 2a, 2b, and 2d virus-like particles, Mycoplasma hyopneumoniae recombinant protein, and inactivated porcine reproductive and respiratory syndrome virus (PRRSV) SCqq strain were diluted to specific concentrations with carbonate buffer at pH 9.6. The concentrations of porcine circovirus type 2a, 2b, and 2d virus-like particles, Mycoplasma hyopneumoniae recombinant protein, and inactivated PRRSV SCqq strain in the mixed antigen were 1.5-24 μg / mL, 3-48 μg / mL, and 3 × 10⁻⁶ μg / mL, respectively. 6.0 -3×10 8.0 TCID 50 Mix in equal proportions of / mL.

[0045] Example 2: Spatial steric hindrance effect when multiple antigens are co-coated 1. Determination of the optimal coating concentration for antigen coating alone Porcine circovirus type 2a, 2b, and 2d virus-like particles, recombinant protein of Mycoplasma hyopneumoniae, and inactivated virus solution of porcine reproductive and respiratory syndrome virus SCqq strain were diluted to different concentrations and individually coated onto ELISA plates. General assay methods were used for detection to determine the optimal coating concentration for each antigen. After coating, the plates were blocked with a washing buffer containing 5% skim milk. After washing, corresponding positive and negative sera were diluted 25, 50, 100, 200, 400, and 800 times and incubated. After washing, the enzyme-labeled secondary antibody was diluted 2000 times and incubated. After color development, termination, and OD value reading, the results are shown in Tables 1-3. In the tables, P and N values ​​represent the detection values ​​of positive and negative sera at the corresponding dilutions, respectively. The P / N value represents the ratio of the detection values ​​of positive and negative sera at the corresponding dilutions. A higher P / N value indicates a better ability to distinguish between positive and negative sera under those conditions.

[0046] Table 1. Determination of optimal coating concentrations for porcine circovirus types 2a, 2b, and 2d virus-like particles.

[0047] Table 2. Determination of the optimal coating concentration of recombinant protein from Mycoplasma hyopneumoniae.

[0048] Table 3. Determination of the optimal coating concentration of inactivated virus solution for porcine reproductive and respiratory syndrome virus (PRRSV) strain SCqq.

[0049] The results in Tables 1-3 indicate that porcine circovirus types 2a, 2b, and 2d virus-like particles exhibited relatively high P / N values ​​at a coating concentration of 2 μg / mL and serum dilutions of 100 or 200 times, suggesting that the optimal coating concentration for this antigen was 2 μg / mL, and the optimal serum dilution was 100 or 200 times. Similarly, recombinant Mycoplasma hyopneumoniae protein showed relatively high P / N values ​​at a coating concentration of 4 μg / mL and serum dilutions of 100 or 200 times, indicating that the optimal coating concentration for this antigen was 4 μg / mL, and the optimal serum dilution was 100 or 200 times. Inactivated porcine reproductive and respiratory syndrome virus (PRRSV) strain SCqq showed relatively high P / N values ​​at a coating concentration of 10 μg / mL. 7.0 TCID 50 The P / N value was highest when serum was diluted 100-fold, indicating that the optimal coating concentration of this antigen was 10. 7.0 TCID 50 / mL, and the optimal serum dilution factor is 100-fold. Based on the above results, the optimal coating concentration for coating the three antigens individually was determined. Considering the optimal serum dilution factor for each antigen and practical operation, the optimal serum dilution factor was determined to be 100-fold. 2. Comparison of single antigen coating and multiple antigen combined coating Under optimal coating concentrations for each antigen, single antigens and mixed antigens (porcine circovirus type 2a, 2b, and 2d virus-like particles + Mycoplasma hyopneumoniae recombinant protein + inactivated swine reproductive and respiratory syndrome virus SCqq strain) were coated separately, and detected using a universal test method to determine the difference in detection values ​​between individual antigen coating and three-antigen mixed coating. Mixed antigens of porcine circovirus type 2 strain 162 + Mycoplasma hyopneumoniae recombinant protein + swine reproductive and respiratory syndrome virus SCqq strain and porcine circovirus type 2a, 2b, and 2d virus-like particles + Mycoplasma hyopneumoniae HP-G strain + swine reproductive and respiratory syndrome virus SCqq strain were used as controls.

[0050] After coating, the cells were blocked with 5% skim milk washing solution. After washing, the corresponding positive and negative sera were diluted 25, 50, 100, 200, 400 and 800 times and incubated. After washing, the enzyme-labeled secondary antibody was diluted 2000 times and incubated. After color development, termination and reading of OD values, the results are shown in Tables 4 and 5.

[0051] Table 4 Results of single antigen and mixed antigen coating detection

[0052] The results in Table 4 show that the detection results of single antigen coating and mixed antigen coating are basically equivalent, indicating that when mixed antigen coating is performed in this invention, no steric hindrance effect is generated between porcine circovirus type 2a, 2b and 2d virus-like particles, porcine mycoplasma pneumoniae recombinant protein and porcine reproductive and respiratory syndrome virus SCqq strain inactivated viral liquid antigen.

[0053] Table 5. Detection results of control group antigen and mixed antigen coating.

[0054] Table 5 shows that when porcine circovirus type 2a, 2b, and 2d virus-like particles were replaced with porcine circovirus type 2 strain 162, the OD value for PCV2 positive serum decreased, the OD value for negative serum increased, and the P / N value decreased. The OD value for Mhp positive serum did not decrease, but the OD value for negative serum increased, and the P / N value decreased. The OD value for PRRSV positive serum decreased, the OD value for negative serum increased, and the P / N value decreased. When the recombinant protein of Mycoplasma hyopneumoniae was replaced with Mycoplasma hyopneumoniae HP-G strain, the OD values ​​for PCV2 positive, Mhp positive, and PRRSV positive sera all decreased, while the OD values ​​for negative sera all increased, and the P / N value all decreased. This indicates that antigen replacement affected the sensitivity and specificity for the corresponding positive sera.

[0055] The above results indicate that only when coated with a mixed antigen consisting of porcine circovirus type 2a, 2b and 2d virus-like particles, recombinant protein of Mycoplasma hyopneumoniae, and inactivated viral fluid of porcine reproductive and respiratory syndrome virus SCqq strain can the steric hindrance effect of multiple antigens be overcome.

[0056] Example 3: Optimizing Detection Conditions 1. Optimization of sealing fluid and optimal sealing conditions Coat the ELISA plate with the mixed antigen, discard the buffer, add 300 μL of washing buffer to each well, wash three times, pat dry, and block with 100 μL of 0.5% gelatin, 5% skim milk, or 2% bovine serum albumin (BSA) per well, respectively, at 37°C for different times. After washing three times, add 100 μL of positive and negative sera diluted 100-fold to each well and incubate at 37°C for 1 hour. After washing, dilute the enzyme-labeled secondary antibody 2000-fold, add 100 μL to each well, and incubate at 37°C for 1 hour. After washing, add 100 μL of substrate chromogenic solution to each well and incubate at room temperature for 15 minutes. Finally, add 50 μL of stop solution to each well and read the OD. 450nm The absorbance values ​​are shown in Table 6-8.

[0057] Table 6 Determination of PCV2 Closure Conditions

[0058] Table 7 Determination of Mhp Closure Conditions

[0059] Table 8 Determination of PRRSV Closure Conditions

[0060] As shown in Tables 6-8, the blocking effect of 2% bovine serum albumin at 37°C for 1.5 hours on PCV2 antigen and Mhp antigen is the best. The blocking effect of 2% bovine serum albumin at 37°C for 1.5 hours on PCV2 antigen and Mhp antigen is the best. The blocking effects of 2% bovine serum albumin at 37°C for 1.5 hours and 2 hours on Mhp antigen are comparable. Based on the above, the blocking condition is determined to be: 2% bovine serum albumin at 37°C for 1.5 hours.

[0061] 2. Optimization of serum incubation time The parameters determined above were fixed to determine the optimal incubation time for serum. The mixed antigen was coated onto the ELISA plate. After discarding the buffer, 300 μL of washing buffer was added to each well, and the plate was washed three times. The plates were then patted dry and blocked with 100 μL of 2% bovine serum albumin (BSA) per well at 37°C for 1.5 h. After three washes, 100 μL of 100-fold diluted positive and negative sera were added to each well and incubated at 37°C for different times. After washing, 100 μL of 2000-fold diluted enzyme-labeled secondary antibody was added to each well and incubated at 37°C for 1 hour. After washing, 100 μL of substrate chromogenic solution was added to each well and incubated at room temperature for 15 minutes. Finally, 50 μL of stop solution was added to each well, and the OD values ​​were read. 450nm The absorbance values ​​are shown in Table 9.

[0062] Table 9 Determination of Optimal Serum Incubation Time

[0063] The results in Table 9 show that all three sera showed the best effect when incubated at 37°C for 1 hour. Therefore, the optimal incubation time for the serum was determined to be 37°C for 1 hour.

[0064] 3. Optimization of the optimal dilution factor and incubation time for the secondary antibody To determine the optimal dilution and incubation time of the secondary antibody, the parameters were fixed. The mixed antigen was coated onto the ELISA plate. After discarding the buffer, 300 μL of washing buffer was added to each well, and the plate was washed three times. The plates were then patted dry and blocked with 100 μL of 2% bovine serum albumin (BSA) per well at 37°C for 1.5 h. After three washes, 100 μL of positive and negative sera diluted 100-fold were added to each well and incubated at 37°C for 1 h. Following washing, the ELISA-labeled secondary antibody was diluted to different factors, with 100 μL added to each well and incubated at 37°C for different times. After washing, 100 μL of substrate chromogenic solution was added to each well and incubated at room temperature for 15 minutes. Finally, 50 μL of stop solution was added to each well, and the OD values ​​were read. 450nm The absorbance values ​​are shown in Tables 10-12.

[0065] Table 10 Determination of Optimal Dilution Factor and Optimal Incubation Time for PCV2 Antibody Detection

[0066] Table 11 Determination of Optimal Dilution Factor and Optimal Incubation Time for Mhp Antibody Detection

[0067] Table 12 Determination of Optimal Dilution Factor and Optimal Incubation Time for PRRSV Antibody Detection

[0068] As shown in Tables 10-12, the optimal secondary antibody dilution for PCV2 and Mhp antibody detection is 2000-fold, and the optimal incubation time is 1 hour at 37°C. The optimal secondary antibody dilution for PRRSV antibody detection is 2000-fold, and the optimal incubation time is 1 hour or 1.5 hours at 37°C. Therefore, the optimal secondary antibody dilution is determined to be 2000-fold, and the optimal incubation time is 1 hour at 37°C.

[0069] Example 4: Stability, sensitivity, and specificity of the kit After determining the parameters of the kit, it is necessary to ensure the stability of the kit and test its sensitivity and specificity.

[0070] 1. Stability and sensitivity testing Based on the above results, the kit parameters were determined as follows: Coat the ELISA plate with the mixed antigen overnight, discard the solution, add 300 μL of washing buffer to each well, wash 3 times, pat dry, and block with 100 μL of 2% bovine serum albumin (BSA) per well at 37°C for 1.5 h. After washing 3 times, add 100 μL of 100-fold diluted positive and negative sera to each well and incubate at 37°C for 1 h. After washing, add 100 μL of 2000-fold diluted enzyme-labeled secondary antibody solution to each well and incubate at 37°C for 1 h. After washing, add 100 μL of substrate chromogenic solution to each well and incubate at room temperature for 15 minutes. Finally, add 50 μL of stop solution to each well and read the OD value. 450nm Absorbance value.

[0071] However, this does not guarantee the long-term activity of biomolecules. The activity of antigens coated on ELISA plates decreases rapidly after drying, and serum controls and ELISA-labeled secondary antibodies diluted with ordinary diluents also quickly become inactive. To solve this technical problem, this invention uses ELISA plate stabilizer I to treat the ELISA plate, protein stabilizer II to dilute the serum control, and HRP conjugate stabilizer I to dilute the HRP-labeled ELISA-labeled secondary antibody, thereby improving the stability of the ELISA plate, serum control, and secondary antibody solution. Specifically: The method for processing ELISA plates is as follows: after blocking with 2% bovine serum albumin, wash three times with washing buffer, then add 100 μL of ELISA plate stabilizer I to each well, incubate overnight at 4°C, spin dry and discard the liquid, air dry at 37°C for 120 minutes, and store the ELISA plates in a sealing bag containing desiccant at 2-8°C. The method for processing serum controls is to dilute serum controls with protein stabilizer II. The method for processing enzyme-labeled secondary antibodies is to dilute HRP-labeled enzyme-labeled secondary antibodies with HRP conjugate stabilizer I.

[0072] After preparing the kit according to the above method, store it at 2-8℃. Take samples monthly to test the stability of the kit. The test results are shown in Tables 13 and 14. Then, dilute the serum at different ratios and test it according to the method determined above. The titer of the positive serum can reflect the sensitivity of the kit to detect positive serum. The results are shown in Table 15.

[0073] Table 13 shows the stability of the kit as detected.

[0074] Table 14 Coefficient of Variation

[0075] The results showed that the kit remained stable after being stored at 2-8℃ for 12 months, and the calculated coefficient of variation was less than 10% (Table 14).

[0076] Table 15 Sensitivity of the Reagent Kit

[0077] Typically, a P / N value ≥ 2.1 is used as the cutoff value for determining the positive or negative status of a sample, and the highest dilution with a P / N value ≥ 2.1 is used as the titer of the serum.

[0078] As can be seen from the results in Table 15, the reagent kit constructed in this invention has a titer of over 1:25600 against PCV2 positive serum, a titer of 1:6400 against Mhp positive serum, and a titer of 1:6400 against PRRSV positive serum, indicating that it has high sensitivity.

[0079] 2. Specific detection Whether the kit can detect other antibodies is an important criterion for determining the specificity of the kit. Positive serum from common swine diseases was selected and tested using this kit. The results are shown in Table 16.

[0080] Table 16 shows the detection results of the kit for positive sera of common swine diseases.

[0081] As shown in Table 16, the kit showed negative results for positive serum tests for common swine diseases other than porcine circovirus type 2, Mycoplasma hyopneumoniae, and porcine reproductive and respiratory syndrome virus antibodies, with P / N values ​​all <2.1 (Table 16), indicating that it has high specificity.

[0082] Example 5: Detection of clinical samples using the kit Twenty clinically collected swine serum samples with varying levels of PCV2, Mhp, and PRRSV antibodies were tested, and the results are shown in Table 17.

[0083] Table 17 Comparison of results between the kit and the standalone detection kit

[0084] As shown in Table 17, serum samples that tested negative with the kit constructed in this invention also tested negative with the commercially available Guangzhou Yueyang Biotechnology Porcine Circovirus Type 2 Antibody ELISA Kit, IDEXX Porcine Mycoplasma Pneumoniae Antibody Kit, and IDEXX Porcine Reproductive and Respiratory Syndrome Virus Antibody Kit. Serum samples that tested positive with the kits showed at least one questionable result when tested with any of the three individual kits. Although the kit cannot distinguish between porcine circovirus type 2, Mycoplasma Pneumoniae, and Porcine Reproductive and Respiratory Syndrome Virus antibodies, it can be used for screening pigs that are triple-negative for these antibodies, and can also be used for disease eradication in non-immunized pig herds. Compared to testing for the three antibodies separately, the kit prepared in this invention is more time- and cost-effective.

[0085] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A kit for simultaneous screening of antibodies against porcine circovirus type 2, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus, characterized in that, The kit comprises a mixed antigen consisting of porcine circovirus type 2a, 2b and 2d virus-like particles, recombinant protein of Mycoplasma hyopneumoniae, and inactivated virus solution of porcine reproductive and respiratory syndrome virus SCqq strain.

2. The kit of claim 1, wherein The concentrations of porcine circovirus types 2a, 2b, and 2d virus-like particles, recombinant protein from Mycoplasma hyopneumoniae, and inactivated virus solution of porcine reproductive and respiratory syndrome virus (PRRSV) strain SCqq in the mixed antigen were 0.5-8 μg / mL, 1-16 μg / mL, and 10 μg / mL, respectively. 6.0 -10 8.0 TCID 50 / mL.

3. The kit of claim 2, wherein The concentrations of porcine circovirus types 2a, 2b, and 2d virus-like particles, recombinant protein from Mycoplasma hyopneumoniae, and inactivated virus solution of porcine reproductive and respiratory syndrome virus (PRRSV) strain SCqq in the mixed antigen were 1.5-3 μg / mL, 2-5 μg / mL, and 10 μg / mL, respectively. 7.0 -10 8.0 TCID 50 / mL.

4. The reagent kit according to claim 1, characterized in that, The kit also includes an antigen-coated plate, a PCV2 positive control, a Mhp positive control, a PRRSV positive control, a PCV2, Mhp, and PRRSV triple antibody negative control, a sample diluent, a washing buffer, an enzyme-labeled secondary antibody solution, a substrate chromogenic solution, and a stop solution.

5. The kit of claim 4, wherein After the antigen-coated plate is blocked with 2-3% bovine serum albumin at 35-37°C for 1-3 hours, it is then incubated overnight at 4°C with ELISA plate stabilizer I.

6. The kit of claim 4, wherein The enzyme-labeled secondary antibody solution is an HRP-labeled goat anti-pig IgG enzyme-labeled secondary antibody solution.

7. The kit of claim 4, wherein The serum control was prepared by diluting the serum with protein stabilizer II.

8. A method of screening for antibodies to porcine circovirus type 2, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus, characterized in that, Includes the following steps: The serum sample to be tested is detected using the kit described in any one of claims 1 to 7, and the presence of the corresponding antibody is determined based on the P / N value.

9. The method of claim 8, wherein, The P / N value represents the ratio of the sample or positive control to the negative control. The P value represents the OD value of the sample or positive control, and the N value represents the OD value of the negative control. A sample is considered positive when the P / N value is ≥2.1, indicating that at least one of the three antibodies, PCV2, Mhp, and PRRSV, is suspected. A sample is considered negative when the P / N value is <2.1, indicating that all three antibodies, PCV2, Mhp, and PRRSV, are negative.

10. Use of the kit according to any one of claims 1 to 7 in the preparation of formulations for screening and purifying PCV2, Mhp, and PRRSV antibody triple-negative animals.

Images